CN115531413A - Medicine for targeted therapy of thyroid cancer and preparation method thereof - Google Patents
Medicine for targeted therapy of thyroid cancer and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a medicine for targeted therapy of thyroid cancer and a preparation method thereof. The inventor discovers for the first time that aluminum sulfate can distinguish and identify normal tissues and cancer tissues of a human body, change the physiological characteristics of cancer cells, inhibit the cancer cells from secreting various proteolytic enzymes, inhibit the metabolic function of mitochondria of the cancer cells and realize the anti-tumor effect, the medicament can be combined into DNA of the cancer cells to promote the cancer cells to be cracked, can quickly and strongly converge and condense glycoprotein on the surfaces of the cells, change the protein structure on the surfaces of the cells, block a signal path, change the microstructures of the cells, combine with succinate dehydrogenase in the mitochondria of the cells and effectively control the proliferation and the metastasis of the cancer cells, and the inhibition rate of the cancer cells reaches 99.8-99.9 percent even 100 percent. The aluminum sulfate has small side effect and high safety, and the oral administration for treating the liver cancer has the advantages of small dosage, quick response, short treatment course and the like, can quickly reduce tumor bodies, induce apoptosis, inhibit proliferation and finally fragment and fall off, and has important practical significance for attacking and overcoming thyroid cancer of human beings.
Description
The patent application of the invention is a divisional application of an invention patent application with the application date of 2017, 9 and 14 and the application number of 201710828803.X, and the name of the invention patent application is 'a medicine for treating thyroid cancer and a preparation method thereof'.
Technical Field
The invention relates to the field of medicines, in particular to a medicine for treating thyroid cancer and a preparation method thereof.
Background
As is well known, malignant tumors are a common disease and frequently encountered diseases that seriously threaten human health, and viral diseases and senile diseases are combined to be the three major challenges of modern medicine, the pathogenesis of the malignant tumors is not completely clarified, and the treatment effect of the malignant tumors is not satisfactory. According to incomplete statistics, the development and treatment cost of tumors in the whole world is not lower than hundreds of billions, and huge losses are brought to the nation, the society and individuals.
Thyroid cancer is the most common malignant tumor in head and neck tumors, the incidence rate is in a continuous and rapid growth trend after 2000 years, the incidence rate of thyroid cancer in 2009 is nearly 3 times higher than that in 1975, the incidence rate is increased from 4.9/10 ten thousand to 14.3/10 ten thousand, and the incidence rate of thyroid cancer in China (Beijing in 2013) in 10 years is nearly 400%. The rank of incidence of thyroid cancer of Chinese females in 2010 is increased to the sixth place, the rank of incidence of thyroid cancer of American females is increased to the fifth place, so that the method has attracted extensive attention of the society, and the diagnosis and treatment of thyroid tumors are more and more emphasized by the society and the medical community.
Since the lymph node is usually the first site of thyroid cancer metastasis at an anatomical position, the metastasis rate is high, and whether the lymph node in the central area of a thyroid cancer patient needs to be cleaned in clinic still has some controversies, including the necessity of cleaning the lymph node in the central area, the scope of cleaning and the like. Professors have thought that, because the incidence of thyroid cancer metastasis is high and the central lymph node is usually the first affected area, central lymph node cleaning is necessary for thyroid cancer patients, and the patients do not clean or clean thoroughly, and the patients will relapse, and the difficulty of performing the operation is increased.
Treatment modalities for thyroid cancer include mainly: surgical therapy, endocrine therapy, radionuclide therapy, and external radiation therapy.
At present, no effective method and medicine for treating thyroid cancer exist in clinic, so an effective anti-cancer medicine is urgently needed to solve the urgent need of cancer patients.
Aluminum sulfate is a common sulfate, is used as a precipitator of rubber materials such as rosin gum, wax emulsion and the like in the paper industry, is used as a flocculating agent in water treatment, can also be used as an internal retention agent of a foam fire extinguisher, and is used for preparing raw materials of alum and aluminum white, a petroleum decoloring agent, a deodorizing agent, auxiliary materials of certain medicines and the like. The first major use of aluminum sulfate, which accounts for about 50% of the total yield, is for papermaking, and the second major use is as a flocculant in the treatment of drinking water, industrial water and industrial wastewater, which accounts for about 40% of the total yield of aluminum sulfate. When aluminum sulfate is added to such water, colloidal aluminum hydroxide flakes are formed which adsorb and precipitate bacteria, colloids and other suspended matter, and are used in drinking water treatment to control the color and taste of the water.
In the aspect of medicine, aluminum sulfate also has a small amount of application, such as Zhou Guoliao, wu Shurong, chen Jinyun, and the like, the research on the composition of compound aluminum sulfate solution [ J ]. The report of the liberty medical college, 1981 (2.), discloses that the intra-tumor injection for treating bladder tumor obtains better curative effect, and the cure rate for early bladder tumor reaches 91.1 percent. Later further research shows that the compound aluminum sulfate injection has a certain treatment effect on bladder tumors, but aluminum sulfate can not be used for treating other tumors for more than 30 years later.
Disclosure of Invention
The invention aims to provide a low-toxicity and high-efficiency medicament for targeted treatment of thyroid cancer, aiming at the technical defects in the prior art, wherein the active ingredient of the medicament is aluminum sulfate or hydrate thereof.
The invention relates to a medicine for treating thyroid cancer, which comprises aluminum sulfate or hydrate thereof and pharmaceutically acceptable auxiliary materials.
The second aspect of the present invention relates to a dosage form of the above-mentioned medicament, which is any pharmaceutically acceptable dosage form.
The dosage form is preferably injection, powder or injection; local injection powder injection is the most preferable.
The third aspect of the present invention relates to a method for producing a pharmaceutical, which is characterized by comprising a step of mixing aluminum sulfate or a hydrate thereof with a pharmaceutically acceptable excipient.
The method for preparing the medicament preferably further comprises the following steps: the preparation method comprises the following steps: dissolving aluminum sulfate, fine filtering, and freeze drying.
The specific preparation method of the aluminum sulfate or the hydrate thereof comprises the following steps: mixing aluminum sulfate with ultrapure water according to the mass ratio of 1 (2-5), after the aluminum sulfate is completely dissolved, fine filtering, cleaning with ultrapure water with the same mass, and freeze-drying the purified liquid to obtain powdery aluminum sulfate or hydrate thereof.
Wherein the mass ratio of the aluminum sulfate to the ultrapure water in the preparation method of the aluminum sulfate or the hydrate thereof is 1:3.
The method for preparing a drug according to the above, preferably further comprising the step of preparing the drug into any dosage form.
The dosage form is preferably injection, powder or injection; the most preferable is local injection powder injection.
The fourth aspect of the invention relates to a use of a composition in preparing a medicament for targeted therapy of thyroid cancer, which is characterized in that the composition comprises aluminum sulfate or hydrate thereof and pharmaceutically acceptable auxiliary materials; and aluminium sulphate or hydrate thereof as an active ingredient thereof; preferably all or only the active ingredient.
The dosage form of the medicament can be any dosage form; preferably injection, powder or injection; the most preferable is local injection powder injection.
A fifth aspect of the invention relates to the use of aluminium sulphate or a hydrate thereof for the manufacture of a medicament for the targeted treatment of thyroid cancer.
The dosage form of the medicament can be any dosage form; preferably injection, powder or injection; the most preferable is local injection powder injection.
The medicament and the preparation method thereof, the use of the composition, and the use of the aluminum sulfate or the hydrate thereof according to the invention are characterized in that the aluminum sulfate or the hydrate thereof is selected from aluminum sulfate octadecahydrate.
And the aluminum sulfate or hydrate thereof according to the present invention is preferably prepared by the aforementioned preparation method. More preferably, the HPLC profile is substantially as shown in FIG. 1.
In order to completely avoid the discharge of three wastes, the invention can also directly adopt high-purity aluminum sulfate (analytically pure) produced by manufacturers. But must be strictly quality controlled from the source of the manufacturer.
The invention is overcome by research for decades, and firstly discovers that aluminum sulfate can distinguish and identify normal tissues and cancer tissues of a human body, so that the cancer tissues can be separated and fall off from the normal tissues, and can distinguish and identify normal cells and cancer cells and selectively starve the cancer cells. The medicine is a leading chemical medicine which has the fastest and most definite curative effect on malignant tumors, has low toxicity and high efficiency, basically has no inhibiting effect on normal cells, can quickly kill cancer cells and reduce the generation of tumor bodies, has a curative effect far superior to that of the traditional anticancer medicine, and can save tens of thousands of patient lives, particularly treat malignant solid cancers.
The invention preferably provides a preparation for local injection for targeted therapy of thyroid cancer and a preparation method thereof. Has the characteristics of micro-dosage, quick response, short treatment course, small side effect and the like, and can quickly reduce tumor body induced apoptosis and inhibit proliferation when used for treating thyroid cancer.
The dosage of the malignant solid cancer targeted local puncture injection (western medicine) is as follows: 1 g of aluminum sulfate purified product is dissolved in 3mL of physiological saline with the concentration of 0.9 percent to obtain a completely dissolved colorless transparent injection. The local direct puncture injection under the guidance of CT positioning B-ultrasonic is as follows:
a first keyword: after the tumor injection is completed, the patient must be prohibited from exercising for 25-30 minutes. The purpose is to avoid the water injected into the tumor needle from being extruded and lost to lose the efficacy.
TABLE 1 local puncture injection dosage for solid cancer of malignant tumor
The invention is formally researched and developed successfully after decades of years and huge investment, and is a leading series of anticancer special-effect new drugs in the world. The curative effect of the medicine is that the medicine can not be realized by human beings for treating malignant tumor solid cancer, radiotherapy and chemotherapy, operation and modern treatment means for hundreds of years, and the malignant tumor solid cancer can be gradually separated from normal tissues of human bodies and fall off after the medicine is administrated.
Can realize the targeted local injection and needle insertion of malignant solid tumor cancer at any part of a human body, for example, the malignant solid tumor volume is about 2cm multiplied by 2cm or 3cm multiplied by 3cm found in early stage, the breakthrough drug effect that the activity of tumor cells is basically completely disappeared in 36h to 72h only by one needle insertion can be realized, and the curative effect of the drug is far superior to that of the traditional anticancer drug at present. If the tumor is too large, the needle can be inserted again.
Pharmacology: the key pathways of apoptosis are as follows: (1) the physiological characteristics of cancer cells can be changed. (2) inhibiting the secretion of various proteolytic enzymes by cancer cells. (3) Inhibiting the metabolic function of cancer cell mitochondria, thereby realizing the anti-tumor effect. (4) The medicine can be combined into DNA of cancer cell to promote its lysis. And (5) the cell surface glycoprotein can be rapidly and strongly converged and condensed. And (6) effectively controlling the proliferation and metastasis of cancer cells. (7) The medicine can effectively change the protein structure on the cell surface. And (8) blocking a signal path. And (9) changing the microstructure of the cells. (10) And binds to succinate dehydrogenase in the mitochondria of cells thereby altering their biological effects. (11) Regular normal cell feeding, and irregular cancer cell feeding. (12) The compound can identify and distinguish normal tissues and abnormal tissues, normal cells and abnormal cells, selectively starve and kill cancer cells, does not inhibit normal cells, and makes malignant tumor solid cancer tissues gradually show important breakthrough of human medicine of fragment shedding. The inhibition rate is as high as 99.8-99.9%, even 100%.
The research further finds that the purified aluminum sulfate can change the physiological characteristics of cancer cells, so that the cancer cells cannot secrete various proteolytic enzymes and cannot express proteins and telomerase activities thereof in embryos. Meanwhile, the purified product can be organically combined with the DNA of the cancer cells, and can rapidly and strongly converge and condense cell surface glycoprotein, the condensation of the cell surface glycoprotein is an effective way for effectively controlling proliferation, metastasis and diffusion of the cancer cells, and forced cell membrane shrinkage inevitably causes blocking and closing of cell absorption channels. The purified substance can effectively change the protein structure on the cell surface, so that signals emitted by the protein and the special growth factor are effectively blocked, meanwhile, the purified substance can permeate cancer cell membranes to influence the normal operation of the physiological function of the cell microstructure, and can be combined with succinate dehydrogenase (succinate dehydrogenase) in a cell line solid to change the biological effect of the purified substance. Mitochondria are one of the important organelles, whether normal or cancerous, and supply the energy required for the cells' vital activities. Mitochondria not only have most of the enzymes in the cell, but are the only sites for oxidative phosphorylation and electron transport within the cell. Mitochondria are therefore sensitive areas for the observation of cellular damage. Biological effects on cancer cell mitochondria once the biological effects of the enzyme are altered, proliferation of the cell is spread and metastasized and cannot be achieved. The purified product has no toxic and side effects with normal cells of human body, thus greatly eliminating the risk of side effects. Particularly, the local injection for treating thyroid cancer and solid cancer thereof has relatively small side effect, and further provides a local injection method for treating thyroid cancer.
Toxicology: the large dose has no obvious toxic or side effect after decades of research and test,
the drug effect is as follows: has the characteristics of micro dosage, quick response, short treatment course, small side effect and the like, and can quickly reduce tumor bodies, induce apoptosis and inhibit proliferation when used for treating the malignant tumors.
The functions and indications are as follows: malignant tumor solid cancer thyroid cancer.
Compared with the prior art, the invention has the beneficial effects that:
the medicine has the characteristics of small dosage, quick response, short treatment course, small side effect and the like in the aspect of local injection for treating the malignant tumor of the thyroid cancer, and the medicine has quick response for treating the malignant tumor; can rapidly reduce tumor body, induce apoptosis, and inhibit proliferation. The preparation method of the medicine is convenient for realizing large-scale production, has relatively low cost of the aluminum sulfate and lower treatment cost, and has important practical significance for developing new local injections for treating thyroid cancer malignant tumors.
Drawings
FIG. 1 is an HPLC chromatogram of purified aluminum sulfate.
Figure 2 is a photograph of an animal undergoing an acute toxicity test.
FIG. 3 is a concentration-inhibition curve of aluminum sulfate against thyroid squamous carcinoma cells SW 579.
FIG. 4 is a concentration-inhibition curve of aluminum sulfate on thyroid ductal carcinoma cells TT.
FIG. 5 is the effect of aluminum sulfate on thyroid cancer cell proliferation, with arrows indicating necrotic cells.
FIG. 6 shows the effect of aluminum sulfate on apoptosis of human thyroid squamous carcinoma cells SW579, wherein A is vehicle control, B is compound 10mg/mL, C is compound 30mg/mL, and D is compound 100 mg/mL.
FIG. 7 shows the effect of aluminum sulfate on apoptosis of human ductal thyroid carcinoma cells TT, where A is vehicle control group, B is compound 10mg/mL, C is compound 30mg/mL, and D is compound 100 mg/mL.
FIG. 8 is the effect of aluminum sulfate on apoptosis in thyroid cancer cells, with arrows indicating shrinkage of the nucleus, suggesting apoptotic cells.
FIG. 9 shows the effect of aluminum sulfate on the cell cycle of human thyroid squamous carcinoma cells SW579, where A is vehicle control, B is compound 10mg/mL, C is compound 30mg/mL, and D is compound 100 mg/mL.
FIG. 10 shows the effect of aluminum sulfate on cell cycle of human thyroid ductal carcinoma cells TT, where A is vehicle control, B is compound 10mg/mL, C is compound 30mg/mL, and D is compound 100 mg/mL.
Detailed Description
Test data for confirming chemical structure
The experiment was entrusted to the animal center of the Hunan province (the center for drug safety evaluation and research in the Hunan province).
1. Name, molecular formula and molecular weight of the compound
The name of Chinese: aluminum sulfate octadecahydrate (hereinafter, unified abbreviated as aluminum sulfate)
The molecular formula is as follows: al (aluminum) 2 (SO 4 ) 3 ·18H 2 O, molecular weight: 666.4.
2. method for confirming chemical structure
1. Moisture determination
1.1 test conditions:
the instrument comprises the following steps: v-30 card type moisture meter
The method comprises the following steps: (first method of 0832 moisture determination method in general rules of the four departments of the edition "Chinese pharmacopoeia" 2015 edition).
1.2 results of measurement
TABLE 2 measurement results of moisture in aluminum sulfate
| Sample volume (mg) | Consumption of Ka's reagent (mL) | Titer (T) | Moisture (%) | Average (%) | Contract trimming |
| 31.08 | 5.59 | 2.6812 | 48.22 | 48.32 | 48.3 |
| 29.24 | 5.28 | 48.42 |
1.3 analysis
The number of crystal water contained in the structure is 18 calculated according to the structure of aluminum sulfate and the moisture content.
Determination of aluminum element
2.1 test conditions
The instrument comprises the following steps: agilent 240-DUO original absorption spectrometer
The method comprises the following steps: and (3) determining the content of the aluminum element in the aluminum sulfate sample by adopting a graphite furnace atomic absorption method.
2.2, test results
TABLE 3 test results of aluminum element in aluminum sulfate samples
The results show that: the average percentage content of aluminum element in the aluminum sulfate sample is 8.15%.
2.3 analysis
According to the above-mentioned moisture measurement, the proportion of the aluminum element in the molecular weight was 8.11% calculated on 18 crystal waters, and it was found that the sample contained the aluminum element and 18 crystal waters in accordance with the content of the aluminum element measured by the atomic absorption.
Determination of sulfate ion
3.1 test conditions
The instrument comprises the following steps: ICS900 ion chromatograph
The method comprises the following steps: 25mM sodium hydroxide is used As leacheate, a chromatographic column is Dionex Ionpac TM As18 (4 x 250 mM), the flow rate is 1.0mL/min, and the sample injection amount is 25ul; anhydrous sodium sulfate was used as a control, and the content was calculated by an external standard method on the peak area.
3.2, test results
TABLE 4 measurement results of sulfate radical content in aluminum sulfate
* Is the actual concentration.
The sample was determined to contain 45.5% sulfate ions.
3.3 analysis
The retention time of the sample chromatogram and the control chromatogram is consistent, which indicates that the sample contains high-concentration sulfate radicals; according to the determination results of the moisture content and the aluminum element content, the sulfate ion content is determined to be 43.4% by adopting an ion chromatography, and the content is basically consistent with the sulfate ion molecular weight accounting for 43.2% in the molecular structure.
4. Conclusion
According to the comprehensive analysis of the results of water content determination, aluminum element content determination and sulfate radical content determination, the molecular formula of the product is Al 2 (SO 4 ) 3 ·18H 2 O。
Purification of aluminium sulphate
1) Dissolving aluminum sulfate in pure water according to the mass ratio of 1:3;
2) Washing with pure water of equal mass, and fine filtering to remove impurities;
3) And (4) freeze-drying the purified liquid after fine filtration to obtain pure white aluminum sulfate purified product.
The HPLC chromatogram of the purified aluminum sulfate is shown in FIG. 1, and it can be seen that the purity of the purified aluminum sulfate is greatly improved compared with that before purification. The aluminum sulfate obtained by freeze drying has better solubility.
In order to completely avoid the discharge of three wastes and avoid purification, high-purity aluminum sulfate (analytically pure or pharmaceutical grade) produced by manufacturers can be directly adopted, but strict quality control must be carried out from the sources of the manufacturers.
Safety tests:
the safety test was conducted by the department of laboratory animals center, zhongshan university.
Experimental design reference:
1. xu Shuyun, third edition of pharmacological Experimental methodology
2.2014 edition of the pharmaceutical safety pharmacological research guidelines.
The specific experimental method is as follows:
experimental Material
Aluminum sulfate, molecular formula: al (Al) 2 (SO 4 ) 3 ·18H 2 O, molecular weight: 666.4.
1. acute toxicity test
1. The purpose of the test is as follows:
whether the aluminum sulfate produces toxic reaction within a certain time after single administration is observed, so that basis is provided for preliminary understanding of the toxic effect of the medicament and toxic target organs thereof and subsequent clinical tests.
2. Test animals and feeding conditions
SPF-level Kunming mice are 40, 20 +/-2 g and half of male and female. Animal production supply units: the central production department of experimental animals of Zhongshan university, the license number of the experimental animals production is: SCXK (Guangdong) 2011-0029, animal quality qualification proves: purchase date of No. 440085000: 2016, month 08, 15; the animal identification method comprises the following steps: coat and dye the fur of different parts of the animal with saturated picric acid to represent different animal numbers, and different animal rearing cages are distinguished by animal rearing information card marks. The breeding temperature is 20-26 ℃; humidity is 40RH% -70 RH%; and (3) ventilation frequency: the breeding room is more than 15 times/hour; feeding density: and (4) group culture, wherein each cage is not more than 6. The feed is used: SPF-grade rat and mouse pellet feed is provided by Australian Co., ltd, beijing Ke. Photographs of animals of the control group and the administration group are shown in FIG. 2.
3. The experimental method comprises the following steps: mice were randomly divided into control and administration groups as follows:
TABLE 5 grouping of acute toxicity tests and dosing
| Group of | Medicine | Administration dose (mg/kg) | Volume of administration | Number of animals |
| Control group | Physiological saline | / | 0.2mL/10g bw | 10 females and 10 males |
| Administration set | Aluminium sulphate solution | 2000 | 0.2mL/10g bw | 10 females and 10 males |
The preparation method of the aluminum sulfate solution comprises the following steps: accurately extracting 5mL of normal saline by using an injector, injecting into an ampule filled with aluminum sulfate, uniformly mixing, standing for 10-20 min, and fully dissolving to obtain the aluminum sulfate-containing water-soluble chitosan.
The administration route is as follows: and (5) performing intragastric administration.
Frequency of dosing and time of observation: the administration is carried out 1 time by intragastric administration, and the observation is carried out for 14 days after the administration.
Detection indexes are as follows: and (3) clinical observation: observing the general symptoms of the animal daily; and (3) measuring the body weight: weighing the animal body weight at the doses D0, D3, D7 and D14; organ coefficient measurement: on day 15, the animals were sacrificed, and the abnormality of the major organs was observed by dissection, and 5 organs, such as heart, liver, spleen, lung, and kidney, were weighed.
Results processing and analysis: the mean body weight and organ coefficients of the two groups of animals were calculated and compared by treatment with statistical software SPSS 24.
The experimental results are as follows:
during the experiment, no mice death and no obvious abnormality were observed in the control group and the administration group.
The body weight of the animals in the administration group was compared with that in the control group, and the differences were not significant, as detailed in Table 6.
TABLE 6 comparison of body weights of acutely toxic mice
The statistical results of organ coefficients are detailed in table 7.
TABLE 7 comparison of organ coefficients in acute toxicity mice
* Compared with the control group, the mice have no obvious difference (P is less than 0.05), and all the mice survive and die.
And (4) conclusion:
the aluminum sulfate has better safety, the administration dosage is 2000mg/kg, 0.2mL/10g bw0 and 2mL/10g bw normal saline, and no obvious toxic or side effect is seen.
Control test for inhibition of growth of thyroid cancer cells
The experiment was entrusted to the experimental animal center in Hunan province (the drug safety evaluation research center in Hunan province).
The research purpose is as follows: the research observes the in-vitro inhibition effect of aluminum sulfate on two human thyroid cancer cell strains, and provides early test basis for further developing the aluminum sulfate into the thyroid cancer resistant medicament.
The research method comprises the following steps: adopts human thyroid squamous carcinoma cell (SW 579) and human thyroid ductal carcinoma cell (TT) which grow in logarithmic phase, and respectively adds aluminum sulfate (0.3-300 mg/mL), cisplatin and 5-fluorouracil with different concentrations,after incubation for 72h, the absorbance value (OD value) of each well is detected by a CCK-8 method, and IC is calculated 50 . SW579 and TT cells are respectively added with aluminum sulfate (10-100 mg/mL) with different concentrations, after incubation for 6 hours, annexin V-FITC and PI double staining methods are used for detecting apoptosis, hoechst 33342 staining is simultaneously adopted, and apoptosis is detected by a fluorescence microscope. SW579 and TT cells are respectively added with aluminum sulfate (10-100 mg/mL) with different concentrations, and after incubation for 6 hours, the cell cycle is detected by flow cytometry.
1 test materials
1.1 test substance: aluminum sulfate, batch number: 20170410, available from Dawn forest, hunan, biotech development Ltd. Compound preparation: adding 9g of aluminum sulfate into 15mL of 0.9% sodium chloride injection, shaking until the aluminum sulfate is completely dissolved to obtain 600mg/mL of compound solution which is the highest-concentration working solution, and carrying out sterilization treatment on the mother solution for later use. The mother solution is prepared into working solutions of 200, 60, 20, 6, 2 and 0.6mg/mL in turn by using 0.9 percent sodium chloride injection.
1.2 Positive control: cisplatin (DDP), lot number: SJMI-IE, tokyo Kasei Kaisha; 5-Fluorouracil (5-FU), batch number: HFBM160120325008, amresco corporation. Preparing a positive control medicament: DDP or 5-FU 2mg is weighed, a fresh complete culture medium is used for preparing a mother solution with the concentration of 100mM, and the mother solution is sequentially prepared into working solutions with the concentration of 200, 60, 20, 6, 2 and 0.6 mu M by using the fresh complete culture medium.
1.3 main materials:
1.4 Main instruments:
2. test method
2.1 Cell culture
Taking confluent SW579 and TT cells, using high-glucose DMEM containing 10% FBS, at 37 deg.C and 5% CO 2 Culture boxAnd (5) medium culture, wherein the cells are subjected to passage or liquid change for 1-2 d according to the growth condition of the cells until the cells reach the logarithmic growth phase for standby.
2.2CCK-8 assay for cell proliferation
Taking SW579, TT cells grown in log phase at 5X 10/well 3 The cells are inoculated in a 96-well cell culture plate, after 12 hours of cell adherence, a solvent control group, a positive control drug (DDP) group, a positive control drug (5-FU) group and an aluminum sulfate group (0.3-300 mg/mL) are arranged, and each group has 5 multiple wells. The vehicle control group incubated the cells with fresh complete DMEM medium, the compound groups incubated the cells with fresh complete DMEM containing compound at a final concentration of 0.3-300mg/mL, respectively, and the DDP and 5-FU groups incubated the cells with fresh complete DMEM containing compound at a final concentration of 100, 30, 10, 3, 1, 0.3. Mu.M, respectively. After incubating the cells for 72h in the above-mentioned manner, 10. Mu.L of CCK-8 was added to each well, and after further incubation for 1h, the absorbance of each well was measured at 450nm using a microplate reader. The OD value of the solvent control group is taken as 100% of cell viability, and the ratio of the OD value of the rest groups to the OD value of the solvent control group is taken as relative viability. The toxicity of the compound on SW579 and TT cells was evaluated based on the cell growth inhibition rate, and when the cell growth inhibition rate was more than 100%, the system error of the apparatus was judged to be 100%.
2.3 detection of apoptosis
2.3.1 Detection of apoptosis by Annexin V-FITC and PI double staining method
Taking SW579 and TT cells in logarithmic growth phase, collecting cells by conventional digestion, and collecting cells at 5 × 10 5 The density per well is inoculated into a 6-well plate, after 12h of cell adherence, a solvent control group and an aluminum sulfate group (10, 30, 100 mg/mL) are set, and each group has 5 multiple wells. The vehicle control group was incubated with fresh complete DMEM medium, and the compound groups were incubated with fresh complete DMEM containing compounds at final concentrations of 10, 30, 100mg/mL, respectively. After 6h, the cells were collected by conventional digestion, 500. Mu.L Binding Buffer was added to resuspend the cells, the cells were transferred to a 1.5mL EP tube, 5. Mu.L Annexin V-FITC and 5. Mu.L PI were added, incubation was carried out for 15min at room temperature in the dark, and apoptosis was detected by flow cytometry.
2.3.2 detection of apoptosis by fluorescent staining
The cells were treated according to 2.3.1, and after 6 hours of treatment with the compound, 1mL of Hoechst 33342 staining solution was added to each well of a 6-well plate to cover the cells sufficiently, and the cells were cultured at 37 ℃ for 20 to 30min. The staining solution was discarded, washed 2-3 times with PBS, and then subjected to fluorescence detection under a fluorescence microscope.
2.4 flow cytometry to detect the Effect of Compounds on thyroid cancer cell cycle
Taking SW579 and TT cells in logarithmic growth phase, collecting cells by conventional digestion, and collecting cells at 5 × 10 5 The density per well is inoculated into a 6-well plate, after 12h of cell adherence, a solvent control group and an aluminum sulfate group (100, 30 and 10 mg/mL) are set, and each group has 5 multiple wells. The vehicle control group was incubated with fresh complete DMEM medium, and the compound groups were incubated with fresh complete DMEM containing compounds at final concentrations of 10, 30, 100mg/mL, respectively. After 6h, cells were collected by conventional digestion, fixed with 70% cold ethanol overnight, 5 μ L of PI was added, incubated at room temperature in the dark for 30min, and cell cycle was checked by flow cytometry.
2.5 statistical analysis
Processing the data with SPSS 16.0 statistical software, and measuring the data
The mean values of two samples are compared by Student T-Test, the mean values among multiple sample groups are compared by One-way ANOVA Test, P < 0.05 shows that the statistical significance is achieved, and P < 0.01 shows that the tested difference has very significant significance.
3 evaluation of results
3.1 Effect of aluminum sulfate on thyroid carcinoma cell proliferation
When the cells are observed under a microscope and treated by compounds with different concentrations, the phenomena of slow cell proliferation speed, increase of cell fragments, increase of cell gaps, sand-shaped vacuoles of the cells and the like appear. The cell state has clear correlation with the co-culture time, and after the co-culture is carried out for 12h, the cells have the phenomena of rounding and shrinking; after the co-culture is carried out for 24h, partial cell swelling occurs, the light transmittance of the cells is poor, and the intercellular space is increased; after the co-culture is carried out for 48 hours, the cells have sand-like vacuoles, and the cells are broken; after 72h of co-culture, the arenaceous vacuole-like cells were almost completely ruptured, and no cells with intact cell morphology were observed at concentrations of 300, 100 and 30mg/mL, and only a small amount of concentration was observed to form black spots. After the cells are co-cultured with a test sample or a positive control drug DDP and 5-FU for 72 hours, the cell proliferation is obviously inhibited, and the cells have a concentration-effect relationship and have a significant difference (P is less than 0.01) compared with a solvent control group. The cell proliferation inhibition results are detailed in table 8.
TABLE 8 Effect of aluminum sulfate on thyroid cancer cell proliferation
Note: * Represents P in comparison with the vehicle control group<0.05, P represents the ratio of the total amount of the composition to the vehicle control group<0.01。IC 50 Indicating the concentration of tumor cells that inhibited 50%, emax indicating the maximum inhibition of tumor cells.
3.2 Effect of aluminum sulfate on apoptosis of thyroid carcinoma cells
And (3) interfering the thyroid cancer cells for 6 hours by adopting compounds with the concentrations of 10, 30 and 100mg/mL, collecting the cells, and detecting apoptosis after Annexin V-FITC/PI double staining. The flow cytometer can divide the double stained cells into 4 groups: Q1-UL represents mechanically damaged cells (Annexin V-/PI +); Q1-UR late apoptotic cells (Annexin V +/PI +); Q1-LL viable cells (Annexin V-/PI-); Q1-LR early apoptotic cells (Annexin V +/PI-). The results show that: the number of the SW579 cells treated by the aluminum sulfate in late apoptosis and necrosis is obviously higher than that of a solvent control group (P is less than 0.05), and the concentration-effect relationship is realized; the number of the TT cells treated by the aluminum sulfate in late apoptosis and necrosis is obviously higher than that of a solvent control group (P is less than 0.05), and the TT cells treated by the aluminum sulfate have a concentration-effect relationship.
TABLE 9 Effect of aluminum sulfate on apoptosis of human thyroid carcinoma cells
Note: * Represents the comparison with the vehicle control group, P is less than 0.05, P is less than 0.01
3.3 Effect of aluminum sulfate on thyroid carcinoma cell cycle
After the thyroid cancer cells are intervened by using compounds with the concentrations of 10, 30 and 100mg/mL for 6 hours, the cells are collected, fixed by 70% cold ethanol, stained by PI, and analyzed by a flow cytometer in the cell cycle. The results show that: the compound obviously increases the ratio of G0/G1 cells and obviously reduces the ratio of G2/M, which shows that the compound has the effect of inhibiting the proliferation of thyroid cancer cells mainly by blocking the thyroid cancer cells in the G0/G1 stage and preventing the thyroid cancer cells from entering the S stage.
TABLE 10 Effect of aluminum sulfate on human thyroid cancer cell cycle
Note: * Represents the comparison with the vehicle control group, P is less than 0.05, P is less than 0.01
4 conclusion and discussion
In conclusion, under the experimental conditions, aluminum sulfate can obviously inhibit the proliferation of two thyroid cancer cells, namely IC 50 The values were SW579 (14.408 mg/mL) and TT (14.116 mg/mL), respectively. And further has obvious apoptosis promoting effect and concentration-effect relationship, and can arrest the cell cycle in the G0/G1 phase and induce apoptosis. Under high concentration condition, cancer cells can be completely killed along with the prolonging of time. The compound has higher cancer cell inhibition concentration, is in a mg/mL level, and is possibly related to a special action mechanism. The compound directly acts on the surface of tumor cells, can change the physiological characteristics of the cancer cells, can effectively change the protein structure on the surface of the cells after being gathered on the surface of the cells, causes the protein precipitation on the surface and the intercellular substance of the cells, leads to the serious reduction of the permeability of the cells, the contraction of the intercellular space, reduces the division capacity of the tumor cells, and effectively controls the proliferation and the metastasis of the cells. After the compound enters into cells, the compound can inhibit cancer cells from secreting various proteolytic enzymes, can be directly combined into DNA of the cancer cells to cause DNA lysis, and effectively inhibit metabolic functions of mitochondria of the cancer cellsThe combination of succinate dehydrogenase in mitochondria can change the biological effect, cause the change of cell microstructure, block signal path, interfere the growth and metabolism of tumor cells, induce the apoptosis of tumor cells and realize the effect of killing cancer cells.
And (4) proposing:
for example, after the injection and the needle feeding are finished in the tumor transplantation maturity of an animal experiment mouse, the needle feeding channel is plugged or closed, and meanwhile, only the animal experiment mouse needs to be fed in an isolated mode, and because the injection of the medicine is finished, the mouse has certain smell and small bulges, and the mice can be bitten by each other if the mice are fed in groups, so that the medicine liquid flows out and the medicine effect is influenced. For example, the human tumor needle insertion does not need to plug the needle insertion channel or seal the needle insertion channel.
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present invention.
Claims (2)
1. The drug for targeted therapy of thyroid cancer is characterized in that the active ingredient is aluminum sulfate octadecahydrate; the pharmaceutical dosage form is injection powder;
the aluminum sulfate octadecahydrate is obtained by purifying the following method:
1) Mixing and dissolving aluminum sulfate and ultrapure water to obtain an aluminum sulfate solution;
2) And (3) carrying out fine filtration on the aluminum sulfate solution, and carrying out freeze drying to obtain the aluminum sulfate octadecahydrate freeze-dried powder.
2. The pharmaceutical according to claim 1, wherein the mass ratio of aluminum sulfate to ultrapure water in the purification of the hydrate of aluminum sulfate is 1:3.
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| 徐风华 等: ""复方硫酸铝注射液体外及小鼠瘤体注射抗肿瘤作用的研究"", 《解放军药学学报》, vol. 21, no. 6, pages 420 - 422 * |
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