Preparation of cancer treatment medicine freeze-dried powder injection

Abstract

The freeze-dried powder injection can effectively control postoperative recurrence of tumors. The freeze-dried powder injection is advanced in preparation process, low in cost, free of pollution and high in effective component extraction rate. The freeze-dried powder injection preparation process is advanced, and trace heat source substances (allergic substances) in the injection can be effectively removed. In the formula of the freeze-dried powder injection, the anti-cancer active ingredient bufonin cantharidin of the venenum bufonis cantharides is unstable in the injection, so that the consistency of the anti-cancer curative effect is difficult to guarantee, and various active ingredients in the freeze-dried powder injection are stable. And the stability investigation accords with pharmacopoeia regulations. The injection prepared from the venenum bufonis and the cantharides has serious irritation to peripheral veins, and the freeze-dried powder has no irritation to the peripheral veins.

Description

[0001] Through more than 10 years of hard work in the research and medication practice of freeze-dried powder injection, we have made many revisions and reforms to the preparation process and use of freeze-dried powder injection. The freeze-dried powder injection has controllable quality, definite efficacy, and safe medication. There are many innovative improvements as follows

[0002] 1. Control tumor recurrence;

[0003] Patients with highly malignant tumors, such as osteosarcoma and pancreatic cancer, are selected. These highly malignant tumors often recur about two years after surgery. The lyophilized powder injection is intravenously dripped in large doses for 3 weeks, with daily fat toad venom + cinobinovein not less than 1mg and cantharidin not less than 1mg. After 3 weeks, the daily dosage of both is not less than 0.8mg. 60 consecutive days is a course of treatment, 3 courses in the first year, and 2 courses in the second year. In the early stage of treatment, a small dose of antimetabolite chemotherapy drugs such as 5-fluoroadenine (5-fu) is used for intravenous drip once a day for 5 consecutive days. Myeloid-derived suppressor cells have a strong inhibitory effect on the tumor immune cell immune system, causing the anti-tumor immune system to completely lose the function of killing and inhibiting tumor cells. Myeloid-derived suppressor cells are extremely sensitive to anti-metabolite chemotherapy drugs and can be completely eliminated at low doses. Adding lyophilized powder has the effect of enhancing the efficacy of postoperative control of recurrence.

[0004] 2. High extraction rate of effective ingredients

[0005] 1000ml is prepared from 100g of red ginseng, 300g of astragalus, 0.1g of toad venom and 2g of blister beetle. According to the original preparation process, red ginseng alcohol extract, astragalus decoction is added with calcium hydroxide milk to pH 12. The viscous liquid formed at this time is extremely difficult to filter, and requires a large amount of pulp, cardboard, and centrifugation, which is time-consuming and consumable. Due to the large amount of mucus encapsulating and adsorbing active ingredients, it is difficult to elute. A large amount of active ingredients are lost. The freeze-dried powder injection preparation process has key improvements. That is, the volume of the decoction must be adjusted to 1:15 and the temperature must be controlled at 65℃±2℃. The pH is greater than 12 when calcium hydroxide milk is added. After being placed for about 2 hours, the liquid becomes clear and the supernatant does not need to be filtered, which solves the great difficulty of preparation filtration. The content of the active ingredients of ginsenosides and astragalosides per milliliter in the original process is only 0.15-0.18 mg. The new improved process not only saves time and materials, but also the active ingredients of ginsenosides and astragalosides in freeze-dried powder injection (diluted to 1000 ml) are 0.6-0.8 mg per milliliter, which is more than 3 times higher.

[0006] 3. Can remove trace pyrogens (allergens) in injections

[0007] The long-standing problem of unsafe clinical use of Chinese medicine injections is that trace allergic substances cannot be completely removed. The freeze-dried powder injection process is to remove the precipitate from the supernatant after calcium hydroxide emulsion precipitation at different pH values. 2PH3.0--4.0. Concentrate to 1:1 and adjust PH8.0 with NaoH (0.1mol) to filter out precipitate. High pressure 115℃ for half an hour. Leave for more than one week. Tested with high sensitivity reagents and all were negative. Tested with high sensitivity reagents and showed negative reaction, which means there is no trace pyrogen (allergenic substance) in the injection.

[0008] 4. The instability of toad venom-based cantharidin, an effective anti-cancer ingredient in toad blister beetles, in injection has long been a huge problem that is difficult to solve. It decomposes during heating and storage. Multiple universities and pharmaceutical research institutions have repeatedly studied this issue for more than 10 years but have been unable to solve it. The freeze-dried powder injection is sterilized and freeze-dried at a certain temperature of 100°C for 30 minutes. The stability tests of all active ingredients in the freeze-dried powder injection all meet the requirements of the pharmacopoeia.

[0009] 5. The injection prepared from toad venom and cantharids, the anti-cancer active ingredients, toad venom base and cantharidin are severely irritating to peripheral veins. The medication needs to be administered through a central vein, which is difficult to use in most hospitals. The lyophilized powder injection repeatedly adjusts the pH value to remove a large number of impurities that are irritating to the blood vessel wall, and the pH value is adjusted to not less than 8 with NaOH. Cantharidic acid, which has the strongest irritation to the blood vessel wall, exists in the injection in the form of sodium salt when the pH value is not less than 8. The inhibitory effect of sodium cantharidic acid on tumors is greater than that of cantharidic acid and it is non-irritating to peripheral veins. When the pH value is not less than 8, the irritation of other ingredients to peripheral superficial veins is also significantly reduced. Technical Field

[0010] The present invention relates to a freeze-dried powder injection composed of ginseng, astragalus, toad venom and mylabris, and a reasonable preparation process. The freeze-dried powder injection can effectively control the recurrence of tumors after surgery, and has reliable efficacy on middle and late stage liver cancer, lung cancer, breast cancer and the like. The technical field specifically relates to prescription combinations and preparation processes. The extraction rate of effective ingredients is high. The preparation process can effectively remove trace pyrogens (allergic substances) in the freeze-dried powder injection. Various components in the freeze-dried powder injection can maintain good stability and have no irritation to peripheral blood vessels. The clinical efficacy is reliable, the quality is controllable, and the medication is safe. It can open up a new way for the treatment of tumors, and is a freeze-dried powder injection anti-tumor therapeutic drug. Technical Background

[0011] In the 1960s, I was the director of the New Medical Department at the Heilongjiang Production and Construction Corps Hospital. Most of the patients admitted to the New Medical Department were patients who did not respond well to conventional treatment or were ineffective, most of whom were patients with advanced cancer. In the 1960s, medicines were extremely scarce, especially antibiotics and analgesics required by advanced cancer patients. Even aspirin painkillers were in short supply, so we had to work hard on Chinese herbal medicines. It was very difficult for most advanced patients to take Chinese herbal decoctions orally, and the effect was not ideal. Chinese medicine fully retains the effective ingredients and is administered intravenously to treat advanced patients, which has a better effect, especially for the stubborn pain of advanced patients. The conventional method of preparing Chinese medicine injections requires a large amount of ethanol, which was also an extremely scarce material at the time. I have developed a method to replace ethanol to extract Chinese medicine injections, namely Ca(HO) 2 -H 2 SO 4 The method is used to prepare Chinese medicine injections for clinical use. A variety of Chinese medicine injections are prepared by formulating a variety of Chinese medicines such as toad venom, blister beetles, ginseng, astragalus, garcinia, aconite, and zedoaria. These Chinese medicine injections have reliable efficacy in prolonging the survival time of patients and relieving the pain of terminal patients. However, the liquid medicine prepared by this process produces smoke and precipitation in about 2 months, and then it is changed to Ca(OH) 2 -SO 2 The method has significantly improved clarity and stability compared with the previous method, and has been used to prepare a variety of Chinese medicine injections for clinical use for many years. It solved the problem of extreme shortage of medicines in remote areas at that time. The preparation process was used to successfully develop Delisheng injection, which was approved as a national medicine for tumor treatment.

[0012] The author has been engaged in clinical practice for 30 years and has been a full-time drug researcher for nearly 20 years. Traditional Chinese medicine can fully retain the effective ingredients for intravenous administration. In most cases, the therapeutic effect is definitely better than oral administration. In some cases, oral administration cannot achieve new therapeutic effects. Traditional Chinese medicine injections have low toxicity. Even for toxic or even highly toxic drugs, as long as the effective dose and safe dose are strictly controlled, there are very few cases of heart, liver, and kidney damage. During the preparation process, the effective ingredients are not lost in large quantities, the formula is appropriate and reasonable, and the efficacy on many diseases is certain.

[0013] At present, the research and development of Chinese medicine injections is in extreme difficulties. Some people in the industry even hope to ban Chinese medicine injections that are fully extracted from ingredients, because Chinese medicine injections occasionally have extremely serious allergic reactions in clinical practice. Although they are extremely rare, severe allergic reactions can occur within 1 minute or 10 minutes of intravenous infusion. The patient's heartbeat and breathing can stop at the same time, and it is difficult for the patient to get a chance to receive treatment. This kind of unsafe clinical drug use has occurred in many Chinese medicine injections, and some have occurred multiple times. The author has conducted hundreds of studies for more than 10 years in the preparation of freeze-dried powder injections for the hypersensitive reactions of very few patients to trace allergens. It was found that the substance that causes severe allergic reactions is the trace pyrogen (allergen) in the injection. In the study, we used 48 kinds of Chinese medicine to prepare 26 kinds of Chinese medicine injections made by ethanol extraction, and conducted (allergen) pyrogen tests. The results were all qualified according to the rabbit pyrogen test method specified in the pharmacopoeia, and no batch had pyrogen reactions. However, 8 batches were positive and 6 batches were weakly positive when tested with high-sensitivity horseshoe crab reagents. 14 batches of freeze-dried powder injections were prepared using the original process, of which 2 batches were positive and 1 batch was weakly positive. The sensitivity of high-sensitivity horseshoe crab reagents to the detection of trace allergens is 10 times that of rabbits (0.06-0.015Eu / ml). These trace pyrogens may not appear in 1,000 patients, but one in 2,000 patients may be extremely sensitive to trace allergens and have extremely serious allergic reactions. After Delisheng injection entered clinical use, a large number of patients occasionally experienced allergic reactions of varying severity. Allergic reactions often occurred within 1 to 15 minutes of intravenous infusion during the first use of the drug. Patients experienced chest tightness, dyspnea, nausea, and vomiting. In particular, the first use of the drug has a high dilution concentration and a fast drip rate, which can easily cause allergic reactions (mild or moderate up to 15%). Therefore, the instructions also state that for the first use of the drug, the dilution concentration should not be lower than 1:10, and no more than 15 drops per minute. If there are no adverse reactions after half an hour, the number of drops can be gradually increased, but no more than 40 drops per minute is appropriate. Allergic reactions are an extremely serious drug safety event. Patients with allergic reactions have no positive reactions in skin tests. In order to solve the allergic reactions, the author conducted multi-faceted research and experiments on the preparation of the freeze-dried powder injection. Finally, all the precipitates produced at different pH were filtered out, and Ca(OH) was added at a certain temperature and a certain volume. 2 After filtering, SO 2 The pH of the concentrated solution prepared by adjusting the pH was then adjusted with NaOH to remove the precipitate. None of the 8 batches of freeze-dried powder injections showed positive or weak positive reactions when tested with a highly sensitive horseshoe crab test kit, and no positive or weak positive reactions were found when tested with a more sensitive colorimetric method. When used clinically, at a high concentration (1:5) of 80 drops per minute, no allergic reactions were seen in patients.

[0014] The main anti-cancer active ingredients of the freeze-dried powder injection are lipobufic toad, cinobucoids, and cantharidin. These anti-cancer active ingredients are all toxic drugs and are also the main anti-cancer ingredients in the freeze-dried powder injection. The effective dose and toxic dose are very close. The quality standard is very rigorous. The daily dosage is 5-6 bottles, which is both a sufficient effective dose and a safe dose in clinical practice. In the injection, lipobufic toad + cinobucoids are 12-18ug / ml, and cantharidin is 14-18ug / ml. The upper and lower limits are only 4ug / ml. It is difficult for inspectors who are not very skilled to accurately measure this extremely narrow data. The error may also exceed this range. Due to the large-scale use of pesticides, the active ingredient cantharidin in artificially raised cantharids has not only decreased in content, but also significantly decreased in stability. Toad venom is also mostly cultivated lipobufic toad and the stability of cinobucoids has also decreased.

[0015] Therefore, it is impossible to maintain the original standard of water injection. The original quality standard has been confirmed by clinical use for many years that the measurement standard of anti-cancer toxic components is both the best effective dose standard and a safe measurement standard. A new dosage form must be developed. The author has been conducting research on freeze-dried powder injection dosage forms since 2001, and used freeze-dried powder injections to treat relatives and friends for free after tumor surgery chemotherapy and control postoperative recurrence of tumors, and conducted clinical observation and follow-up. Among those who used the freeze-dried powder injection, no patient had allergic reactions or other adverse reactions, even the first intravenous injection at a rate of 80 drops per minute.

[0016] The freeze-dried powder was tested for stability and the effective ingredients could be kept for more than two years. All effective ingredients were within the standard range (3 months of heating test and 2 years at room temperature). Liposobacillus, Cinobufacini and Cantharidin were all stable within the quality standard range.

[0017] Implementation

[0018] Specific implementation scheme of the freeze-dried powder injection

[0019] 1. Preparation process

[0020] 1. Take the prescribed amount of toad venom and grind it into 60 mesh size. Add appropriate amount of water and stir into a paste. Add 25 times amount of ethanol and soak overnight. Filter and rinse the residue with a small amount of ethanol (the residue is placed in the refrigerator as residue ①). Adjust the ethanol concentration to 85% with ethanol solution. Leave it for 24 hours. Filter with 0.3 microporous membrane. Recover ethanol from the filtrate until there is no ethanol smell. Place it in a refrigerator at 0℃ (1).

[0021] 2. Crush the blister beetles (60 mesh), add 50 times the amount of acetone and adjust the pH to 3 with HCl, soak overnight, filter (the residue is placed in the refrigerator, residue ②), recover acetone from the filtrate, wash the oily residue with petroleum ether until white crystals are obtained, place in the refrigerator (2), recover the petroleum ether oil from the petroleum ether washing solution, place in the refrigerator, residue ③.

[0022] 3. Add 65% ethanol to ginseng + residue ②③ and reflux for 3 times, the first time for 1.5 hours, the second and third times for 1 hour each time, add 8 times the amount of 65% ethanol each time, combine the extracts, recover the ethanol until there is no ethanol smell, and put it in the refrigerator; add water to Astragalus + residue ① and boil it twice, each time adding 8 times the amount of water, each time boiling for 1.5 hours, combine the decoctions and add ginseng ethanol extract, adjust the volume to 1:15 and heat to 65℃, add calcium hydroxide milk until the pH is greater than 12, stir, filter with cotton cloth, pass sulfur dioxide through the filtrate until precipitation occurs (pH is about 7.5-8.0), filter, pass SO 2 Filter to pH 4.0, concentrate the filtrate to 2:1, adjust pH 8.0 with 10% NaOH and filter, seal the filtrate in a 500ml saline bottle, sterilize at 115°C for 30 minutes, let stand for one week, add acetone extract of Mylabris blister and ethanol extract of Toad Venenum, filter, let stand for 1 week, filter and pack, and place in a low-temperature refrigerator to precool to -25~-30°C. Quickly transfer to a freeze dryer, turn on the vacuum pump when the temperature reaches -25°C, and the vacuum degree reaches 660Pa. Start heating the shelf, keep it at -20°C for 2 hours, -15°C for 1.5 hours, and -10°C for 2 hours. No crystal water is observed by naked eyes, and the shelf temperature is kept at 26°C for about 2 hours. After freeze drying, fill with nitrogen and seal.

[0023] (1) Good reconstitution: Take the freeze-dried powder injection, add 10 ml of water for injection to each vial, and gently shake it 2 to 3 times to completely dissolve it. Alternatively, after injecting water, let it stand for 20 seconds to completely dissolve it and the solution will be clear.

[0024] (2) Appearance: Light yellow clear liquid

[0025] Technology highlights

[0026] 1. It can effectively remove allergic substances in the injection solution. The freeze-dried powder injection preparation process is advanced and reasonable, which can completely remove trace allergic substances in traditional Chinese medicine injections, solving the problem of unsafe use of traditional Chinese medicine injections that has long plagued the clinical use of traditional Chinese medicine injections.

[0027] 2. The freeze-dried powder injection can effectively control the postoperative recurrence of tumors. This is a breakthrough in tumor treatment and opens a new path for tumor treatment.

[0028] 3. The freeze-dried powder injection preparation process is advanced and reasonable. It can not only effectively remove trace allergic substances in the injection powder, but also retain various effective ingredients to the maximum extent. All effective ingredients have good stability. The freeze-dried powder is non-irritating to peripheral veins.

[0029] Typical Cases

[0030] Typical cases of freeze-dried powder injection

[0031] Case

[0032] Zhou, female, 25 years old, from Anyue, Sichuan, developed left abdominal pain in November 2003, which gradually worsened. She was diagnosed with left ovarian teratoma in Chengdu People's Hospital, Sichuan, and underwent surgical resection. The pathology was grade 3. No other treatment was performed after the operation. From June 2004, she began to use 5-6 injections of the freeze-dried powder + 250-500 ml diluted with saline solution, intravenous drip, once a day, for 45 days as a course of treatment. After 45 days, the drug was stopped for 3 days and the second course of treatment was carried out. There were 4 consecutive courses of treatment, and 3 courses of treatment each year in the second and third years. The patient is still healthy and has negative tumor markers.

[0033] Zhang, female, 29 years old, from Gu'an County, Hebei Province, was diagnosed with osteosarcoma in 2014. She was treated with the freeze-dried powder injection after the operation, 5-6 injections per day diluted with 250ml of saline and then dripped intravenously for 45 days as a course of treatment. She was treated for 4 courses in the first year, and 3 courses each year for 5 consecutive years. She was healthy and had no recurrence during the follow-up in 2017.

[0034] Triple-negative breast cancer and grade III brain glioma have not recurred for more than 8 years after surgery. Grade III teratoma and osteosarcoma, which are extremely malignant tumors, often recur in about 2 years after surgery regardless of any treatment methods. Triple-negative breast cancer and grade III brain glioma also often recur within 3 to 5 years. No tumor patients treated with this freeze-dried powder injection had a recurrence.

[0035] Freeze-dried powder injection quality inspection

[0036] [Properties] This product is a light yellow-brown clear liquid.

[0037]

Identification

[0038] (1) Take 10 ml of this product and shake and extract twice with n-butanol saturated with water, 20 ml each time, combine the n-butanol extracts, evaporate to dryness, dissolve the residue with 3 ml of water, add it to a neutral alumina column (100-120 mesh, 5 g, inner diameter 0.9 cm), elute with 50 ml of 40% methanol, collect the eluate, evaporate to dryness, and dissolve the residue with 1 ml of methanol as the test solution. Separately, take the ginsenoside Re reference substance, ginsenoside Chi reference substance, and astragaloside IV reference substance, add methanol to make a mixed solution containing 1 mg of each per 1 ml, as the reference substance solution. According to the thin layer chromatography method (Appendix VI B of Part I of the 2005 edition of the Chinese Pharmacopoeia), 5% of each of the above two solutions were taken and spotted on the same silica gel G thin layer plate, and the lower layer solution placed below 10°C in chloroform-methanol-water (13:7:2) was used as the developing agent. The plate was developed, taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots were clearly colored, and examined under sunlight and ultraviolet light (365nm) respectively. In the chromatogram of the test sample, at the corresponding position of the chromatogram of the reference sample, spots of the same color appeared under sunlight; and fluorescent spots of the same color appeared under ultraviolet light.

[0039] (2) Take 30 ml of this product and shake and extract it twice with chloroform, 30 ml each time. Combine the chloroform extracts, evaporate to dryness, and dissolve the residue in 0.5 ml of chloroform to prepare a mixed solution containing 1 mg of each in 1 ml of chloroform. Use this solution as the reference solution. According to the thin layer chromatography method (Appendix VI B of Part I of the Chinese Pharmacopoeia 2005 Edition), take 10 ml of the test solution and add chloroform to the residue. M 5jJ of the k reference solution were spotted on the same silica gel G thin layer plate, developed with chloroform-acetone-cyclohexane-ethyl acetate (4:2.2:5:1) as the developing solvent, taken out, dried, sprayed with 10% sulfuric acid ethanol solution, and heated until the spots were clearly colored. In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions of the chromatogram of the reference substance.

[0040]

examine

[0041] The pH value should be 6.5-8.0 (Appendix VIIIG of Part I of the Chinese Pharmacopoeia 2005 edition).

[0042] Batch number: 20180705 7.5% Batch number: 20180807 7.7%

[0043] Batch No.: 20180819 7.3%

[0044] Ignition residue: Accurately measure 2 ml of the product and place it in a crucible that has been ignited to constant weight, evaporate to dryness, and inspect according to the law (Appendix IX J of Part I of the Chinese Pharmacopoeia 2005 Edition) from "slowly ignite until completely carbonized". The residue shall not exceed 0.65%.

[0045] Batch number: 20180705 0.48% Batch number: 20180807 0.50%

[0046] Batch No.: 20180819 0.42%

[0047] Heavy metals: The residue under the ignition residue inspection item shall be inspected in accordance with the law (Appendix IX E Method 2 of Part I of the 2005 edition of the Chinese Pharmacopoeia). The heavy metal content shall not exceed 10 parts per million.

[0048] Batch number: 20180705 (-) Batch number: 20180807 (-)

[0049] Batch No.: 20180819 (-)

[0050] Arsenic salt Take 1 ml of this product, add 1 g of calcium hydroxide, mix well, evaporate to dryness, first burn with low heat to carbonize, then burn at 500-600℃ to completely ash, cool, add 5 ml of hydrochloric acid and 23 ml of water to the residue to dissolve, and check according to the law (Appendix IX F Method 1 of Part I of the 2005 edition of the Chinese Pharmacopoeia). The arsenic content of this product shall not exceed 2 parts per million.

[0051] For protein, take 1 ml of this product, add 1 ml of newly prepared 30% sulfosalicylic acid test solution, mix well, and let stand for 5 minutes. No turbidity should appear. For tannin, take 1 ml of this product and check according to the law (Appendix IX S of Part I of the 2005 edition of the Chinese Pharmacopoeia). It should meet the regulations.

[0052] Batch number: 20180705 (-) Batch number: 20180807 (-)

[0053] Batch No.: 20180819 (-)

[0054] Take 5 ml of this product from the resin and inspect it according to the law (Appendix IX S of Part I of the 2005 edition of the Chinese Pharmacopoeia). It should meet the requirements.

[0055] Batch number: 20180705 (-) Batch number: 20180807 (-)

[0056] Batch No.: 20180819 (-)

[0057] Oxalate Take 5 ml of this product and check it according to the law (Appendix IX S of Part I of the Chinese Pharmacopoeia 2005 Edition). It should meet the requirements.

[0058] Batch number: 20180705 Complies with regulations Batch number: 20180807 Complies with regulations

[0059] Batch number: 20180819 Complies with regulations

[0060] Potassium ion: Accurately measure 2 ml of this product and check it according to the law (Appendix IX S of Part I of the 2005 edition of the Chinese Pharmacopoeia). It should meet the regulations.

[0061] Take this product for pyrogen and check it according to the law (Appendix XIIIA of Part I of the 2005 edition of the Chinese Pharmacopoeia). The dosage is 3 ml per kg of body weight of rabbits. It should comply with the regulations.

[0062] Batch number: 20180705 (-) Batch number: 20180807 (-)

[0063] Batch No.: 20180819 (-)

[0064] Others should comply with the relevant provisions under injection (Appendix IU of Part I of the Chinese Pharmacopoeia 2005 Edition).

[0065] [n-Butanol extract] Accurately measure 25 ml of this product, place it in a separatory funnel, and shake with chloroform to extract 3 times, 30 ml each time. Combine the chloroform extracts, wash twice with water, 10 ml each time, add the washings to the above aqueous solution, shake and extract 3 times with water-saturated n-butanol, 30 ml each time, combine the n-butanol extracts, dry to a constant weight in an evaporating dish, evaporate to dryness, and dry at 105°C for 3 hours. Cool in a desiccator for 30 minutes, and quickly weigh the weight to obtain the product.

[0066] This product contains not less than 0.15% n-butanol extract.

[0067] Batch number: 20180705 0.28% Batch number: 20180807 0.29%

[0068] Batch No.: 20180819 0.276%

[0069]

Content determination

[0070] Total solids: Take 10 ml of this product accurately, place it in an evaporating dish that has been dried to a constant weight, evaporate to dryness, dry at 105°C for 3 hours, cool in a desiccator for 30 minutes, and weigh it quickly. This product should contain no less than 20 mg of total solids per 1 ml.

[0071] Batch number: 20180705 24mg Batch number: 20180807 23mg

[0072] Batch number: 20180819 28mg

[0073] Total polysaccharides

[0074] Preparation of reference solution: Take 80 mg of anhydrous glucose reference substance dried to constant weight at 80°C, accurately weigh it, place it in a 100 ml volumetric flask, dissolve it with water and dilute it to the mark, shake it well, accurately measure 5 ml, place it in a 50 ml volumetric flask, add water to the mark, shake it well, and the solution is obtained (each 1 ml contains 80 ug of anhydrous glucose).

[0075] Preparation of standard curve: 0.2, 0.4, 0.6, 0.8, 1.0 ml of reference solution were accurately measured and placed in 10 ml stoppered test tubes respectively. Water was accurately added to each tube to make 1 ml. 4 ml of anthrone sulfate solution (50 mg of anthrone was dissolved in 25 ml of water and 75 ml of sulfuric acid: prepared before use) was accurately added in an ice bath, shaken well, heated in a boiling water bath for 10 minutes, and cooled for 40 minutes; the corresponding solution was used as blank. According to the UV-visible spectrophotometry method (Appendix VA of Part I of the Chinese Pharmacopoeia 2005 Edition), the absorbance was measured at a wavelength of 625 nm, and the standard curve was drawn with absorbance as the ordinate and concentration as the abscissa.

[0076] Preparation of the test solution: Precisely measure 50 ml of the product, concentrate it in a water bath to about 5 ml, cool it, transfer it to a 10 ml volumetric flask, add water to the mark, and shake it well. Precisely measure 1 ml, accurately add 10 ml of anhydrous ethanol, seal it, shake it well, let it stand for 4 hours, centrifuge it, discard the supernatant, dissolve the precipitate with water, transfer it to a 50 ml volumetric flask, dilute it with water to the mark, shake it well, accurately measure 5 ml, place it in a 50 ml volumetric flask, add water to the mark, shake it well, and you have it.

[0077] Determination method: Accurately measure 1 ml of the test solution and place it in a 10 ml stoppered test tube. According to the method under the standard curve, starting from "accurately add 4 ml of anthrone sulfate solution in an ice bath", determine the absorbance according to the law, and read the amount of polysaccharide in the test solution from the standard curve to obtain.

[0078] This product contains total polysaccharide in anhydrous glucose (C 6 H 12 O 6 ) shall not be less than 3.5 mg.

[0079] Batch number: 20180705 4.8mg Batch number: 20180807 4.3mg

[0080] Batch number: 20180819 5.01mg

[0081] The content of toad venom was determined by high performance liquid chromatography (Appendix VI D of Part I of the Chinese Pharmacopoeia 2005 edition).

[0082] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel as filler; n-propanol-methanol-0.04% potassium dihydrogen phosphate solution (2:3:7) as mobile phase; detection wavelength is 300nm. The theoretical plate number calculated based on the base peak of cinona toad venom should not be less than 2500.

[0083] Preparation of reference solution: Take appropriate amount of resin toad venom based reference substance and cinobucini based reference substance respectively. Weigh accurately. Add methanol to make a mixed solution containing 40ug of each per 1ml.

[0084] Preparation of test solution: Accurately measure 20 ml of the product, place in a separatory funnel, and extract with chloroform by shaking for 4 times, 30 ml each time. Filter the extract through filter paper covered with a small amount of anhydrous sodium sulfate. Combine the filtrates and evaporate to dryness. Dissolve the residue with methanol and transfer to a 5 ml volumetric flask. Add methanol to the mark and shake well to obtain the solution.

[0085] The determination method is to accurately pipette 10ul of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and determine them.

[0086] Each 1ml of this product contains toad venom and resinobufagin (C 24 H 32O 4 ) and cinobucoid toxin (C 26 H 34 O 6 ) should be 12 to 18 ug.

[0087] Batch number: 20180705 17.8ug Batch number: 20180807 16.96ug

[0088] Batch number: 20180819 17.30ug

[0089] The content of Mylabris was determined by gas chromatography (Appendix VIE of Part I of the Chinese Pharmacopoeia 2005 edition).

[0090] The chromatographic conditions and system suitability test used methyl silicone rubber (SE-30) as the stationary liquid, with a coating concentration of 3.5% and a column temperature of 165 ± 10°C. The theoretical plate number calculated based on the cantharidin peak should not be less than 2500.

[0091] Preparation of reference solution: Take an appropriate amount of cantharidin reference substance, weigh accurately, and add chloroform to make a solution containing 0.32 mg per 1 ml.

[0092] Preparation of test solution: Accurately measure 50 ml of the product, adjust the pH to 3-4 with 5% sulfuric acid solution, and extract 5 times with water-saturated chloroform by shaking, 40 ml each time. Combine the chloroform extracts, filter through filter paper covered with a small amount of anhydrous sodium sulfate, combine the filtrates, evaporate the chloroform to about 10 ml on a water bath below 60°C, and then evaporate naturally to 2-3 ml, transfer to a 5 ml volumetric flask, add chloroform to the scale, shake well, and obtain.

[0093] The determination method is to accurately pipette 2ul of the reference solution and 4ul of the test solution respectively, inject them into the gas chromatograph, and determine the result.

[0094] Each 1ml of this product contains cantharidin (C W H 12 O 4 ) should be 14 to 18ug.

[0095] Batch number: 20180705 17.0ug Batch number: 20180807 17.2ug

[0096] Batch number: 20180819 17.6ug

[0097] [Function and indications] It can replenish qi and strengthen the body, eliminate symptoms and disperse nodules. It is used for the syndrome of qi deficiency and stasis in middle and late stage primary liver cancer, with symptoms of right rib and abdominal mass, constant pain, abdominal distension, loss of appetite, fatigue and weakness.

[0098] [Usage and Dosage] Intravenous drip. Dilute 40-60ml of this product with 500ml 5% glucose injection or sodium chloride injection, and the drip rate should not exceed 60 drops per minute, once a day. The first dose of each course of treatment is halved, and diluted with 500ml 5% glucose injection or sodium chloride injection, and dripped slowly, with a drip rate of no more than 15 drops per minute. If there is no adverse reaction, the drip rate can be gradually increased after half an hour, and it is preferably not more than 60 drops per minute. A course of treatment is 45 days. The next course of treatment can be carried out after one week of discontinuation of the drug, or follow the doctor's advice.

[0099]

Note

[0100] [Specification] 10ml per bottle

[0101]

Storage

[0102] [Validity period] 24 months

[0103] Study on the stability of freeze-dried powder injection

[0104] 1. Observe at 395℃ for three months (placed in constant temperature phase)

[0105] Three batches produced in 2014

[0106] (1) Results of the first batch heating test

[0107] Batch number: 20190305 First month

[0108] Check items Quality standard stipulates the content per ml Measured results Butanol extract Not less than 0.15% 0.25％ Total polysaccharides No less than 3.5mg per ml 4.9mg Resin + Cinobufacini Each ml should be 12-18ug 16.92ug Cantharidin Each ml should be 14-18ug 17.3ug

[0109] Batch number: 20190305 February

[0110] Check items The quality standard stipulates the content per ml Measured results Butanol extract Not less than 0.15% 0.28％ Total polysaccharides No less than 3.5mg per ml 4.6mg Resin + Cinobufacini Each ml should be 12-18ug 16.02ug Cantharidin Each ml should be 14-18ug 16.92ug

[0111] Batch number: 20190305 March

[0112] Check items Quality standard stipulates the content per ml Measured results Butanol extract Not less than 0.15% 0.26％ Total polysaccharides No less than 3.5mg per ml 4.0mg Resin + Cinobufacini Each ml should be 12-18ug 15.6ug Cantharidin Each ml should be 14-18ug 16.70ug

[0113] The above heating experiments investigate the stability of each effective ingredient.

[0114] 2. Stability test after 2 years of natural storage

[0115] Batch number: 20150306

[0116]

[0117] Freeze-dried powder for postoperative treatment of tumors

[0118] With the improvement of people's living standards and tumor diagnosis technology, more and more patients can undergo surgery. Despite conventional chemotherapy and radiotherapy after surgery, most patients will eventually relapse. This is especially true for patients with highly malignant tumors and those with residual trace cancerous tissue that has not been completely removed. These patients will relapse soon after surgery and quickly enter the late stage of the tumor, spending the last days of their lives in pain. The effective control of tumor recurrence after surgery is a breakthrough in tumor treatment.

[0119] Tumors occur, proliferate, and grow to the point where they can be discovered by us. Most of them have already metastasized to distant sites under a microscope. Moreover, some tumors are multicentric in nature. The tumors that can be discovered have been removed, but the tiny lesions that cannot be discovered still exist. No matter how high the dose of chemotherapy and radiotherapy is after surgery, as long as life exists, there will always be 6 million tumor cells, tiny lesions, and residual cancer cells. However, most of these cancer cells have escaped immune surveillance and become escaped cancer cells. These escaped cancer cells may remain dormant for a long time due to the low malignancy of the cancer cells, the recovery of the body's immune function, and the improvement of the patient's internal environment. Therefore, they can remain recurrent for a long time, and even the antigens of the cancer cells are re-expressed and completely cleared by the immune system without recurrence for life. However, most escaped cancer cells, especially highly malignant cancer cells, interact with tumor-related inflammatory cells and tumor-related inflammatory factors, as well as tumor cells secreting tumor-related inflammatory factors to form a tumor microenvironment suitable for the survival, proliferation, and growth of tumor cells. Under the action of inflammatory cells and cytokines in the tumor microenvironment, cancer cells continue to proliferate and grow in the microenvironment, and can always remain in an escaped state, escaping the capture of the immune system, and eventually causing tumor recurrence.

[0120] The freeze-dried powder injection prescription is composed of: ginseng, astragalus, toad venom, and blister beetle. Pharmacological experiments have shown that the freeze-dried powder injection has a very significant anti-inflammatory effect, with an inhibition rate of up to 65% on mouse transplanted tumors, and can promote cancer cell differentiation and apoptosis.

[0121] After the tumor is removed, the myeloid-derived suppressor cells (MDSC) that have a strong inhibitory effect on the immune system gradually decrease in the body with the removal of the tumor. The freeze-dried powder injection can also effectively inhibit the effect of MDSC. When combined with a small dose of antimetabolites, it can effectively restore the body's immune function to normal. The toad venom base in the freeze-dried powder injection has a multi-target anti-cancer effect, which effectively inhibits the occurrence of tumors, the proliferation of tumor cells, the invasion and metastasis of tumors, tumor-related inflammatory cells of various targets, inflammatory factors and inflammatory factors secreted by tumor cells, [1-11] thereby blocking the survival and proliferation of tumor cells. The toad venom base is not easily absorbed by the gastrointestinal tract after oral administration. It has an effect on the growth of mouse transplanted tumors S 180 , the inhibition rates of solid liver cancer were 36.82% and 41.5% respectively, while oral administration was only 15.3% and 16.89%.

[0122] A concentration of 2.5nm of toad venom can reduce the expression level of cox2 in human lung cancer, inhibit the expression and phosphorylation of NF-kB, inhibit the activation and expression of matrix metalloproteinases (MMP) in various cancer cells, resulting in the inhibition of the number and expression of inflammatory factors, the inability to form a tumor microenvironment, and the residual cancer cells have no conditions for proliferation, growth, invasion and metastasis. Jiang reported that a very small amount of 100-200nm of toad venom can induce mitochondrial dysfunction in cancer cells, interfere with the cell cycle, and lead to apoptosis of cancer cells. Experiments have shown that when toad venom and mylabris are combined, they have a broad-spectrum inhibitory effect on various tumors. Mylabris can directly kill tumor cells and promote tumor cell apoptosis. Tumor-related inflammation is controlled, and a tumor microenvironment suitable for tumor cell survival, proliferation, invasion, metastasis, and recurrence cannot be formed. With the removal of the tumor, myeloid suppressor cells are reduced in the body, and freeze-dried powder injections can quickly restore immune function to normal. When tumor cells lose the inflammatory cells that they rely on for survival, proliferation, and development, the cytokine antigenicity re-appears and is completely eliminated by the normal immune cells in the body. Cancer cells have no environment to survive in, and postoperative recurrence is completely controlled.

[0123] The freeze-dried powder injection was used to treat 9 patients after tumor surgery, and no recurrence occurred in the 8-year follow-up. A new theory was proposed based on the latest research on tumors and inflammation, immunity and inflammation, and multi-target anticancer pharmacology of toxic anticancer Chinese medicine. The freeze-dried powder injection can control the theory of tumor recurrence after surgery, and the mechanism is consistent with the clinical results. The freeze-dried powder injection controls tumor recurrence after surgery, and the same results are expected to be obtained after clinical trials.

[0124] References

[0125] 1.Haus J, Dimitoxin is a potentinl ant carseraqent for several types of carer[J]med Hypotheses[I]199953[6]; 548-548

[0126] 2.TianD DimiieverRIetal protein kinasezeta regulation ofalkATPase,apersistentneuroproctiremechanismofischemicprecconditren in hippocrnpal slicecultre[j]Brain Res 2008,1213;127-139

[0127] 3.LeeD,Yasudam,YamotoT,et al life su 1997,60(2);127-134

[0128] 4.Hem CM Tsai Y,etalBufalim induce G2 / m phase arest andtirqqerautophgy Via the TNFJNK BFCNI and AT c8pathway in hman hepatomacells[J]oncol 201343[1]228-248

[0129] 5.Zhang c,yoshidaT,Kuroiwa T,et ofelife Sci 199251(1);17-24

[0130] 6.WatabeM,MasudaY,Nakajos,etal[J]Biolhem 1996,271(4)14067-14072

[0131] 7.Kiml,xuReedJC,celldeathd tndoplasmicreticulum iseaserelevance andtherapeuticopporunities[J]Nat rerDrugDiscov2008,7(12);1013-1030

[0132] 8.Chen YY,LU HF,Bufalin inhibitsigrtion and invasicnin humanhepatocelluarcarinomask-Hepl cells throuqtthe inhibitions of NF-kB and matrixmetalloprotianast-z-p-siqnalingthways[J]nvron joxical,2015,30(1)74;82.

[0133] 9.QinDz,zhangZJ,WUwZ et at Bufalin acomponent in chansu,inhibitsproliferation and invasion of hepatocelluluar carcinoma cell[J]BNCcomplementAITER med 2013,13;185

[0134] 10.HONG SH kim cy etalBufalin prevent the mlgratran and invasicn oft24 bladder carcinoma cells the inactivatioomatirc metalloproteinase andmodulativnoftiqhtjunetion(J)Onelo 2013,42(1);277-286

[0135] 11.XIE CM,clan wy,etal Bufalin induce autophuyy mediated cell deathin human colon cance cells throuqh reactive oxyqen species qenerafion JNKactivation[J]Free RalicBicl med 2011,51(7);1365-1357。

Claims

1. A Chinese medicine injection, characterized in that The active ingredients include 500-1000 parts by weight of ginseng, 1500-3000 parts by weight of astragalus, 10-20 parts by weight of mylabris and 2-4 parts by weight of toad venom.

2. The Chinese medicine combination according to claim 1, characterized in that: Its active ingredients include 250 parts of ginseng, 600 parts of astragalus, 4 parts of blister beetles, and 0.8 parts of toad venom.

3. A pharmaceutical preparation, made from the Chinese medicine composition according to any one of claims 1 to 2 alone, or composed of the Chinese medicine composition according to any one of claims 1 to 2 and a pharmaceutically acceptable carrier.

4. The pharmaceutical preparation according to claim 3, characterized in that The pharmaceutical preparation is a freeze-dried powder injection.

5. The pharmaceutical preparation according to claim 4, characterized in that The medicine is a freeze-dried powder injection, and the preparation method comprises ginseng ethanol extraction, astragalus water decoction extraction, cantharides acetone extraction, residue ethanol extraction, toad venom ethanol extraction, and residue water decoction extraction. The extracts of ginseng and astragalus are purified by the Ca(OH)-SO2 method, and then added with toad venom and mylabris extracts, sterilized, finely filtered, packaged, and freeze-dried to obtain the product.

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