AU2021100812A4 - Medicament for treating liver cancer and thyroid cancer by local injection, and preparation method thereof - Google Patents

Abstract

DCC- 902/2021 ABSTRACT The present disclosure discloses a medicament for treating liver cancer and thyroid cancer by local injection, and a preparation method thereof. Through long-term research, the inventors found for the first time that aluminum sulfate can distinguish between normal human tissues and cancer tissues, and can change the physiological characteristics of cancer cells, inhibit the secretion of various proteolytic enzymes by cancer cells and suppress the metabolic function of mitochondria in cancer cells to achieve anti-tumor effects. The medicament can bind to DNA of cancer cells and promote the lysis thereof, and can quickly and efficiently converge and condense glycoproteins on the cell surface, change protein structures on the cell surface, block signaling pathways, change cell microstructures, and bind to succinate dehydrogenase (SDH) in mitochondria. Malignant solid tumors with the highest incidence among human beings, the highest mortality, the highest treatment difficulty, and the worst prognosis (including lymphatic metastasis with a relatively-high mortality) can be treated by western medicines (specific), with a cure rate as high as 99.8% to 99.9% or even 100%. The present disclosure has important practical significance for humans to conquer liver cancer and thyroid cancer by local injection. 21160142.1:DCC -12/03/2021 3/20 A 120 IC50=14.408mg/ml Emax=100% 0o, 100 CO 0 -0 -o 40 20 0 1 0 1 2 3 Logarithmicconcentration FIG. 3 B 120 IC50=14.116mg/ml Emax=100% 100 so. aD 4 CU80 0 40 -0o 0 -1 2 3 Logarithmic concentration FIG. 4

Description

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3/20

A 120 IC50=14.408mg/ml Emax=100% 0o, 100

CO 0-0 -o

40

20

0

1 0 1 2 3 Logarithmicconcentration FIG. 3

B 120 IC50=14.116mg/ml Emax=100% 100

so. aDCU80 4

0 40

-0o

0

-1 2 3

Logarithmic concentration

FIG. 4

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MEDICAMENT FOR TREATING LIVER CANCER AND THYROID CANCER BY LOCAL INJECTION, AND PREPARATION METHOD THEREOF

TECHNICAL FIELD The present disclosure relates to the field of medicine, and in particular to a medicament for treating liver cancer and thyroid cancer by local injection, and a preparation method thereof. BACKGROUND It is well known that malignant tumors are a class of frequently-occurring and common diseases that seriously threaten human health, whose pathogenesis has not been fully figured out. No satisfactory therapeutic effect has been achieved for malignant tumors. Malignant tumors, viral diseases and senile diseases are known as three major challenges for modem medicine. According to incomplete statistics, no less than tens of billions of money is expended on the research and treatment of tumors on average each year globally, causing huge losses to the country, society and individuals. Liver cancer is a malignant tumor in the liver, which includes two categories: primary and secondary. Primary hepatic malignant tumors originate from the epithelial or mesenchymal tissues of the liver. A primary hepatic malignant tumor originating from the epithelial tissue is called primary liver cancer, which is a malignant tumor with high incidence and great harm in China; and a primary hepatic malignant tumor originating from the mesenchymal tissue is called sarcoma, which is uncommon compared with the primary liver cancer. Secondary or metastatic liver cancer refers to invasion of malignant tumors originating from various other organs of the body into the liver, which is common in liver metastases of malignant tumors in stomach, biliary tract, pancreas, colorectum, ovary, uterus, lung, breast, and other organs. The pathogeny and exact molecular mechanism of primary liver cancer are not fully understood. At present, the pathogenesis of primary liver cancer is considered to be a multi-factor and multi-step complex process, which is affected by both the environment and the diet. Epidemiological and experimental research data show that hepatitis B virus (HBV) and hepatitis C virus (HCV) infection, aflatoxin, drinking water pollution, alcohol, liver cirrhosis, sex hormones, nitrosamines, trace elements, and the like are all correlated with the onset of liver cancer. Secondary liver cancer (metastatic liver cancer) can be

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formed through different ways, such as metastasis with blood and lymph or direct infiltration into the liver. 1. Primary liver cancer (1) Symptoms: Early liver cancer often shows no specific symptoms, while moderate or advanced liver cancer shows many symptoms, including common clinical manifestations: liver pain, abdominal distension, poor appetite, fatigue, weight loss, progressive hepatomegaly, upper abdominal masses, or the like. Some patients have low-grade fever, jaundice, diarrhea, and upper gastrointestinal bleeding. Acute abdomen or the like occurs after the rupture of liver cancer. There are also symptoms that are not obvious or only manifest as metastases. (2) Signs: Early liver cancer often leads to no obvious positive signs or only leads to signs similar to that of liver cirrhosis. Signs of hepatomegaly, jaundice, ascites and the like usually appear in patients with moderate or advanced liver cancer. In addition, liver cancer patients with liver cirrhosis often have liver palms, spider nevi, gynecomastia, lower limb edema, and the like. When extrahepatic metastasis occurs, corresponding signs may appear at each metastatic site. (3) Common complications include upper gastrointestinal bleeding, liver cancer rupture and bleeding, liver and kidney failure, and so on. 2. Secondary liver cancer (1) The clinical manifestations of a primary tumor are mainly seen in patients with no history of liver disease, and hepatic metastases are still at an early stage and cause no corresponding symptoms, but most symptoms of the primary tumor obviously appear at middle and late stages. The secondary liver cancer of such patients is mostly found in the examination and follow-up of the primary therapy. (2) The clinical manifestations of secondary liver cancer, often self-reported by patients, include stuffiness or dull pain in upper abdomen or liver, and as the disease progresses, patients develop fatigue, poor appetite, weight loss, fever, or the like. During physical examination, an enlarged liver or hard nodules with a stiff texture and tenderness can be palpable in the upper middle abdomen, and anemia, jaundice, ascites or the like may appear in patients with advanced liver cancer. The clinical manifestations of such patients are similar to that of patients with primary liver cancer, but generally develops relatively slowly and are less severe. Most of the tumors are suspected to metastasis during

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various liver examinations, and the primary tumor can be found during further examination or surgical exploration. The primary cancer focus cannot be found in some patients through various examinations. (3) The clinical manifestations of both a primary tumor and the secondary liver cancer may be mainly seen when both the primary tumor and the liver metastasis are not at an early stage. In addition to the symptoms and signs similar to that of primary liver cancer in the liver, patients also have the clinical manifestations caused by the primary tumor, for example, the hepatic metastasis of colorectal cancer (CRC) may be accompanied by changes in bowel habits and stool characteristics, hematochezia, and so on. Individualized comprehensive treatment can be applied as appropriate according to different stages of liver cancer, which is the key to improving a curative effect; and treatment methods include surgery, hepatic artery ligation (HAL), hepatic artery chemoembolization (HACE), radiofrequency (RF) therapy, cryotherapy, laser therapy, microwave therapy, chemotherapy, radiotherapy, and so on. Biotherapy and therapies using traditional Chinese medicine (TCM) are also used in the treatment of liver cancer. 1. Surgical treatment Surgery is a preferred choice and the most effective way to treat liver cancer. Surgical methods include radical hepatectomy, palliative hepatectomy, and so on. For unresectable liver cancers, intraoperative HAL, HACE, RF therapy, cryotherapy, laser therapy, microwave therapy and other therapies can be applied based on practical conditions to achieve some curative effects. Primary liver cancer is also one of the indications for liver transplantation. 2. Chemotherapy When a laparotomy reveals that the cancer cannot be resected, or as follow-up treatment of palliative tumor resection, a hepatic artery and/or portal vein pump (a subcutaneously embedded perfusion device) can be used for regional chemoembolization. Patients whose tumors are estimated to be unresectable can also be subjected to interventional radiotherapy, where, selective cannulation is conducted via the femoral artery to the hepatic artery, and embolic agents (such as commonly used iodized oil) and anti-cancer medicaments are injected for chemoembolization, and some patients can thus get the opportunity of surgical resection.

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3. Radiotherapy Radiation-based comprehensive treatment can be adopted for patients who have preferable general conditions and fair liver functions without liver cirrhosis, jaundice, ascites, hypersplenism, esophageal varices and distant metastasis, have relatively limited tumors, and are not suitable for surgical resection or may be subjected to relapse after surgery. 4. Biotherapy Immune ribonucleic acid (iRNA), interferon, interleukin-2, thymosin, etc. are commonly used, which can be used in combination with chemotherapy. 5. Therapies using TCM These therapies are treatment methods based on an overall analysis of a patient's illness and conditions that combine tonification and purgation, which are often used in conjunction with other therapies. These therapies can improve the body's resistance to diseases, improve the general conditions and symptoms, and alleviate adverse reactions caused by chemotherapy and radiotherapy. Existing treatment methods have the disadvantages of high cost, long course of treatment, and poor effect. Therefore, there is an urgent need for a medicament for treating liver cancer to alleviate pressing demands of cancer patients. Thyroid cancer is the most common malignant tumor among head and neck tumors, with an incidence showing a continuous and rapid growth trend after 2000. The incidence of thyroid cancer in 2009 increased by nearly three times compared with that in 1975, from 4.9/100,000 to 14.3/100,000. The 10-year incidence of thyroid cancer increased by nearly 400% in China (Beijing, 2013). In 2010, the incidence of thyroid cancer among Chinese women rose to the sixth place, and the incidence of thyroid cancer among American women rose to the fifth place, which attracted widespread attention from the society. The diagnosis and treatment of thyroid tumors has received more and more attention from the society and medical community. Lymph nodes are often the first site of thyroid cancer metastasis in anatomical location, which have a relatively high probability to develop metastases. So in clinics, there are still some controversies about whether central lymph nodes in patients with thyroid cancer need to be dissected, including necessity to dissect central lymph nodes, dissection scope, etc. Professor Gao Li believes that, given that the incidence of thyroid cancer metastasis is high

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and the central lymph nodes are often the first affected areas, it is very necessary to conduct a central lymph node dissection for patients with thyroid cancer. If not dissected or incompletely dissected, the cancer will definitely relapse, and meanwhile, it is more difficult to conduct surgery. The treatment methods of thyroid cancer mainly include: surgical therapy, endocrine therapy, radionuclide therapy, and external radiation therapy. At present, there are no effective methods and medicaments to treat thyroid cancer clinically. Therefore, there is an urgent need for an effective anti-cancer medicament to alleviate the pressing demands of cancer patients. Aluminum sulfate is a common sulfate, which can be used as a precipitating agent for rosin size, wax emulsion and other sizing materials in the paper industry, as a flocculant in water treatment, as an internal retention agent for foam extinguishers, as a raw material for producing alum and aluminum white, and as an adjuvant for petroleum decolorization, deodorization, and some medicaments. About 50% of the total aluminum sulfate output is used in papermaking, as the first major use, and about 40% of the total aluminum sulfate output is used as a flocculant in treatment of drinking water, industrial water and industrial wastewater, as the second major use. When aluminum sulfate is added to these types of water, colloidal aluminum hydroxide flakes can be produced to adsorb and precipitate bacteria, colloids and other suspended solids. Aluminum sulfate can be used in drinking water treatment to control the color and taste of water. SUMMARY The present disclosure is intended to provide a low-toxic and highly-efficient medicament for treating liver cancer and thyroid cancer by local injection, where, an active ingredient in the medicament is aluminum sulfate or a hydrate thereof. A first aspect of the present disclosure relates to a medicament for treating liver cancer and thyroid cancer by local injection, where, the medicament includes aluminum sulfate or a hydrate thereof and pharmaceutically acceptable adjuvants. A second aspect of the present disclosure relates to the medicament that is a powder or an injection, and preferably a powder injection administered by local injection. A third aspect of the present disclosure relates to a method for preparing the medicament, and the method includes the step of mixing the aluminum sulfate or a hydrate thereof with the pharmaceutically acceptable adjuvants.

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The method for preparing the medicament described above may preferably further include a step of preparing the aluminum sulfate or a hydrate thereof: dissolving aluminum sulfate, and subjecting a resulting solution to fine filtration and lyophilization to obtain the aluminum sulfate or a hydrate thereof. The method for preparing the aluminum sulfate or a hydrate thereof may specifically include: mixing aluminum sulfate with distilled water at a mass ratio of 1:2; after the aluminum sulfate is completely dissolved, subjecting a resulting solution to finefiltration, and washing a resulting filter cake with the same mass of distilled water; and subjecting a resulting filtrate to lyophilization to obtain powdered aluminum sulfate or a hydrate thereof. The medicament may preferably be a powder or an injection, and more preferably a powder injection administered by local injection. A fourth aspect of the present disclosure relates to use of a composition in the preparation of a medicament for treating liver cancer and thyroid cancer by local injection, where, the composition includes aluminum sulfate or a hydrate thereof and pharmaceutically acceptable adjuvants; and the aluminum sulfate or a hydrate thereof is an active ingredient, and preferably all active ingredients or the sole active ingredient. The medicament may preferably be a powder or an injection, and more preferably a powder injection administered by local injection. A fifth aspect of the present disclosure relates to use of aluminum sulfate or a hydrate thereof in the preparation of a medicament for treating liver cancer and thyroid cancer by local injection. The medicament may preferably be a powder or an injection, and more preferably a powder injection administered by local injection. According to the medicament and the preparation method thereof, the use of the composition, the use of aluminum sulfate or a hydrate thereof provided in the present disclosure, the aluminum sulfate or a hydrate thereof may be selected from aluminum sulfate octadecahydrate. Moreover, the aluminum sulfate or a hydrate thereof according to the present disclosure may preferably be prepared by the aforementioned preparation method. The high-performance liquid chromatography (HPLC) chromatogram is more preferably basically as shown in FIG. 1.

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In order to completely eliminate emission of the "three wastes" (waste gas, waste water, and waste residues), high-purity aluminum sulfate (analytical purity) produced by a manufacturer can also be directly used according to the present disclosure. However, strict quality control must be conducted from the source of the manufacturer. After decades of research and exploration, the present disclosure found for the first time that aluminum sulfate can distinguish between normal human tissues and cancerous tissues and promote the volunteer separation and exfoliation of the cancerous tissues from the normal tissues, and can also distinguish between normal cells and cancer cells and selectively starve cancer cells. So far, aluminum sulfate is a leading chemical medicament that can achieve the fastest and most accurate treatment for malignant tumors, with low toxicity and high efficiency. Aluminum sulfate has basically no inhibitory effect on normal cells, can quickly kill cancer cells and reduce tumor volumes, and exhibits a therapeutic effect far better than that of a traditional anti-cancer medicament. The advent of this medicament can save tens of thousands of lives, which is especially suitable for the treatment of malignant solid tumors. The present disclosure preferably provides a preparation for treating liver cancer and thyroid cancer by local injection, and a preparation method thereof. The preparation has the characteristics of small dosage, quick onset, short course of treatment, small side effects, etc. The preparation for treating liver cancer and thyroid cancer by local injection can quickly reduce tumor volumes, induce apoptosis, and inhibit proliferation. Usage and dosage for local injection into malignant solid tumors (western medicine): 1 g of purified aluminum sulfate is dissolved in 3 mL x 0.9% normal saline (NS) to obtain a colorless and transparent injection with aluminum sulfate being completely dissolved. Local direct injection under the guidance of CT localization and B-scan ultrasonography is as follows: Key word 1: After the injection into tumors, a patient must be prohibited from exercising for 25 min to 30 min, which is intended to prevent the injected medicament from being squeezed out of the tumor and thus failing to exert its efficacy. Conclusion: The present disclosure is a major milestone in human history. The present disclosure can make an inestimable outstanding contribution to the treatment of severe human diseases. Refractory malignant solid tumors with the highest incidence among human beings, the highest mortality, the highest treatment difficulty, and the worst

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prognosis (including lymphatic metastasis) can be treated by local injection of western medicines (specific), with a cure rate as high as 99.8% to 99.9% or even 100%. Once the commercialization is successful, tens of thousands of lives can be saved every year. The medicament of the present disclosure has been painstakingly pursued by humans for decades. One injection can be administered for inactivation, which will surely astonish the world. The advent of the medicament can rewrite the history that human tumors emerge endlessly, require a large amount of time and money for treatment, and may not be cured after a long time of treatment. Pharmacology: The key pathways of cancer cell apoptosis after administration are as follows: 1. The medicament can quickly and efficiently converge and condense glycoproteins on the surface of cancer cells. 2. The medicament can change the physiological characteristics of cancer cells. 3. The medicament can inhibit the secretion of various proteolytic enzymes by cancer cells. 4. The medicament can inhibit the metabolic function of mitochondria in cancer cells. 5. The medicament can bind to DNA in cancer cells and promote the lysis thereof. 6. The medicament can effectively change a protein structure on the surface of cancer cells. 7. The medicament can block the signaling pathway. 8. The medicament can quickly and forcefully close the cancer cell feeding channel. 9. The medicament can change cell microstructures. 10. The medicament can bind to succinate dehydrogenase (SDH) in mitochondria to change the biological effect thereof. 11. Normal cells are fed regularly while cancer cells are fed irregularly. 12. The medicament selectively attacks abnormal cancer cells and does not attack normal cells. 13. The medicament selectively attacks abnormal cancer tissues and does not attack normal tissues. 14. With the continuous penetration of the medicament injected into malignant solid tumors, abnormal malignant cancer tissues attached to normal tissues and organs of the human body are gradually separated and exfoliated volunteerly.

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Notes: For a mouse tumor volume of 3 cm x 3 cm and at a dosage of 300 mg/mL, the tumor is scabbed at 6 h and disappears the next day. At a dosage of 400 mg/mL to 500 mg/mL, the tumor activity disappears more quickly. Toxicology: From decades of research and testing, no obvious toxic and side effects have been seen at high dosages. Efficacy: The medicament has the characteristics of small dosage, quick onset, short course of treatment, small side effects, etc. The medicament to treat the following malignant solid tumors is the latest compound and TCM that humans most hope to find and discover so far. The treatment of the following severe diseases does not vary from person to person, and consistency can be achieved. Notices: 1. Before any tumor at an early, middle or late stage (malignant solid tumor) is injected with the medicament, general examination needs to be conducted by positron emission tomography-computed tomography (PET/CT). 2. Routine blood examination. 3. All carcinoembryonic indicators are fully examined, needle biopsy is conducted after a treatment period ends, and examination results before and after the treatment are compared. 4. An imaging agent is injected according to a traditional therapy. The puncture injection should avoid blood vessels and the medicament is injected into the core of a tumor. 5. If a malignant tumor is wrapped by organs in a cavity, it is inconvenient for local percutaneous injection. A laparotomy can be conducted to achieve injections at multiple sites at one time, and then the abdominal cavity is closed. For tumors (malignant tumors) at a late stage that are too large in size, multi-site injection can be conducted by inserting needles at one time, so as to avoid the need to insert needles multiple times. The medicament must be injected into the core of a tumor rather than the surface of the tumor, otherwise, no effects will be achieved. 6. In short, it is simple, fast and thorough in treating human malignant solid tumors. 7. For tumors at early and middle stages, only a single injection is required; and for tumors at a late stage, multi-site injection is conducted at one time, and the tumor can be observed to shrink rapidly the next day. The course of treatment is 6 days and the

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examination method must be a biopsy. If other methods are used for examination, it is a misjudgment that observing a shadow does not refer to the presence of cancer. Practice has proved that all cancers are not formed by accumulation of cancer cells. Most tumors only include 45% to 55% of cancer cells, and the rest are cells whose autoimmune repair is damaged by cancer cells. Therefore, it is critical to adopt a needle biopsy after the treatment period ends, which can truly, objectively, fairly and scientifically reflect the results. Scirrhous carcinoma: with few cancer cells but much stroma, and a hard texture. Simple carcinoma: with a ratio of parenchyma to stroma: approximately 1:1. Atypical medullary carcinoma: with more parenchyma but less stroma, a soft texture, large cancer cells, obvious atypia, and common mitotic figures, where, there is generally no lymphocytic infiltrate in the stroma and the prognosis of a traditional treatment is poor. Solid cancer: a general term for cancer cells that grow into a solid, where, the cancer nest is solid, which has no glandular cavity-like structures but has high atypia and many mitotic figures; and solid cancers include early liver cancer and thyroid cancer, which have different ratios of parenchyma to stroma. Research findings: 1. Malignant tumors and lesion neoplasms thereof are not controlled by the human central nervous system macroscopically and microscopically, so there is basically no pain sense at these sites when injection. Because the human body's normal skin and flesh tissues are controlled by the central nervous system macroscopically and microscopically, there is pain sense at these sites. 2. Therefore, injection of the medicament into normal skin and muscle tissues of the human body should be avoided as far as possible when injection. If the medicament penetrates into the normal skin and muscle tissues of the human body, there will not be a serious problem, and the pain sense will voluntarily disappear about 12 h to 24 h later without other auxiliary treatments.

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Table 1 Different dosages for local injection into malignant solid tumors with different volumes Notes: each (powder injection): 4,000 mg diluted in 8 mL of 0.9% sodium chloride NS. No. Tumor volume (cm) Dosage x0.9% sodium Injection method chloride injection Solid cancer: 2 cm x 2 cm 2 mL local percutaneous injection into a 1 to 3 cm x 3 cm malignant solid tumor Solid cancer: 4 cm x 4 cm 4 mL local percutaneous injection into a 2 to 5 cm x 5 cm malignant solid tumor 6 mL local percutaneous injection into a 3 Solid cancer: 6 cm x 6 cm to 7 cm x 7 cm malignant solid tumor Solid cancer: 8 cm x 8 cm 8 mL local percutaneous injection into a 4 to 9 cm x 9 cm malignant solid tumor 10 mL local percutaneous injection into a 5 Solid cancer: 10 cm x 10 cmto 11 cm x 11 cm malignant solid tumor Solid cancer: 12 cm x 12 12 mL local percutaneous injection into a 6 cm to 13 cm x 13 cm malignant solid tumor Notes: The onset time of the medicament is 3 min to 5 min, namely, cancer cell apoptosis begins after that time. A patient is prohibited from walking within 25 min to 30 min after injection to avoid loss of the medicament and reduction of the therapeutic effect. The medicament of the present disclosure has be formally successfully developed through several decades of research and huge investment, which is one of the world's most cutting-edge and leading anti-cancer specific novel medicaments. The medicament exhibits a therapeutic effect that was not achieved by humans through radiotherapy, chemotherapy, surgery and any modern treatment method for treating malignant solid tumors before. After administration of the medicament, a malignant solid tumor can be gradually separated and exfoliated volunteerly from normal human tissues. Functions and indications: For local injection of malignant solid tumors of liver cancer and thyroid cancer, if the malignant tumors are found to be about 2 cm x 2 cm x 2 cm or 3 cm x 3 cm x 3 cm at an early stage, only a single injection of the medicament can enable the tumor cell activity to basically disappear at 36 h to 72 h. The breakthrough efficacy is far superior to that of the current traditional anti-cancer medicaments. If the tumor is too large, another injection can be administered again. The medicament of the present disclosure has the characteristics of small dosage, quick onset, short course of treatment, small side effects, etc. in the treatment of liver cancer and thyroid cancer by local injection. The medicament can quickly reduce tumor volumes, induce apoptosis, and inhibit proliferation. The preparation method of the medicament of the present disclosure is suitable for large-scale production. Moreover, aluminum sulfate is relatively cheap, so the treatment cost is low, which has important

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practical significance for the development of a novel medicament for treating liver cancer and thyroid cancer by local injection. BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is an HPLC chromatogram of purified aluminum sulfate. FIG. 2 shows pictures of animals in an acute toxicity test. FIG. 3 is a concentration-inhibition rate curve of aluminum sulfate on squamous thyroid cancer cells SW579. FIG. 4 is a concentration-inhibition rate curve of aluminum sulfate on thyroid ductal cancer cells TT. FIG. 5 shows the effect of aluminum sulfate on the proliferation of thyroid cancer cells, where, the arrows indicate necrotic cells. FIG. 6 shows the effect of aluminum sulfate on the apoptosis of human squamous thyroid cancer cells SW579, where, A is for a vehicle control group, B is for an aluminum sulfate group with a concentration of 10 mg/mL, C is for an aluminum sulfate group with a concentration of 30 mg/mL, and D is for an aluminum sulfate group with a concentration of 100 mg/mL. FIG. 7 shows the effect of aluminum sulfate on the apoptosis of human thyroid ductal cancer cells TT, where, A is for a vehicle control group, B is for an aluminum sulfate group with a concentration of 10 mg/mL, C is for an aluminum sulfate group with a concentration of 30 mg/mL, and D is for an aluminum sulfate group with a concentration of 100 mg/mL. FIG. 8 shows the effect of aluminum sulfate on the apoptosis of thyroid cancer cells, where, the arrows indicate shrinkage of a nucleus, namely, indicating apoptotic cells. FIG. 9 shows the effect of aluminum sulfate on the cycle of human squamous thyroid cancer cells SW579, where, A is for a vehicle control group, B is for an aluminum sulfate group with a concentration of 10 mg/mL, C is for an aluminum sulfate group with a concentration of 30 mg/mL, and D is for an aluminum sulfate group with a concentration of 100 mg/mL.

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FIG. 10 shows the effect of aluminum sulfate on the cycle of human thyroid ductal cancer cells TT, where, A is for a vehicle control group, B is for an aluminum sulfate group with a concentration of 10 mg/mL, C is for an aluminum sulfate group with a concentration of 30 mg/mL, and D is for an aluminum sulfate group with a concentration of 100 mg/mL. FIG. 11 shows concentration-inhibition rate curves of aluminum sulfate on different liver cancer cells, where, A and B are concentration-inhibition rate curves for SMMC-7721 and MHCC-97H cells, respectively. FIG. 12 shows the effect of aluminum sulfate on the proliferation of liver cancer cells, where, the arrows indicate necrotic cells. FIG. 13 shows the effect of aluminum sulfate on the apoptosis of human liver cancer cells SMMC-7721, where, A is for a vehicle control group, B is for an aluminum sulfate group with a concentration of 10 mg/mL, C is for an aluminum sulfate group with a concentration of 30 mg/mL, and D is for an aluminum sulfate group with a concentration of 100 mg/mL. FIG. 14 shows the effect of aluminum sulfate on the apoptosis of human high-metastatic liver cancer cells SGC-7901, where, A is for a vehicle control group, B is for an aluminum sulfate group with a concentration of 10 mg/mL, C is for an aluminum sulfate group with a concentration of 30 mg/mL, and D is for an aluminum sulfate group with a concentration of 100 mg/mL. FIG. 15 shows the effect of aluminum sulfate on the apoptosis of liver cancer cells, where, the arrows indicate shrinkage of a nucleus, namely, indicating apoptotic cells. FIG. 16 shows the effect of aluminum sulfate on the cycle of human liver cancer cells SMMC-7721, where, A is for a vehicle control group, B is for an aluminum sulfate group with a concentration of 10 mg/mL, C is for an aluminum sulfate group with a concentration of 30 mg/mL, and D is for an aluminum sulfate group with a concentration of 100 mg/mL. FIG. 17 shows the effect of aluminum sulfate on the cycle of human high-metastatic liver cancer cells MHCC-97H, where, A is for a vehicle control group, B is for an aluminum sulfate group with a concentration of 10 mg/mL, C is for an aluminum sulfate group with a concentration of 30 mg/mL, and D is for an aluminum sulfate group with a concentration of 100 mg/mL.

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DETAILED DESCRIPTION Experimental data to confirm the chemical structure The experiment was entrusted to Hunan Experimental Animal Center (Hunan Drug Safety Evaluation Research Center). This experiment was conducted in accordance with the relevant technical guidelines for preclinical pharmacodynamics; this report faithfully reflected the experimental materials, methods and results; and the factors affecting the experimental results were minimized or avoided in this experiment. Research purpose: This study observed the in vitro inhibitory effect of a compound XL-011 on two human liver cancer cell lines, so as to provide a preliminary experimental basis for further developing the compound into an anti-liver-cancer medicament. I. Name, molecular formula and molecular weight of the compound Name: aluminum sulfate octadecahydrate (hereinafter referred to as aluminum sulfate); molecular formula: A1 2 (SO 4 )r18H20; and molecular weight: 666.4. II. Method to confirm the chemical structure 1. Moisture determination 1.1 Determination conditions: Instrument: V-30 Karl Fischer moisture analyzer Method: The first method of Moisture Determination 0832 in the fourth general rule of the Chinese Pharmacopoeia, 2015 edition. 1.2 Determination results Table 2 Results of determination of moisture in aluminum sulfate Sample Consumption of a Karl Titer Moisture Average Rounding quantity (mg) Fischer reagent (mL) (T) (%) (%) value 31.08 5.59 2.6812 48.22 48.32 48.3 29.24 5.28 48.42 1.3 Analysis According to the structure and moisture content calculation of aluminum sulfate, there are 18 waters of crystallization in the structure. 2. Aluminum determination 2.1 Determination conditions

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Instrument: Agilent 240-DUO atomic absorption spectrometer Method: The graphite furnace atomic absorption spectrometry (GFAAS) was used to determine the content of aluminum in aluminum sulfate samples. 2.2 Determination results Table 3 Results of determination of aluminum in aluminum sulfate samples

Sample Determined mass Percentage Average Name No. quantity Absorbance concentration content percentage (ng/mL) () content (mg)

Blank 1 / 0.0865 / /

/ 2 / 0.0837 / /

/ 1 25.39 0.3717 18.1852 7.16 Test 2 25.39 0.4031 20.1776 7.95 substance 25.39 0.4297 21.8655 8.61 4 24.78 0.4096 20.5901 8.31 8.15 Test 5 24.78 0.4113 20.6979 8.35 substance 6 24.78 0.4177 21.1040 8.52 Results showed that the average percentage content of aluminum in aluminum sulfate samples was 8.15%. 2.3 Analysis According to the above moisture determination, namely, as calculated based on 18 waters of crystallization, the percentage of aluminum in the molecular weight is 8.11%, which is consistent with the aluminum content as determined by the above GFAAS, further indicating that the sample includes aluminum and 18 waters of crystallization. 3. Sulfate ion determination 3.1 Determination conditions Instrument: ICS900 ion chromatograph Method: the following chromatographic conditions were adopted: eluent: 25 mM sodium hydroxide, chromatographic column: Dionex IonpacTM As18 (4 x 250 mm), flow rate: 1.0 mL/min, and injection volume: 25 ul; anhydrous sodium sulfate was used as a reference substance; and based on the peak area, an external standard method was used to calculate the content.

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3.2 Determination results Table 4 Results of determination of the sulfate content in aluminum sulfate Sample Peak Determined mass Percentage Average percentage Name No. quantity concentration content content (mg) area (ug/mL) (%) (%) Blank 1 /

/ Reference 1 10.06 5.047 3.37* substance 2 5.026 Test 1 7.54 3.30 43.843.4 substance 1 2 _____ 4.915 3.3043. Test 7.15 3.25 4.853 43.0 substance 2 5 4.848 _________________________ _____

* represents an actual concentration.

As determined, the sample has a sulfate content of 45.5%. 3.3 Analysis The retention time (RT) in the sample chromatogram is consistent with that in the reference chromatogram, indicating that the sample includes a high concentration of sulfate. The sulfate ion content determined by ion chromatography is 43.4%, which is basically consistent with the percentage of sulfate ion in the molecular weight (43.2%) according to the results of determination of moisture and aluminum. 4. Conclusion According to comprehensive analysis of the results of moisture, aluminum and sulfate content determination, the molecular formula of this product is A12 (SO4)r 18H 2 0. Purification of aluminum sulfate 1) Aluminum sulfate and distilled water were mixed at a mass ratio of 1:10 for dissolution; 2) a resulting solution was subjected to fine filtration and a resulting filter cake was washed with the same mass of distilled water to remove impurities; and 3) a filtrate obtained from the fine filtration was subjected to lyophilization to obtain a purified white aluminum sulfate powder. The HPLC chromatogram of the purified aluminum sulfate is shown in FIG. 1. It can be seen that the purity of the purified aluminum sulfate has been greatly improved compared with that before the purification. The aluminum sulfate obtained from lyophilization has better solubility. In order to completely eliminate emission of the "three wastes" (waste gas, waste water, and waste residues), high-purity aluminum sulfate (analytical purity or

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pharmaceutical grade) produced by a manufacturer can be directly used, but strict quality control must be conducted from the source of the manufacturer. Safety experiment: The safety experiment was entrusted to the Laboratory Department of the Laboratory Animal Center of Sun Yat-sen University. Experimental design references: 1. Xu Shuyun, Methodology of Pharmacological Experiment, 3th edition, 2. Technical Guidelines for Drug Safety Pharmacology Research, 2014 edition. The specific experimental method was as follows: Experimental materials Aluminum sulfate: molecular formula: A2(SO4)r 18H 2 0; and molecular weight: 666.4. I. Acute toxicity test 1. Test purpose: Whether a toxic reaction produces within a specified period of time after a single administration of aluminum sulfate was observed to preliminarily understand the toxic effects and target organs of the toxicity of the medicament, thereby providing a basis for subsequent clinical trials. 2. Test animals and feeding conditions 40 SPF Kunming mice, 20 2 g, half male and half female. Animal production and supply unit: Production Department of the Laboratory Animal Center of Sun Yat-sen University; laboratory animal production license No.: SCXK (Guangdong) 2011-0029; animal quality certificate: No. 440085000; purchase date: August 15, 2016; animal labelling method: furs at different parts of the animal were dyed with saturated picric acid to represent different animal numbers, and different animal cages were labelled with different animal feeding information cards for distinguishing. Feeding temperature: 20°C to 26°C; humidity: 40RH% to 70RH%; number of air changes: more than 15 times/h in a feeding room; feeding density: group feeding, no more than 6 mice per cage. Feed used: SPF-grade pellet feed for rats and mice, provided by Beijing Keao Co., Ltd. FIG. 2 shows the pictures of animals in the control groups and the administration groups. 3. Test method: The mice were randomly divided into control groups and administration groups, specifically as follows:

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Table 5 Grouping and dosage for the acute toxicity test

Medicament Dosage Administration Number of Group (mg/kg) volume animals Control group NS / 0.2 mL/10 g bw 0 male and 10

Administration Aluminum 2,000 0.2mL/10gbw 10 male and 10 group solution female A preparation method of an aluminum sulfate solution: 5 mL of NS was accurately extracted and injected into an ampule filled with aluminum sulfate using a syringe, and a resulting mixture was thoroughly mixed and stood for 10 min to 20 min for sufficient dissolution to obtain the aluminum sulfate solution. Administration route: intragastric administration. Administration frequency and observation time: intragastric administration once, and observing for 14 days after administration. Detection indicators: clinical observation: general symptoms of the animals were observed every day; weight measurement: the body weight of the animals was measured on D 0, D 3, D 7, and D 14 after the administration; organ coefficient determination: the animals were sacrificed on D 15, abnormalities in main organs were observed by anatomy, and the 5 organs of heart, liver, spleen, lung and kidney were collected and weighed. Processing and analysis of results: The statistical software SPSS 24 was used to calculate and compare the average body weights and organ coefficients of two groups of animals. Test results: During the test, no deaths and no obvious abnormalities were observed in the control and administration groups. The body weight of the animals in the administration group showed no significant difference from that of the animals the control group. Details were shown in Table 6. Table 6 Comparison of body weights of mice in the acute toxicity test

Time Female in the Male in the Female in the Male in the control group/g control group/g administration group/g administration group/g n= 10/9 10 10/9 10/9 D0 20.6 ±1.4 20.7 ±1.3 20.7 ±1.2 20.6 ±1.1 D3 24.8 ±1.7 24.7 ±1.8 23.7 ±1.7 23.7 ±1.6 D7 31.2 ±2.2 31.3 ±2.2 31.3 ±2.1 31.2 ±2.2 D 14 37.3 ±2.6 38.4 ±2.9 38.4 ±2.8 38.4 ±2.7

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The statistical results of organ coefficients were shown in Table 7. Table 7 Comparison of organ coefficients of mice in the acute toxicity test

. Female in the Male in the Female in the Male in the Time control group control group administration group administration group n= 9 10 9 9

0.65 ±0.09 0.65 0.09 0.56 ±0.04 0.55 0.03 coeffcient

5.71 ±0.55 5.7 0.56 5.7 ±0.57* 5.6 0.56 coefficient Slecent 0.34 ±0.09 0.35 0.08 0.35 ±0.08 0.34 0.07 coeffcient Lung coefficient 0.76 ±0.06 0.75 0.05 0.75 ±0.04 0.74 0.03 Left kidney 0.71 ±0.04 0.71 0.04 0.67 ±0.04 0.66 0.0 coefficient Right kidney 0.74 ±0.05 0.7 0.05 0.69 ±0.06 0.68 0.05 coefficient * indicates that there is no significant difference as compared with the control group (P < 0.05). All the mice survive without death. Conclusion: Aluminum sulfate has excellent safety, and exhibits no significant toxic and side effects when administered at dosages of 2,000 mg/kg, 0.2 mL/10g bw0, 2 mL/10 g bw NS. Liver cancer cell growth inhibition control test The experiment was entrusted to Hunan Experimental Animal Center (Hunan Drug Safety Evaluation Research Center). This experiment was conducted in accordance with the relevant technical guidelines for preclinical pharmacodynamics; this report faithfully reflected the experimental materials, methods and results; and the factors affecting the experimental results were minimized or avoided in this experiment. Research purpose: This study observed the in vitro inhibitory effect of a compound XL-011 on two human liver cancer cell lines, so as to provide a preliminary experimental basis for further developing the compound into an anti-liver-cancer medicament. 1. Experimental materials 1.1 Test substance: compound No.: XL-011, batch No.: 20170410, provided by Hunan Xiaolin Biological Technology Development Co., Ltd. Preparation of aluminum sulfate solutions: 9 g of aluminum sulfate was weighed and

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added to 15 mL of a 0.9% sodium chloride injection, and a resulting mixture was shaken until the aluminum sulfate was completely dissolved to give a 600 mg/mL aluminum sulfate solution, which was a working solution with the highest concentration. The stock solution was sterilized for later use. Working solutions of 200 mg/mL, 60 mg/mL, 20 mg/mL, 6 mg/mL, 2 mg/mL, and 0.6 mg/mL were successively prepared from the stock solution using a 0.9% sodium chloride injection. 1.2 Positive control medicaments: Cisplatin (DDP), batch No.: SJJMI-IE, Tokyo Chemical Industry Co., Ltd.; and 5-fluorouracil (5-FU), batch No.: HFBM160120325008, Amresco. Preparation of positive control medicament solutions: 2 mg of DDP or 5-FU was weighed and prepared into a 100 mM stock solution using fresh complete medium, and then working solutions of 200 M, 60 M, 20 M, 6 M, 2 M, and 0.6 M were prepared from the stock solution using fresh complete medium. 1.3 Main materials: Main materials Source Batch No.

Human liver cancer cells SMMC-7721 Chinese Academy of Sciences Cell Bank Human high-metastatic liver cancer ATCC Cell Bank cells MHCC-97H High-glucose DMEM medium HyClone, USA, 500 mL/bottle AB10201637 FBS Sciencell, USA, 500 mL/bottle M048-6 CCK-8 Bimake, USA, 100 mL/bottle B34304 Annexin V-FITC/PI apoptosis detection Bimake, USA, 100 times 3211713 kit

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1.4 Main instruments:

Main Instruments No. Manufacturer 3111 CO 2 incubator 024 Thermo, USA DMIL inverted microscope 027 Leica, Germany DM2500 fluorescence microscope 018 Leica, Germany MR-96A microplate reader 007 Mindray, Shenzhen, China Accrui C6 flow cytometer 329 BD, USA

CJ-1F medical clean bench 176 Fengshi Laboratory Animal Equipment Co.,Ltd, Suzhou, China 2. Experimental method 2.1 Cultivation of cells SMMC-7721 and MHCC-97H cells at confluence were collected and cultivated in an incubator at 37°C and 5% C02 using a high-glucose DMEM complete medium with 10% FBS; and depending on the cell growth, the cells were passaged or the medium was replaced 1 d to 2 d later, and cells at logarithmic growth phase would be used for later experiment.

2.2 Detection of the proliferation of cells by the CCK-8 method SMMC-7721 and MHCC-97H cells at logarithmic growth phase were inoculated into a 96-well cell culture plate at 5 x 103 cells/well; and after the cells grew adherently 12 h later, a vehicle control group, DDP (positive control medicament) groups, 5-FU (positive control medicament) groups, and aluminum sulfate groups (0.3 mg/mL to 300 mg/mL) were set, with 5 replicates for each group. In the vehicle control group, the cells were incubated in fresh complete DMEM medium; in the aluminum sulfate groups, the cells were incubated in fresh complete DMEM media including the aluminum sulfate at final concentrations of 0.3 mg/mL to 300 mg/mL; and in the DDP and 5-FU groups, the cells were incubated in fresh complete DMEM media including the aluminum sulfate at final concentrations of 100 M, 30 M, 10 M, 3 M, 1 M, and 0.3 M. After the cells were incubated for 72 h in the above treatment modes, 10 [ of CCK-8 was added to each well, and the cells were further cultivated for 1 h. Then the absorbance was determined for each well at 450 nm with a microplate reader. The OD value of the vehicle control group is set as 100% cell viability, and the ratio of the OD value of each of the other groups to the OD value of the vehicle control group represents the relative viability. The inhibition rate for cell proliferation is used to evaluate the toxicity of aluminum sulfate on SMMC-7721 and

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MHCC-97H cells. If there is an inhibition rate for cell proliferation > 100%, it is considered as a system error of the instrument, and the inhibition rate is counted as 100%. 2.3 Detection of apoptosis 2.3.1 Detection of apoptosis by Annexin V-FITC and PI double staining The SMMC-7721 and MHCC-97H cells at logarithmic growth phase were collected by routine digestion and then inoculated into a 6-well plate at a density of 5 x 105 cells/well; and after the cells grew adherently 12 h later, a vehicle control group and aluminum sulfate groups (10 mg/mL, 30 mg/mL and 100 mg/mL) were set, with 5 replicates for each group. In the vehicle control group, the cells were incubated in fresh complete DMEM medium; and in the aluminum sulfate groups, the cells were incubated in fresh complete DMEM media including the aluminum sulfate at final concentrations of 10 mg/mL, 30 mg/mL, and 100 mg/mL. 6 h after cultivation, the cells were collected by routine digestion, resuspended with 500 1 of Binding Buffer, and then transferred to a 1.5 mL EP tube; 5 l of Annexin V-FITC and 5 1 of PI were added, and a resulting mixture was incubated for 15 min at room temperature in the dark; and then the apoptosis was determined by flow cytometer. 2.3.2 Detection of apoptosis by fluorescent staining The cells were treated according to the method in 2.3.1; 6 h after the aluminum sulfate treatment, 1 mL of Hoechst 33342 staining solution was added to each well of the 6-well plate to fully cover the cells, and a resulting mixture was incubated at 37°C for 20 min to min; the staining solution was removed, and the cells were washed 2 to 3 times with PBS; and then fluoroscopic examination was conducted under a fluorescence microscope. 2.4 Effect of aluminum sulfate on the cycle of liver cancer cells as detected by flow cytometry The SMMC-7721 and MHCC-97H cells at logarithmic growth phase were collected by routine digestion and then inoculated into a 6-well plate at a density of 5 x 1005 cells/well; and after the cells grew adherently 12 h later, a vehicle control group and aluminum sulfate groups (10 mg/mL, 30 mg/mL and 100 mg/mL) were set, with 5 replicates for each group. In the vehicle control group, the cells were incubated in fresh complete DMEM medium; and in the aluminum sulfate groups, the cells were incubated in fresh complete DMEM media including the aluminum sulfate at final concentrations of 10 mg/mL, 30 mg/mL, and 100 mg/mL. 6 h after cultivation, the cells were collected by

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routine digestion, and fixed overnight with 70% cold ethanol; then 5 1 of PI was added, and a resulting mixture was incubated for 30 min at room temperature in the dark; and the cell cycle was detected by a flow cytometer. 2.5 Statistical analysis The SPSS 16.0 statistical software was used to process the data, and measurement data were expressed in tables. The comparison of means of two samples adopted the Student's t-test, and the comparison of means of multiple sample groups adopted the One-way ANOVA. P < 0.05 indicates statistical significance, and P < 0.01 indicates that the difference tested is very significant. 3. Evaluation of results 3.1 Effect of aluminum sulfate on the proliferation of liver cancer cells The cells treated with different concentrations of aluminum sulfate were observed under a microscope, and it was found that the cell proliferation rate was reduced, the cell debris increased, the cell gap increased, and sand-like vacuoles appeared in cells. There is a clear correlation between the cell status and the co-cultivation time. About 12 h after the co-cultivation, the cells became round and shrunk. 24 h after the co-cultivation, some cells swelled, the cells exhibited deteriorated light permeability, and the intercellular space increased. 48 h after the co-cultivation, sand-like vacuoles appeared in the cells, and cell rupture and the like occurred. 72 h after the co-cultivation, cells with sand-like vacuoles were almost completely ruptured, and there was no cells with an intact cell shape at concentrations of 30 mg/mL, 100 mg/mL, and 300 mg/mL, only with a small number of cells shrinking into black spots. After the cells were co-cultivated with the test substance or the positive control medicaments DDP and 5-FU for 72 h, the cell proliferation was significantly inhibited, and there was a concentration-effect relationship, exhibiting a significant difference as compared with the vehicle control group (P< 0.01). The results of cell proliferation inhibition were shown in Table 7.

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Table 7 Effects of different compounds on the proliferation of liver cancer cells SMMC-7721 MHCC-97H substc Concentration Inhibition 5 substance oInhibition rate IC5 0 Emax rate IC50 Emax

0.3 mg/mL 2.1 0.5 1 mg/mL 3.4 3.2 aluminum 3mg/mL 22.6** 28.860 100 6.** 14.465 100 sulfate 30 mg/mL 51.4** mg/mL 59.0** mg/mL 100 mg/mL 81.3** 87.8** 300 mg/mL 101.4** 100.1** 0.3 pM 8.7 0.2 1 tM 18.9** 9.6 DDP 3 pM 37.2** 3.967 94.6 30.5** 7.556 95.2 10 pM 76.5** pM 54.0** pM 30 pM 92.1** 83.6** 100 pM 94.6** 95.2** 0.3 pM 1.4 0.1 1 M 18.4** 15.0 5-FU 3 pM 53.5** 3.906 93.7 51.1** 4.525 93.5 10 pM 78.4** pM 73.5** pM 30 pM 86.6** 86.7** 100 pM 93.7** 93.5** Notes: *means P < 0.05 as compared with the vehicle control group, and ** means P < 0.01 as compared with the vehicle control group. ICo represents the concentration at which 50% of tumor cells are inhibited, and Emax represents the maximum inhibition rate for tumor cells. FIG. 3 and FIG. 4 show concentration-inhibition rate curves of aluminum sulfate on liver cancer cells, where, A and B are concentration-inhibition rate curves for SMMC-7721 and MHCC-97H cells, respectively. FIG. 4 shows the effect of aluminum sulfate on the proliferation of liver cancer cells, where, the arrows indicate necrotic cells. It can be clearly seen from the figure that aluminum sulfate can promote the necrosis of liver cancer cells. 3.2 Effect of aluminum sulfate on the apoptosis of liver cancer cells Aluminum sulfate solutions with concentrations of 10 mg/mL, 30 mg/mL, and 100 mg/mL were used to intervene liver cancer cells for 6 h, then the cells were collected, and apoptosis was detected by Annexin V-FITC/PI double staining. A flow cytometer can divide the double-stained cells into 4 groups: Qi-UL: mechanically-damaged cells (Annexin V-/PI+); QI-UR: advanced apoptotic cells (Annexin V+/PI+); Q1-LL: survival cells (Annexin V-/PI-); and Q1-LR: early apoptotic cells (Annexin V+/PI-). The

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experimental results were shown in Table 8: Table 8 Effect of aluminum sulfate on the apoptosis of human liver cancer cells

Cells Concentration Early apoptotic Necrotic cells and advanced Total apoptotic cells apoptotic cells cells (%) (mg/mL) QI-LR (%) QI-UR (%) 0 7.75 1.57 4.82 2.38 12.57 10 35.66 5.92** 13.30 1.25** 48.96** SMMC-7721 30 46.37 12.32** 14.43 1.80** 60.80** 100 42.15 4.72** 19.88 3.23** 62.03** 0 1.58 1.39 1.84 1.32 3.42 10 40.26 12.30** 20.52 ±2.22** 60.78** MHCC-97H 30 25.52 6.83** 39.09 ±2.86** 64.61** 100 21.50 5.12** 64.12 ±1.85** 85.62** Notes: * means P < 0.05 as compared with the vehicle control group, and ** means P < 0.01 as compared with the vehicle control group. The results showed that the number of advanced apoptotic and necrotic cells in SMMC-7721 cells treated with aluminum sulfate was significantly higher than that in the vehicle control group (P < 0.05), and there was a concentration-effect relationship; the number of advanced apoptotic and necrotic cells in SMMC-7721 cells treated with aluminum sulfate was significantly higher than that in the vehicle control group (P < 0.05), and there was a concentration-effect relationship; and the number of advanced apoptotic and necrotic cells in MHCC-97H cells treated with aluminum sulfate was significantly higher than that in the vehicle control group (P < 0.05), and there was a concentration-effectrelationship. FIG. 5 shows the effect of aluminum sulfate on the apoptosis of human liver cancer cells SMMC-7721, where, A is for a vehicle control group, B is for an aluminum sulfate group with a concentration of 10 mg/mL, C is for an aluminum sulfate group with a concentration of 30 mg/mL, and D is for an aluminum sulfate group with a concentration of 100 mg/mL. FIG. 6 shows the effect of aluminum sulfate on the apoptosis of human high-metastatic liver cancer cells MHCC-97H, where, A is for a vehicle control group, B is for an aluminum sulfate group with a concentration of 10 mg/mL, C is for an aluminum sulfate group with a concentration of 30 mg/mL, and D is for an aluminum sulfate group with a concentration of 100 mg/mL.

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FIG. 7 shows the effect of aluminum sulfate on the apoptosis of liver cancer cells, where, the arrows indicate shrinkage of a nucleus, namely, indicating apoptotic cells. 3.3 Effect of aluminum sulfate on the cycle of liver cancer cells Aluminum sulfate solutions with concentrations of 10 mg/mL, 30 mg/mL, and 100 mg/mL were used to intervene liver cancer cells for 6 h; then the cells were collected, fixed with 70% cold ethanol, and stained with PI; and the cell cycle was analyzed by a flow cytometer. The experimental results were shown in Table 9. Table 9 Effect of aluminum sulfate on the cycle of human liver cancer cells

Cells Concentration Cells at Go/G1 phase Cells at S phase Cells at G 2/M phase (mg/mL) (%) (%) (%) 0 64.05 ±8.34 1.22 ±0.51 14.91 ±6.07 10 70.29 ±6.21 1.92 ±0.63 24.08± 9.47** SMMC-7721 30 72.80 3.94* 2.64 ±0.44** 19.70 ±7.36 100 81.07 6.72** 1.37 ±0.77 7.54 ±8.10** 0 71.04 ±8.00 1.66 ±0.89 13.77 ±3.67 10 75.00 ±9.17 2.79 ±1.00** 20.46 ±9.05 MHCC-97H 30 80.44 4.66* 1.36 ±0.62 12.61 ±8.25 100 83.10 9.39** 2.21 ±0.78 10.36 ±5.88 Notes: * means P < 0.05 as compared with the vehicle control group, and ** means P < 0.01 as compared with the vehicle control group. The results showed that, after the cells were treated with aluminum sulfate, the proportion of cells at Go/Gi phase increased significantly, and the proportion of cells at G2 /M phase decreased significantly, indicating that the aluminum sulfate inhibited the proliferation of liver cancer cells mainly by blocking liver cancer cells at Go/G1 phase and preventing them from entering the S phase. FIG. 8 shows the effect of aluminum sulfate on the apoptosis cycle of human liver cancer cells SMMC-7721, where, A is for a vehicle control group, B is for an aluminum sulfate group with a concentration of 10 mg/mL, C is for an aluminum sulfate group with a concentration of 30 mg/mL, and D is for an aluminum sulfate group with a concentration of 100 mg/mL. FIG. 9 shows the effect of aluminum sulfate on the apoptosis cycle of human high-metastatic liver cancer cells MHCC-97H, where, A is for a vehicle control group, B is for an aluminum sulfate group with a concentration of 10 mg/mL, C is for an aluminum

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sulfate group with a concentration of 30 mg/mL, and D is for an aluminum sulfate group with a concentration of 100 mg/mL. In summary, under the experimental conditions, the aluminum sulfate can significantly inhibit the proliferation of two kinds of liver cancer cells, exhibiting a concentration-effect relationship, and can completely kill cancer cells over time at high concentrations, which can block the cell cycle at Go/Gi phase and induce apoptosis. The compound exerts an inhibitory effect on cancer cells at a relatively-high concentration, reaching the mg/mL level, which may be related to a specific active mechanism of the compound. The compound may directly act on the surface of tumor cells, can change the physiological characteristics of cancer cells, and can effectively change the protein structure on the cell surface after being aggregated on the cell surface, thus causing protein precipitation on the cell surface and in the intercellular matrix, significantly reducing the cellular permeability, resulting in shrinkage of the intercellular matrix, decreasing the division ability of tumor cells, and effectively controlling the proliferation and metastasis of cells. The compound, after entering cells, can inhibit the secretion of various proteolytic enzymes by cancer cells, directly bind to DNA of cancer cells to cause DNA lysis, and effectively inhibit the metabolic function of mitochondria in cancer cells, namely, binding to SDH in mitochondria and changing the biological effect thereof, which causes changes in the cell microstructure, blocks the signaling pathway, interferes with the growth and metabolism of tumor cells, and induces the apoptosis of tumor cells, thus achieving the effect to kill cancer cells. Recommendations: For example, after the tumors transplanted into mice in the animal experiment are matured and the administration by injection is completed, the injection delivery channel should be embolized or closed, and the animals must be separated from each other and raised alone. Because there are somewhat a smell and a small bulge after the medicament is injected, the mice, if raised in groups, will bite each other so that the medicament solution will flow out and thus the efficacy will be compromised. For example, the injection delivery channel needs not to be embolized or closed after the medicament is injected into a human tumor.

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Related experiments: 6 h after the medicament was administered at about 10 am (2018.05.24), it was observed that scabs appeared on the tumor surface in some animals of a low-dosage group; scabs appeared on the tumor surface in all animals of the medium-dosage and high-dosage groups; and tissues around the tumor in some animals were necrotic. In the morning (2018.05.25), it was observed that the tumors in the medium-dosage group (2/5) were completely scabbed, and the tumors in the high-dosage group (4/5) were completely scabbed. Today, only some animals with tumors were administered with 0.02 mL of the medicament, and the animals without tumors were not administered with the medicament. Some of the medicament that was administered after the tumor was scabbed leaked to the surface of the tumor. Results are shown in Figs 18 and 19 and Attachments 1 and 2.

3D medicament-sensitivity-test results: No. Sampling time Name Sex Age Disease Tester November 12, Male 45 Hepatocellular carcinoma Xiongfeng 2015 Introduction of a technical system: The center adopts the only three-dimensional (3D) multi-cell cocultivation system for in vitro detection worldwide, which mainly includes two layers. A first layer is a stromal cell gel layer, which is closely attached to the bottom of a culture dish, and vascular endothelial cells and tumor cells are suspended in the gel layer. A second layer is a cell culture medium layer, which is on top of the first layer. The system can simulate the whole process of tumor occurrence and development. By observing the regeneration level of tumor cells and vascular endothelial cells, the personalized treatment and medicament-resistance of cancer patients can be detected. Tested medicaments: sorafenib, betel nut, a rectal cancer-specific medicament, docetaxel, pemetrexed, paclitaxel, 5-FU, carboplatin, etc. Effective medicaments: betel nut and a rectal cancer-specific medicament exhibit the best effect, and docetaxel and paclitaxel exhibit the secondary effect. Ineffective medicaments: sorafenib, pemetrexed, 5-FU, and carboplatin

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Thyroid cancer cell growth inhibition control test The experiment was entrusted to Hunan Experimental Animal Center (Hunan Drug Safety Evaluation Research Center). Research purpose: This study observed the in vitro inhibitory effect of aluminum sulfate on two human thyroid cancer cell lines, so as to provide a preliminary experimental basis for further developing the aluminum sulfate into an anti-thyroid cancer medicament. Research method: different concentrations of aluminum sulfate (0.3 mg/mL to 300 mg/mL), DDP and 5-FU were added to human squamous thyroid cancer cells (SW579) and human thyroid ductal cancer cells (TT), separately; the cells were incubated for 72 h; and the absorbance value (OD value) of each well was detected by the CCK-8 method, and the IC 5 0 was calculated. Different concentrations of aluminum sulfate (10 mg/mL to 100 mg/mL) were added to SW579 and TT cells, separately; after the cells were incubated for 6 h, cell apoptosis was detected by Annexin V-FITC and PI double staining; and at the same time, the cells were stained with Hoechst 33342, and cell apoptosis was observed under a fluorescence microscope. Different concentrations of aluminum sulfate (10 mg/mL to 100 mg/mL) were added to SW579 and TT cells, separately, and after the cells were incubated for 6 h, the cell cycle was detected by flow cytometry. 1. Experimental materials 1.1 Test substance: aluminum sulfate, batch No.: 20170410, provided by Hunan Xiaolin Biological Technology Development Co., Ltd. Preparation of aluminum sulfate solutions: 9 g of aluminum sulfate was added to 15 mL of a 0.9% sodium chloride injection, and a resulting mixture was shaken until the aluminum sulfate was completely dissolved to give a 600 mg/mL aluminum sulfate solution, which was a working solution with the highest concentration. The stock solution was sterilized for later use. Working solutions of 200 mg/mL, 60 mg/mL, 20 mg/mL, 6 mg/mL, 2 mg/mL, and 0.6 mg/mL were successively prepared from the stock solution using a 0.9% sodium chloride injection. 1.2 Positive control medicaments: DDP, batch No.: SJJMI-IE, Tokyo Chemical Industry Co., Ltd.; and 5-FU, batch No.: HFBM160120325008, Amresco. Preparation of positive control medicament solutions: 2 mg of DDP or 5-FU was weighed and prepared into a 100 mM stock solution using fresh complete medium, and then working solutions of 200 M, 60 M, 20 M, 6 M, 2 M, and 0.6 M were prepared from the stock solution using fresh complete medium.

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1.3 Main materials: Main materials Source Batch No. Human squamous thyroid cancer cells Chinese Academy of Sciences Cell SW579 Bank Chinese Academy of Sciences Cell Human thyroid ductal cancer cells TT Bank High-glucose DMEM medium HyClone, USA, 500 mL/bottle AB10201637 FBS Sciencell, USA, 500 mL/bottle M048-6 CCK-8 Bimake, USA, 100 mL/bottle B34304 Annexin V-FITC/PI apoptosis detection Bimake, USA, 100 times 3211713 kit

1.4 Main instruments:

Main Instruments No. Manufacturer

3111 CO 2 incubator 024 Thermo, USA

DMIL inverted microscope 027 Leica, Germany

DM2500 fluorescence microscope 018 Leica, Germany

MR-96A microplate reader 007 Mindray, Shenzhen, China

Accrui C6 flow cytometer 329 BD, USA

176 Fengshi Laboratory Animal CJ-1F medical clean bench Equipment Co.,Ltd, Suzhou, China

2. Experimental method 2.1 Cultivation of cells SW579 and TT cells at confluence were collected and cultivated in an incubator at 37°C and 5% C02 using a high-glucose DMEM complete medium with 10% FBS; and depending on the cell growth, the cells were passaged or the medium was replaced 1 d to 2 d later, and cells at logarithmic growth phase would be used for later experiment. 2.2 Detection of the proliferation of cells by the CCK-8 method SW579 and TT cells at logarithmic growth phase were inoculated into a 96-well cell culture plate at 5 x 103 cells/well; and after the cells grew adherently 12 h later, a vehicle

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control group, DDP (positive control medicament) groups, 5-FU (positive control medicament) groups, and aluminum sulfate groups (0.3 mg/mL to 300 mg/mL) were set, with 5 replicates for each group. In the vehicle control group, the cells were incubated in fresh complete DMEM medium; in the aluminum sulfate groups, the cells were incubated in fresh complete DMEM media including the aluminum sulfate at final concentrations of 0.3 mg/mL to 300 mg/mL; and in the DDP and 5-FU groups, the cells were incubated in fresh complete DMEM media including the aluminum sulfate at final concentrations of 100 M, 30 M, 10 M, 3 M, 1 M, and 0.3 [M. After the cells were incubated for 72 h in the above treatment modes, 10 [ of CCK-8 was added to each well, and the cells were further cultivated for 1 h. Then the absorbance was determined for each well at 450 nm with a microplate reader. The OD value of the vehicle control group is set as 100% cell viability, and the ratio of the OD value of each of the other groups to the OD value of the vehicle control group represents the relative viability. The inhibition rate for cell proliferation is used to evaluate the toxicity of aluminum sulfate on SW579 and TT cells. If there is an inhibition rate for cell proliferation > 100%, it is considered as a system error of the instrument, and the inhibition rate is counted as 100%. 2.3 Detection of apoptosis 2.3.1 Detection of apoptosis by Annexin V-FITC and PI double staining The SW579 and TT cells at logarithmic growth phase were collected by routine digestion and then inoculated into a 6-well plate at a density of 5 x 105 cells/well; and after the cells grew adherently 12 h later, a vehicle control group and aluminum sulfate groups (10 mg/mL, 30 mg/mL and 100 mg/mL) were set, with 5 replicates for each group. In the vehicle control group, the cells were incubated in fresh complete DMEM medium; and in the aluminum sulfate groups, the cells were incubated in fresh complete DMEM media including the aluminum sulfate at final concentrations of 10 mg/mL, 30 mg/mL, and 100 mg/mL. 6 h after cultivation, the cells were collected by routine digestion, resuspended with 500 [ of Binding Buffer, and then transferred to a 1.5 mL EP tube; 5 l of Annexin V-FITC and 5 1 of PI were added, and the mixture was incubated for 15 min at room temperature in the dark; and then the apoptosis was determined by a flow cytometer. 2.3.2 Detection of apoptosis by fluorescent staining The cells were treated according to the method in 2.3.1; 6 h after the aluminum sulfate treatment, 1 mL of Hoechst 33342 staining solution was added to each well of the 6-well

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plate to fully cover the cells, and a resulting mixture was incubated at 37C for 20 min to min; the staining solution was removed, and the cells were washed 2 to 3 times with PBS; and then fluoroscopic examination was conducted under a fluorescence microscope. 2.4 Effect of aluminum sulfate on the cycle of thyroid cancer cells as detected by flow cytometry The SW579 and TT cells at logarithmic growth phase were collected by routine digestion and then inoculated into a 6-well plate at a density of 5 x 105 cells/well; and after the cells grew adherently 12 h later, a vehicle control group and aluminum sulfate groups (10 mg/mL, 30 mg/mL and 100 mg/mL) were set, with 5 replicates for each group. In the vehicle control group, the cells were incubated in fresh complete DMEM medium; and in the aluminum sulfate groups, the cells were incubated in fresh complete DMEM media including the aluminum sulfate at final concentrations of 10 mg/mL, 30 mg/mL, and 100 mg/mL. 6 h after cultivation, the cells were collected by routine digestion and fixed overnight with 70% cold ethanol; then 5 L of PI was added, and a resulting mixture was incubated for 30 min at room temperature in the dark; and the cell cycle was detected by a flow cytometer. 2.5 Statistical analysis The SPSS 16.0 statistical software was used to process the data, and measurement data were expressed in tables. The comparison of means of two samples adopted the Student's t-test, and the comparison of means of multiple sample groups adopted the One-way ANOVA. P < 0.05 indicates statistical significance, and P < 0.01 indicates that the difference tested is very significant. 3. Evaluation of results 3.1 Effect of aluminum sulfate on the proliferation of thyroid cancer cells The cells treated with different concentrations of aluminum sulfate were observed under a microscope, and it was found that the cell proliferation rate was reduced, the cell debris increased, the cell gap increased, and sand-like vacuoles appeared in cells. There is a clear correlation between the cell status and the co-cultivation time. About 12 h after the co-cultivation, the cells became round and shrunk. 24 h after the co-cultivation, some cells swelled, the cells exhibited deteriorated light permeability, and the intercellular space increased. 48 h after the co-cultivation, sand-like vacuoles appeared in the cells, and cell rupture and the like occurred. 72 h after the co-cultivation, cells with sand-like vacuoles

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were almost completely ruptured, and there was no cells with an intact cell shape at concentrations of 30 mg/mL, 100 mg/mL, and 300 mg/mL, only with a small number of cells shrinking into black spots. After the cells were co-cultivated with the test substance or the positive control medicaments DDP and 5-FU for 72 h, the cell proliferation was significantly inhibited, and there was a concentration-effect relationship, exhibiting a significant difference as compared with the vehicle control group (P< 0.01). The results of cell proliferation inhibition were shown in Table 8. Table 8 Effect of aluminum sulfate on the proliferation of thyroid cancer cells SW579 TT Test substance Concentration Inhibition rate IC5 o Emnax Inhibition rateICo Emax 0.3 mg/mL 1.0 -1.6 1 mg/mL 2.2 12.3 3 mg/mL 4.6 14.408m9. aluminum 130mg/mL 42.2** 100 37.2** 14.116 100 sulfate 30 mg/mL 73.5** g/mL 63.6** mg/mL 100 mg/mL 92.3** 94.9** 300 mg/mL 100.0** 100.7** 0.3 pM -1.1 22.9** 1 tM 10.1 40.8** DDP 3 pM 48.1** 4.758 M 90.9 58.5** 1.706 92.9 10 tM 69.7** 478M 909 77.3** t 30 pM 89.4** 93.0** 100 pM 90.9** 92.9** 0.3 pM 1.2 1.3 1 pM 5.0 19.7** 5-FU 3 pM 22.0** 9.341 pM 91.7 56.9** 4.273 92.5 10 pM 56.2** 67.5** pM 30 pM 79.8** 85.7** 100 pM 91.7** 92.5** Notes: * means P < 0.05 as compared with the vehicle control group, and ** means P < 0.01 as compared with the vehicle control group. ICo represents the concentration at which 50% of tumor cells are inhibited, and Emax represents the maximum inhibition rate for tumor cells. 3.2 Effect of aluminum sulfate on the apoptosis of thyroid cancer cells Aluminum sulfate solutions with concentrations of 10 mg/mL, 30 mg/mL, and 100 mg/mL were used to intervene thyroid cancer cells for 6 h, then the cells were collected, and apoptosis was detected by Annexin V-FITC/PI double staining. A flow cytometer can divide the double-stained cells into 4 groups: Q1-UL: mechanically-damaged cells (Annexin V-/PI+); Q1-UR: advanced apoptotic cells (Annexin V+/PI+); Q1-LL: survival cells (Annexin V-/PI-); and Q1-LR: early apoptotic cells (Annexin V+/PI-). The results

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showed that the number of advanced apoptotic and necrotic cells in SW579 cells treated with aluminum sulfate was significantly higher than that in the vehicle control group (P < 0.05), and there was a concentration-effect relationship; and the number of advanced apoptotic and necrotic cells in TT cells treated with aluminum sulfate was significantly higher than that in the vehicle control group (P < 0.05), and there was a concentration-effect relationship. Table 9 Effect of aluminum sulfate on the apoptosis of human thyroid cancer cells

Cells Concentration Early apoptotic Necrotic cells and advanced Total apoptotic cells cells apoptotic cells (%) (mg/mL) Q1-LR (%) QI-UR (%) 0 11.42 1.84 1.20 ±1.33 12.62 10 15.14 11.65 4.10 ±2.08** 19.24* SW579 30 24.42 ±5.19** 7.72 ±1.86** 32.14** 100 31.25 ±8.08** 16.38 1.77** 47.63** 0 4.01 ±1.51 3.09 1.56 7.10 10 34.59 10.38** 7.78 1.89* 42.37** TT 30 34.27 9.06** 7.45 1.51* 41.72** 100 29.03 7.36** 17.77 3.26** 46.80** Notes:* means P < 0.05 as compared with the vehicle control group, and ** means P < 0.01 as compared with the vehicle control group. 3.3 Effect of aluminum sulfate on the cycle of thyroid cancer cells Aluminum sulfate solutions with concentrations of 10 mg/mL, 30 mg/mL, and 100 mg/mL were used to intervene thyroid cancer cells for 6 h; then the cells were collected, fixed with 70% cold ethanol, and stained with PI; and the cell cycle was analyzed by flow cytometry. The results showed that, after the aluminum sulfate treatment, the proportion of cells at Go/Gi phase increased significantly, and the proportion of cells at G2/M phase decreased significantly, indicating that the aluminum sulfate inhibited the proliferation of thyroid cancer cells mainly by blocking thyroid cancer cells at Go/Gi phase and preventing them from entering the S phase.

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Table 10 Effect of aluminum sulfate on the cycle of human thyroid cancer cells

Cells Concentration Cells at Go/G1 phase Cells at S phase Cells at G 2/M phase (mg/mL) (%) (%) (%) 0 65.55 ±7.23 1.97 ±0.94 23.93 ±6.61 10 68.42 ±9.42 3.27 ±0.92** 22.47 ±7.81 SW579 30 79.74 ±5.91** 1.13 ±1.17 12.68 9.47** 100 84.92 ±8.48** 1.75 ±0.83 7.42 4.35** 0 65.71 ±3.58 0.8 0.58 20.47 ±9.56 10 70.77 ±5.35 1.28 0.60** 23.27 ±5.44 TT 30 73.26 ±5.80** 1.28 0.86** 19.29 ±8.02 100 92.01 ±5.46** 0.64 ±1.14 2.22± 7.35** Notes:* means P < 0.05 as compared with the vehicle control group, and ** means P < 0.01 as compared with the vehicle control group. 4. Conclusion and discussion In summary, under the experimental conditions, aluminum sulfate can significantly inhibit proliferation of the two types of thyroid cancer cells, with IC5 o values of SW579 (14.408 mg/mL) and TT (14.116 mg/mL), respectively. Furthermore, aluminum sulfate shows a significant pro-apoptotic effect, with a concentration-effect relationship, which can block the cell cycle at the G/G1 phase and induce cell apoptosis. Under high concentration conditions, cancer cells can be completely killed over time. The compound exerts an inhibitory effect on cancer cells at a relatively-high concentration, reaching the mg/mL level, which may be related to a specific active mechanism of the compound. The compound directly acts on the surface of tumor cells, can change the physiological characteristics of cancer cells, and can effectively change the protein structure on the cell surface after being aggregated on the cell surface, thus causing protein precipitation on the cell surface and in the intercellular matrix, significantly reducing the cellular permeability, resulting in shrinkage of the intercellular matrix, decreasing the division ability of tumor cells, and effectively controlling the proliferation and metastasis of cells. The compound, after entering cells, can inhibit the secretion of various proteolytic enzymes by cancer cells, directly bind to DNA of cancer cells to cause DNA lysis, and effectively inhibit the metabolic function of mitochondria in cancer cells, namely, binding to SDH in mitochondria and changing the biological effect thereof, which causes changes in cell microstructures, blocks signaling pathways, interferes with the growth and metabolism of

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tumor cells, and induces the apoptosis of tumor cells, thus achieving the effect to kill cancer cells. The above descriptions are merely preferred implementations of the present disclosure. It should be noted that a person of ordinary skill in the art may further make several improvements and modifications without departing from the principle of the present disclosure, but such improvements and modifications should be deemed as falling within the protection scope of the present disclosure.

Claims (5)

1. 21160142.1:DCC - 9/02/2021

   What is claimed is: 1. A medicament for treating liver cancer and thyroid cancer by local injection, comprising aluminum sulfate or a hydrate thereof and pharmaceutically acceptable adjuvants; wherein, the aluminum sulfate or a hydrate thereof is an active ingredient in the medicament; the medicament is a powder injection; and the aluminum sulfate or a hydrate thereof is selected from aluminum sulfate octadecahydrate.
2. 2. A method for preparing the medicament according to claim 1, comprising the steps of mixing the aluminum sulfate or a hydrate thereof with the pharmaceutically acceptable adjuvants and preparing a resulting mixture into the medicament, wherein,

   the aluminum sulfate or a hydrate thereof is prepared as follows: dissolving aluminum sulfate, and subjecting a resulting solution to fine filtration and lyophilization to obtain the aluminum sulfate or a hydrate thereof.
3. 3. The method for preparing the medicament according to claim 2, wherein, the method for preparing the aluminum sulfate or a hydrate thereof specifically comprises: mixing aluminum sulfate with distilled water at a mass ratio of 1:10; after the aluminum

   sulfate is completely dissolved, subjecting a resulting solution to fine filtration, and washing a resulting filter cake with the same mass of distilled water; and subjecting a resulting filtrate to lyophilization to obtain powdered aluminum sulfate or a hydrate thereof; and

   the medicament is a powder or an injection, and preferably a powder injection administered by local injection.
4. 4. Use of a composition in the preparation of a medicament for treating liver cancer and thyroid cancer by local injection, wherein, the composition comprises aluminum sulfate or a hydrate thereof and pharmaceutically acceptable adjuvants.
5. 5. Use of aluminum sulfate or a hydrate thereof in the preparation of a medicament for treating liver cancer and thyroid cancer by local injection, wherein, the aluminum sulfate or a hydrate thereof is an active ingredient in the medicament; the medicament is a powder or an injection, and preferably a powder injection administered by local injection; and the aluminum sulfate or a hydrate thereof is selected from aluminum sulfate octadecahydrate.

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   1/20 2021100812

   Response (S)

   Time (min)

   FIG. 1

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   Acute toxicity test for medicament 1 Acute toxicity test for medicament 1 (20160901) (20160901) Control group-female Control group-male 2021100812

   Female animals in the control group Male animals in the control group

   Acute toxicity test for medicament 1 Acute toxicity test for medicament 1 (20160901) (20160901) Administration group-female Administration group-male

   Female animals in the control group Male animals in the control group

   FIG. 2

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   Inhibition rate (%) 2021100812

   Logarithmic concentration FIG. 3 Inhibition rate (%)

   Logarithmic concentration

   FIG. 4

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   Vehicle control-SW579 300 mg/mL-SW579

   Vehicle control-TT 300 mg/mL-TT

   FIG. 5

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   FIG. 6

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   FIG. 7

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   Vehicle control-SW579 300 mg/mL-SW579

   Vehicle control-TT 300 mg/mL-TT

   FIG. 8

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   FIG. 9

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   FIG. 10

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   Inhibition rate (%) 2021100812

   Logarithmic concentration Inhibition rate (%)

   Logarithmic concentration

   FIG. 11

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   Vehicle control-SMMC-7721 300 mg/mL-SMMC-7721

   Vehicle control-MHCC-97H 300 mg/mL-MHCC-97H

   FIG. 12

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   FIG. 13

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   FIG. 14

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   Vehicle control-SMMC-7721 300 mg/mL-SMMC-7721

   Vehicle control-MHCC-97H 300 mg/mL-MHCC-97H

   FIG. 15

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   FIG. 16

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   FIG. 17

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   FIG. 18

   Detection unit: a people's hospital in Hunan province November 15, 2015 3D tumor personalized medicament-sensitivity-test report

   FIG. 19

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   Attachment 1: groups with the best efficacy Betel nut extract Control group for betel nut extract 2021100812

   Rectal cancer-specific medicament on Control group for the rectal cancer-specific liver metastasis cells medicament on liver metastasis cells

   Docetaxel Paclitaxel

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   Attachment 2: groups with the worst efficacy Control Sorafenib 2021100812

   Pemetrexed 5-FU

   Carboplatin

   Note: Imatinib functions best among the additional medicaments

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   Control Imatinib 2021100812

   A medicament safety evaluation center. Local injection experiment in mice (malignant solid tumors). That is, the tumor is scabbed at 6 h and disappears the next day.