Application of electrolytic silver ions in preparation of medicine for treating liver cancer

Abstract

The invention provides application of electrolytic silver ions in preparation of a medicine for treating liver cancer, and belongs to the technical field of medical preparations. According to the application of the electrolytic silver ions in preparation of the medicine for treating the liver cancer, the electrolytic silver ions are high-valence electrolytic silver ion solutions, the concentration of the positive bivalent silver ions is 50-120 ppm, and the pH is 9.5 + / -1.5. Animal experiments preliminarily prove that the electrolytic silver ions can be used for treating the liver cancer and are expected to be developed and applied in preparation of the medicine for treating the liver cancer.

Description

Technical Field

[0001] The invention belongs to the technical field of medical preparations, and in particular relates to the application of electrolytic silver ions in the preparation of drugs for treating liver cancer. Background Art

[0002] Cancer is a common and frequently occurring disease that poses a serious threat to human life and health.

[0003] There are three main types of cancer treatment drugs in my country. One is conventional chemotherapy drugs, which treat cancer by blocking DNA replication and inhibiting protein synthesis, such as docetaxel, paclitaxel, cisplatin, etc. The other is targeted drugs, which inhibit or kill cancer cells by specifically binding to carcinogenic sites and vascular proliferation targets in cancer cells, such as gefitinib, sorafenib, etc. The third is immunotherapy drugs, which automatically identify cancer cells in the body and eliminate them under the action of the immune system by enhancing the patient's own immune function, such as pembrolizumab, nivolumab, toripalimab, etc.

[0004] Although the above existing drugs have made certain contributions to the treatment of cancer, most of them can only inhibit cancer cells. Once the drug is stopped, the cancer will spread, metastasize and recur in a short period of time. They cannot completely cure cancer and are expensive. Therefore, it is urgent to research and develop new anticancer drugs that can completely inactivate cancer cells and have mild toxicity to the body.

[0005] The antibacterial and antiviral properties of electrolytic silver ions have been widely recognized in the academic community. However, there is no consensus in the scientific community as to whether electrolytic silver ions have anticancer effects. This uncertainty mainly stems from the lack of animal cancer model treatment research based on electrolytic silver ions. Due to the lack of relevant experimental data, we are unable to accurately and objectively evaluate the anticancer potential of electrolytic silver ions. In order to fill this gap, the present invention studies the anticancer effect of electrolytic silver ions by independently designing and implementing corresponding animal cancer model treatment experiments. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide application of electrolytic silver ions in preparing drugs for treating liver cancer in view of the deficiencies of the prior art.

[0007] To solve the above technical problems, the technical solution adopted by the present invention is: application of electrolytic silver ions in the preparation of drugs for treating liver cancer, wherein the electrolytic silver ions are high-valence electrolytic silver ion solutions, wherein the concentration of divalent silver ions is 50 to 120 ppm and the pH is 9.5±1.5.

[0008] Optionally, the use of the electrolytic silver ions in the preparation of drugs for treating liver cancer is carried out in animal experiments using athymic nude mice.

[0009] Optionally, the dosage for the athymic nude mice is 150-300 ug / kg body weight, and when the concentration of the divalent silver ions in the electrolytic silver ions is 50 ppm, the dosage is 35-60 ul / 10 g body weight.

[0010] Optionally, the administration route for the athymic nude mice is intraperitoneal injection.

[0011] Optionally, a treatment group and a control group are set up in the experiment using athymic nude mice, the drug used in the treatment group is electrolytic silver ions, and the drug used in the control group is physiological saline.

[0012] Optionally, the athymic nude mice in the treatment group are intraperitoneally injected with electrolytic silver ions once a day, with 70-120ul of electrolytic silver ions with a divalent silver ion concentration of 50ppm each time, for 4-5 consecutive days as one course of treatment, and a second course of treatment is carried out after an interval of 16 days, for a total of two courses of treatment. After the two courses of treatment, the experimental animals are allowed to rest and recover for 10 days, for a total of 50-55 days, and then the experimental animals are killed for toxicity testing.

[0013] Optionally, before killing the experimental animals, record the living status of all experimental animals, as well as whether their diet, defecation, urination, mental state, etc. are abnormal. If there is an accidental death, perform an autopsy to find out the cause of death and take films for preservation.

[0014] Optionally, from the beginning to the end of the experiment, the tumor size of the athymic nude mice was measured every 4 days, and records were made and photographs were taken for preservation.

[0015] Optionally, after the experimental animals are killed, sufficient blood is collected for liver and kidney function tests, and then the lung, liver, and kidney tissues and organs are removed for pathological section testing.

[0016] Optionally, all sacrificed experimental animals were subjected to autopsy, and the subcutaneous tumor sites of the mice were completely peeled off to observe whether there were any metastatic and recurrent lesions, and the images were recorded.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention preliminarily confirms through animal experiments that electrolytic silver ions can be used to treat liver cancer and is expected to be developed and applied in the preparation of drugs for treating liver cancer.

[0019] In the experimental method of the present invention, the drug administration method for animals is only one method, which is simple to operate, easy to administer, and accurate in dosage compared to oral or intravenous administration.

[0020] In the experimental method of the present invention, athymic nude mice are used as a cancer model, and the experimental results are authoritative and highly reliable.

[0021] In the experimental method of the present invention, electrolytic silver ions are used to treat athymic nude mouse tumors, and only two courses of treatment are required, which is short in time and has a good effect.

[0022] In the experimental method of the present invention, the tumor size is recorded every 4 days, important changes are filmed and retained, liver and kidney function tests are performed after the animals are killed, and the lungs, livers and kidneys of the animals are taken for pathological examination and autopsy. The experimental data collection is comprehensive and complete, with high reliability and accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 1 are pathological sections of nude mice in each group of the present invention, wherein A1, A2, and A3 represent HE staining images of the lung, kidney, and liver of mouse No. 1, respectively; B1, B2, and B3 represent HE staining images of the lung, kidney, and liver of mouse No. 3, respectively; and C1, C2, and C3 represent HE staining images of the lung, kidney, and liver of mouse No. 5, respectively.

[0025] Figure 2 These are comparison photos of the changes of nude mouse tumor foci before and after treatment in the present invention.

[0026] Figure 3 This is a nude mouse autopsy diagram of the present invention, from left to right: mouse No. 2, mouse No. 4, mouse No. 1, mouse No. 3 and mouse No. 5. DETAILED DESCRIPTION

[0027] In order to better understand the present invention, the content of the present invention is further clearly set forth in conjunction with the embodiments below, but the protection content of the present invention is not limited to the following embodiments. In the following description, a large number of specific details are provided in order to provide a more thorough understanding of the present invention. However, it is obvious to those skilled in the art that the present invention can be implemented without one or more of these details.

[0028] In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limiting. Therefore, other examples of the exemplary embodiments may have different values.

[0029] Unless otherwise specified, all raw materials are from commercially available products and do not contain other unspecified components except inevitable impurities unless otherwise specified.

[0030] Embodiment 1:

[0031] 1. Test products:

[0032] Electrolytic silver ions with a positive divalent silver ion concentration of 50 ppm, colorless transparent liquid, batch number 221117-3, produced by Luoyang Guanyin Biotechnology Co., Ltd. The production method of electrolytic silver ions is a prior art, please refer to the records of the embodiments in the public / announcement text of patent number ZL202010395234.6.

[0033] 2. Experimental animals:

[0034] Strain: BALB / c-nu athymic nude mice, sex: male, age: 4-6 weeks, source: Shanghai Shengchang Biotechnology Co., Ltd., quantity: 5, animal number: footmark. The experimental animals were kept in the SPF barrier facility environment of Shanghai Shengchang Biotechnology Co., Ltd., using independent ventilation cages IVC. Before the experiment, the experimental animals must undergo 3-7 days of adaptive feeding. The temperature and humidity of the barrier system are controlled in the following range: temperature 22+2℃, humidity 40-70%, and day and night alternation time 12h / 12h. Cage: Made of PEI, volume 320mmx200mmx135mm. Wood shavings are sterilized and cleaned by high pressure sterilization of bedding, and changed 1-2 times a week. Each cage has a cage label indicating the number of animals, sex, strain, receipt time, group and start time of the experiment. Feed and drinking water: Normal breeding feed for nude mice (sterilized and sealed packaging) was purchased from Shanghai Proton Biotechnology Co., Ltd., and drinking water was purified water provided by Shanghai Zhihuakui Biotechnology Co., Ltd., which was sterilized. The animals had free access to sterile food and water.

[0035] 3. Experimental instruments: as shown in Table 1.

[0036] Table 1 Experimental instruments

[0037] Reagent / Instrument Name Supply Unit Part Number Storage conditions Electronic balance Shanghai Jingtian Electronic Instrument Co., Ltd. JT201N Room temperature Disposable syringe Shanghai Kindly Enterprise Development Group Co., Ltd. K20190705 Room temperature

[0038] 4. Experimental methods:

[0039] 50ppm electrolytic silver ions are used directly without adding any other materials. They are drawn up with a disposable syringe and injected intraperitoneally.

[0040] Table 2 Animal grouping

[0041]

[0042]

[0043] 5. Experimental plan:

[0044] Five male nude mice aged 4 to 6 weeks were selected and subcutaneously loaded with HepG2 liver model, which was prepared by Shanghai Zhihuakui Biotechnology Co., Ltd. The five nude mice were divided into three groups: the first group had two mice, which was a low-dose treatment group; the second group had two mice, which was a high-dose treatment group; and the third group had one mouse, which was a control group.

[0045] Low-dose group: On the 1st to 4th day, 75ul of electrolytic silver ions were injected intraperitoneally each day, and 4 days was a course of treatment, with a total of 300u1 of intraperitoneal injection, which is equivalent to 180ml of electrolytic silver ions per day for an adult weighing 60 kg, and a total of 720ml for 4 days. Since the elimination of electrolytic silver ions in the body is slow and takes about 16 days to be emptied, the animals in this experiment rested on the 5th to 20th day, and the second course of treatment was carried out on the 21st to 24th day, with the dose still being 75ul each time per day, and resting on the 25th to 40th day. After the electrolytic silver ions were emptied, the body was allowed to rest and recover for 10 days. On the 50th day, the animals were killed for autopsy, and one of the two nude mice was randomly selected. After the killing, sufficient blood was collected for liver and kidney function tests, and then the lung, liver, and kidney tissues and organs were removed for pathological section tests.

[0046] High-dose group: The administration method is the same as the low-dose group, only the dosage is different. On the 1st to 4th day, 110ul of electrolytic silver ions are injected intraperitoneally each day, and a total of 440ul is given in 4 days, which is equivalent to 264ml of electrolytic silver ions per day for an adult weighing 60 kg, and a total of 1056ml in 4 days. The dosage of the second course of treatment is the same as the first course of treatment. The animals are killed on the 50th day, and the detection method is the same as the low-dose group.

[0047] Control group: physiological saline was used instead of electrolytic silver ions, and the usage, dosage and other procedures were the same as the low-dose group.

[0048] All treated nude mice were autopsied, with a focus on examining the subcutaneous tissues and tissues and organs for metastasis, spread, and recurrence of cancer cells.

[0049] 6. Analysis of experimental results:

[0050] 6.1 Changes in tumor size in nude mice during the experiment

[0051] During the experiment, the changes in the size of nude mouse tumors were measured by a third party, Shanghai Zhihuakui Biological Co., Ltd. The results are shown in Table 3.

[0052] In the table, L length , L short and V represent the length (mm), width (mm) and volume (mm) of the tumor, respectively. 3 ).

[0053] Table 3 Changes of tumor size in nude mice of different groups over time

[0054]

[0055]

[0056] Combined with the results in Table 3, the effect of electrolytic silver ions on the size of nude mouse tumors is analyzed as follows: From June 28, the nude mice were intraperitoneally injected with electrolytic silver ions. By July 23, the tumors of the four nude mice in the treatment group had all disappeared. The total number of days was 25, and the tumor disappearance rate of nude mice was 100%. At this time, the tumor volume of the nude mice in the control group was 319.994mm 3 , it can be seen that the effect of electrolytic silver ions in inactivating cancer cells is very obvious.

[0057] 6.2 Changes in nude mouse body weight during the experiment

[0058] The weight changes of nude mice during the experiment were measured by a third party, Shanghai Zhihuakui Biotechnology Co., Ltd. The results are shown in Table 4.

[0059] Table 4 Nude mouse weight test record

[0060]

[0061] Combined with the data in Table 4, the results of mouse weight test were analyzed: During the drug administration period, the weight of mice was recorded. From the weight data, the weight of mice in each group increased in an orderly manner with growth, and there was no significant difference in weight between the high-concentration drug administration group and the low-concentration drug administration group; combined with the daily status of the mice, neither the high-concentration drug administration group nor the low-concentration drug administration group affected the normal growth and eating of the mice, and overall, the weight of the drug administration group tended to be higher than that of the control group. Although it was not obvious, it can be seen that drug administration is more conducive to the recovery of the body functions of mice. It is also possible that with the treatment effect, the tumor in the drug administration group disappeared, resulting in the overall trend of mouse weight being better than that of the control group.

[0062] 6.3 Observation of the toxicity of electrolytic silver ions

[0063] The toxicity observation of electrolytic silver ions was measured by a third party, Shanghai Zhihuakui Biotechnology Co., Ltd., and the results are shown in Table 5.

[0064] Table 5 Results of liver and kidney function tests in nude mice

[0065]

[0066] Combined with the data in Table 5, the common indicators of liver and kidney function in nude mice are analyzed as follows: Alanine aminotransferase (ALT): the normal range is generally around 10-100u / L; Aspartate aminotransferase (AST): the normal range is usually around 20-100u / L. From the experimental results in Table 5, it can be seen that the nude mice in each group tested in this test are all within the normal range. From the results of serum tests, there was no liver and kidney damage to nude mice after administration.

[0067] 6.4 Results and analysis of nude mouse lung, liver and kidney pathology

[0068] The nude mouse lung, liver and kidney pathological tests were commissioned to a third party, Shanghai Zhihuakui Biotechnology Co., Ltd., and the results were as follows: Figure 1 shown.

[0069] See also Figure 1 , Pathological examination results of lung, liver and kidney of nude mice. At the end point sampling, the lungs, kidneys and livers of nude mice in each group were sampled and HE staining was performed. The HE results of lungs showed that the alveolar ducts were connected to a large number of alveoli, and their surfaces were covered with a single layer of cubic or flat epithelium. The alveolar sacs were connected to the alveolar ducts, and each alveolar duct branched into 2 to 3 alveolar sacs. The alveoli were polyhedral vesicles with openings, which opened in the alveolar sacs. The structures of various tissues were normal, and no adverse reconstruction and immune cell increase were observed. The two concentrations of electrolytic silver ion drugs did not cause toxic adverse effects on the lungs after administration. The results of kidney HE staining showed that the kidney was shaped like a broad bean, with a clear renal hilum, and the outer surface of the kidney was covered with a fibrous capsule. The outer layer was the cortex, which contained glomeruli, proximal tubules, and distal tubules; the inner layer was the medulla, which contained proximal tubules, distal tubules and collecting ducts. The structures of nude mice in each group were clear, and no adverse reconstruction was found. Therefore, it was speculated that the electrolytic silver ion drugs did not cause irreversible toxic damage to the kidneys of nude mice. The results of liver HE showed that the hepatocytes were arranged in a single row in plate-like shape. The liver plates were uneven and roughly radial. The adjacent liver plates were anastomosed to form a labyrinth-like structure. The hepatic alveoli were composed of 1 / 6 of each of the two adjacent hepatic lobules, and their volume was 1 / 3 of the hepatic lobule. The hepatic alveoli were divided into three zones. The cells near the axial blood vessels had active metabolism and normal morphology. Judging from the liver tissue structure, there were no necrotic areas in the hepatocytes. Therefore, it was speculated that the electrolytic silver ion drug was not toxic to the liver of nude mice.

[0070] 6.5 Results and analysis of changes in nude mouse tumor foci before and after treatment

[0071] The changes in nude mouse tumor foci before and after treatment were measured by a third party, Shanghai Zhihuakui Biotechnology Co., Ltd. The results are as follows Figure 2 .

[0072] See also Figure 2 , Figure 2 The results show the changes in the tumor foci of nude mouse No. 3 before and after treatment. This result more intuitively proves that electrolytic silver ions have a significant therapeutic effect on nude mouse liver cancer.

[0073] 6.6 Nude Mouse Autopsy

[0074] See also Figure 3When sampling, the subcutaneous tissue of the nude mice was completely stripped, and no metastatic lesions were found in the four nude mice treated with intraperitoneal injection of electrolytic silver ions. The tumors had all disappeared and did not recur. Only the nude mice in the control group injected with normal saline still had tumor tissue masses, which further proved that electrolytic silver ions have a significant inactivating effect on cancer cells.

[0075] 7. Conclusion;

[0076] According to the above experimental results, it can be seen that electrolytic silver ions have a deactivating effect on cancer cells and have no toxic side effects on the lungs, liver, and kidneys of animals.

[0077] The experimental method of treating rat liver cancer with electrolytic silver ions of the present invention can be further implemented and studied according to the following scheme.

[0078] Embodiment 2:

[0079] Fifteen athymic nude mice aged 4 to 6 weeks were obtained from Shanghai Shengchang Biotechnology Co., Ltd. The subcutaneous liver-loaded model HepG2 was prepared by Shanghai Zhihuakui Biotechnology Co., Ltd. The 15 nude mice were divided into 3 groups: the first group of 5 mice was the low-dose treatment group; the second group of 5 mice was the high-dose treatment group; and the third group of 5 mice was the control group. Low-dose group: On the 1st to 4th day, 80ul of electrolytic silver ions were injected intraperitoneally each day, and 4 days was a course of treatment, with a total of 320u1 injected intraperitoneally. Since the electrolytic silver ions are eliminated slowly in the body and take about 16 days to be emptied, the animals rested on the 5th to 20th day, and the second course of treatment was carried out on the 21st to 24th day, with the dosage still being 80ul each time per day. The animals rested on the 25th to 40th day, and the body was allowed to rest and recover for 15 days after the electrolytic silver ions were emptied. On the 55th day, the animals were killed for autopsy, and one nude mouse was randomly selected from 5. After the killing, a sufficient amount of blood was collected for liver and kidney function tests, and then the lung, liver, and kidney tissues and organs were removed for pathological section testing and autopsy. High-dose group: The administration method is the same as the low-dose group, only the dosage is different. On the 1st to 4th day, 120ul of electrolytic silver ions are injected intraperitoneally each day, a total of 480u for 4 days. The dosage of the second course of treatment is the same as the first course of treatment. The animals are killed on the 55th day, and the other procedures are the same as the low-dose group. Control group: Physiological saline is used instead of electrolytic silver ions, and the usage, dosage and other procedures are the same as the low-dose group.

[0080] Embodiment three:

[0081] Thirty athymic nude mice aged 4 to 6 weeks were obtained from Shanghai Shengchang Biotechnology Co., Ltd., and the subcutaneous liver-loaded model HepG2 was prepared by Shanghai Zhihuakui Biotechnology Co., Ltd. The 30 nude mice were divided into 3 groups: the first group of 10 mice was the low-dose treatment group; the second group of 10 mice was the high-dose treatment group; and the third group of 10 mice was the control group. Low-dose group: On the 1st to 4th day, 70ul of electrolytic silver ions were injected intraperitoneally each day, and 4 days was a course of treatment, with a total of 280u1 injected intraperitoneally. Since the electrolytic silver ions are eliminated slowly in the body and take about 16 days to be emptied, the animals rested on the 5th to 20th day, and the second course of treatment was carried out on the 21st to 24th day, with the dosage still being 80ul each time per day. The animals rested on the 25th to 40th day, and the body was allowed to rest and recover for 15 days after the electrolytic silver ions were emptied. On the 55th day, the animals were killed for autopsy, and one nude mouse was randomly selected from 5. After the killing, a sufficient amount of blood was collected for liver and kidney function tests, and then the lung, liver, and kidney tissues and organs were removed for pathological section testing and autopsy. High-dose group: The administration method is the same as the low-dose group, only the dosage is different. From the 1st to the 4th day, 110ul of electrolytic silver ions are injected intraperitoneally each day, and a total of 440ul is injected in 4 days. The dosage of the second course of treatment is the same as the first course of treatment. The animals are killed on the 55th day, and the other procedures are the same as the low-dose group. Control group: Physiological saline is used instead of electrolytic silver ions, and the usage, dosage and other procedures are the same as the low-dose group.

[0082] The present invention preliminarily confirms through animal experiments that electrolytic silver ions can be used to treat liver cancer and is expected to be developed and applied in the preparation of drugs for treating liver cancer.

[0083] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Other modifications or equivalent substitutions made to the technical solution of the present invention by ordinary technicians in the field should be included in the scope of the claims of the present invention as long as they do not depart from the spirit and scope of the technical solution of the present invention.

Claims

1. The application of electrolytic silver ions in the preparation of drugs for treating liver cancer is characterized by: The electrolytic silver ion is a high-valence electrolytic silver ion solution, wherein the concentration of divalent silver ions is 50-120 ppm and the pH is 9.5±1.

5.

2. The use of electrolytic silver ions in the preparation of a drug for treating liver cancer as claimed in claim 1, characterized in that: Animal experiments were performed using athymic nude mice.

3. The use of electrolytic silver ions in the preparation of a drug for treating liver cancer as claimed in claim 2, characterized in that: The dosage for the athymic nude mice is 150-300 ug / kg body weight. When the concentration of divalent silver ions in the electrolytic silver ions is 50 ppm, the dosage is 35-60 ul / 10 g body weight.

4. The use of electrolytic silver ions in the preparation of a drug for treating liver cancer as claimed in claim 3, characterized in that: The administration route for the athymic nude mice is intraperitoneal injection.

5. The use of electrolytic silver ions in the preparation of a drug for treating liver cancer as claimed in claim 4, characterized in that: In the experiment using athymic nude mice, a treatment group and a control group were set up. The drug used in the treatment group was electrolytic silver ions, and the drug used in the control group was normal saline.

6. The use of electrolytic silver ions in the preparation of a drug for treating liver cancer as claimed in claim 5, characterized in that: The athymic nude mice in the treatment group were intraperitoneally injected with electrolytic silver ions once a day, with 70-120ul of electrolytic silver ions with a divalent silver ion concentration of 50ppm each time, for 4-5 consecutive days as one course of treatment, and a second course of treatment was carried out after an interval of 16 days, for a total of two courses of treatment. After the two courses of treatment, the experimental animals were allowed to rest and recover for 10 days, for a total of 50-55 days, and then the experimental animals were killed for toxicity testing.

7. The use of electrolytic silver ions in the preparation of a drug for treating liver cancer as claimed in claim 6, characterized in that: Before killing the experimental animals, record the living status of all experimental animals, as well as whether their diet, defecation, urination, mental state, etc. are abnormal. If there is an accidental death, perform an autopsy to find out the cause of death and take films for preservation.

8. The use of electrolytic silver ions in the preparation of a drug for treating liver cancer as claimed in claim 7, characterized in that: From the beginning to the end of the experiment, the tumor size of the athymic nude mice was measured every 4 days, and records were made and photographs were taken for preservation.

9. The use of electrolytic silver ions in the preparation of a drug for treating liver cancer as claimed in claim 8, characterized in that: After the experimental animals are killed, sufficient blood is collected for liver and kidney function tests, and then the lung, liver, and kidney tissues and organs are removed for pathological section testing.

10. The use of electrolytic silver ions in the preparation of a drug for treating liver cancer as claimed in claim 9, characterized in that: All experimental animals were autopsied and the subcutaneous tumor sites of the mice were completely peeled off to observe whether there were any metastatic and recurrent lesions, and the images were taken and recorded.

Images