CN1788723A - Liposome microsphere injection liquid containing demethylate disodium cantharidinate and its preparation method - Google Patents

Abstract

The invention relates to a lipid microsphere injection containing demethylcantharidinate sodium and a composition of an oil-in-water emulsion, and also relates to a preparation method thereof. The preparation applies the principle of interfacial membrane drug loading, increases the drug loading amount of sodium desmethylcantharidinate in the emulsion, reduces its toxicity and enables targeted drug release in vivo. It comprises sodium demethylcantharidate, fat-soluble medium, water, surfactant and isotonic regulator, wherein said fat-soluble medium is used in an amount of 5% to 30% in terms of preparation, and sodium demethylcantharidate is calculated in preparation The dosage is 0.01% to 5%, the dosage of the surfactant is 0.5% to 5%, the dosage of the isotonic regulator is 0.5% to 5%, and the balance is water. Wherein, sodium demethylcantharidate is distributed to a certain extent in the interfacial film of the oil-water two phases, and the remaining part of sodium demethylcantharidate is distributed in the water phase and the oil phase. This preparation is a preparation of antineoplastic drugs, and the main characteristics in the body after injection are low toxicity and liver targeting.

Description

Lipid microsphere injection containing sodium demethylcantharidinate and preparation method thereof

technical field

The invention relates to the technical field of medicine, in particular to a lipid microsphere injection (oil-in-water emulsion) containing sodium demethylcantharidinate and a preparation method thereof.

Background technique

In the past half a century, medical science has made great progress. Human beings have basically controlled the infectious diseases that mainly threatened human health in the past with the three major prevention and treatment methods of chemistry, immunity, and antibiotic therapy. Non-infectious diseases such as tumors, cardiovascular and cerebrovascular diseases, etc. Diseases have gradually become the main enemy of human health, especially the harm of tumors. Over the years, although people have made a lot of efforts and made great progress, the harm of tumors has not been effectively curbed so far. Statistics show that in 2002, the number of deaths from malignant tumors and cancers in China accounted for 25.37% of the total number of deaths in that year, and tumors have become the number one killer that endangers human health. In the past two years, the incidence of tumors has shown an upward trend, and the age of onset has also dropped significantly from the average age of 55. It can be predicted that in the next 20 to 30 years, the morbidity and mortality of cancer will continue to rise. Therefore, the development of antitumor drugs is the top priority of many pharmaceutical workers. In addition, due to the serious adverse reactions of most antineoplastic drugs, they cause great physical and mental harm to patients during the treatment process. Therefore, how to improve the efficacy of drugs while reducing the irritation and toxicity of drugs, thereby improving the quality of life of patients has gradually become the current anticancer drug. New direction and focus of tumor drug research.

Mylabris is an insect of the family Coleoptera, which has been used as medicine for more than 2,000 years. It has been clearly recorded in the Southern Song Dynasty that mylabris can treat cancer, and it was used to treat primary liver cancer in the 1970s. Mylabris is a highly poisonous drug. If it is abused, excessively used, used together with wine and garlic, used raw (or improperly brewed), used externally on a large area, accumulated, liver and kidney dysfunction, or mixed with water, it will cause poisoning. The poisoning dose is 0.6g , the lethal dose is 1.5g; cantharidin 0.14μg can induce skin blistering, 10mg can cause severe poisoning or death, which limits its clinical application. In recent years, through long-term research and experiments, people have synthesized derivatives or analogs of some cantharidin, such as demethylcantharidin, sodium cantharidinate and hydroxycantharidinine and other anticancer drugs to replace cantharidin. Experiments show that they The pharmacological effects of cantharidin are similar, but the toxicity is much smaller than that of cantharidin. In 1989, my country first synthesized a new anticancer drug, Norcantharidin, which was obtained by removing 1 and 2 methyl groups from cantharidin. Sodium demethylcantharidinate is the sodium salt obtained by direct hydrolysis of norcantharidin. It is only dissolved in a small number of organic solvents (such as acetone, ethyl acetate) and hot water, and its solubility in cold water and oil is very low.

Compared with cantharidin, sodium demethylcantharidinate has greatly reduced the stimulating effect on the urinary system, and at the same time has a more significant effect on increasing white blood cells than cantharidin. In vitro experiments have shown that sodium demethylcantharidinate can destroy or inhibit the morphology or proliferation of cell lines such as liver cancer, esophageal cancer, laryngeal cancer, and cervical cancer. For patients with liver cancer, the survival period can be prolonged, AFP can be decreased, the tumor body and enlarged liver can shrink, and the texture becomes soft, which can relieve pain and ascites in the liver area; for patients with esophageal cancer, gastric cancer, and cardia cancer, it can be seen that the tumor focus is stable or shrinks, and the pain And obstruction relieved, eating improved. In pathological specimens, different degeneration or necrosis of the above-mentioned cancers can be seen, and lymphocyte infiltration of cancer nests can be seen at the same time. Experiments in animals show that sodium demethylcantharidinate has a certain inhibitory effect on mouse Ehrlich ascites carcinoma and S180 liver cancer solid type, and can improve the respiratory control law and lysosomal enzyme activity of ascites liver cancer H22 and cancer cell mitochondria, and interfere with cancer cells. Cell division, inhibition of DNA synthesis, destruction of the cancer cell skeleton (microfilaments, microtubules), affecting its ultrastructure, resulting in damage to mitochondria, microvilli and plasma membranes. This product has little effect on normal cells, can antagonize bone marrow suppression, and increase white blood cells. This product is a cell cycle-specific drug that blocks the M cycle and affects its cycle speed. Clinically, it is mainly used for preoperative medication or combined chemotherapy for liver cancer, esophageal cancer, gastric cancer, cardia cancer, etc. It can be used for leukopenia and hepatitis B. The study of sodium demethylcantharidinate tablets by intragastric administration of mice showed that this product was quickly distributed in various tissues after absorption, and had a high concentration in the liver, kidney, stomach, intestine, heart, lung, salivary gland, thyroid gland and tumor of the animal. 15 minutes after administration, the concentration of the drug reaches the peak in the liver and cancer tissues, and the concentration drops significantly after 6 hours. After 24 hours, most of it is excreted by the kidneys, and there is less accumulation in the body. After intravenous injection of this product to animals, it can be quickly distributed in various tissues, and there are higher drug concentrations in liver, kidney, lung, stomach, intestine, heart, salivary gland, thyroid, and cancer tissues. The drug is mainly excreted through the urinary system. After intravenous injection, the drug concentration in the urine reaches a peak within 0.5 to 1 hour, and most of it is excreted in the urine within 24 hours.

At present, sodium desmethylcantharidate is mainly used in clinical practice with tablet and sodium desmethylcantharidate injection. Although sodium demethylcantharidinate significantly reduced the toxicity of cantharidin, it did not completely eliminate it. The results of animal experiments showed that when the dosage increased to a certain extent, pathological changes appeared in the kidney and liver tissues. The strict limitation of dosage also seriously hinders the exertion of drug curative effect. In clinical use, some patients have adverse reactions when intravenous administration exceeds 20 mg per day or oral administration exceeds 30 mg per day. Oral adverse reactions mainly manifest as nausea, vomiting, dizziness and other symptoms. Because the injection is an alkaline solution, its pH value is about 8, which is much higher than the blood pH value (7.35-7.45), so it is more irritating to the blood vessel wall and easily causes chemical phlebitis; and because the drug itself has a strong irritant , can also cause vasospasm, blood reduction, relative local concentration of sodium cantharidinate increases, aggravating phlebitis, and if the infusion rate is greater than the blood flow rate, the incidence of phlebitis is significantly increased. The main adverse reactions are easy to cause venous inflammation, red, swollen, hot, and painful veins, redness along the veins in a long strip, if there is exudation, it will be red and swollen into lumps, and the activity is limited. It will be cured after hot compress physical therapy. Therefore, when it is clinically used for peripheral superficial vein infusion, the ratio of superficial phlebitis is quite high, and some patients refuse to use the drug for treatment, which affects the normal practicality of the drug in clinical practice.

Therefore, in view of the above-mentioned characteristics, in order to make demethylcantharidinate sodium exert its clinical curative effect better, research and develop new dosage forms, give full play to demethylcantharidate sodium antitumor activity and reduce toxicity with the advantages of high efficiency and low toxicity of new dosage forms. Undoubtedly, this will further promote and popularize the clinical application of demethylcantharidin. At present, there are very limited reports on new formulations of demethylcantharidin, including microcapsules, albumin microspheres, and conjugated targeting materials.

It has been nearly half a century since fat emulsion has been used clinically as an important way of supplying parenteral energy. From 1920 to 1930, Japanese scholars used castor oil as raw material and lecithin as emulsifier, and first artificially synthesized fat emulsion for trial use on animals; in the 1950s, the United States launched Lipomul based on cottonseed oil and applied it to patients, but all due to severe toxicity and failed to promote. In 1964, the United States stopped the production and application of fat emulsion. However, in Europe, a group of scholars represented by Wretling have been tirelessly researching and applying Introlipid, a fat emulsion based on soybean oil, with egg yolk phospholipid as an emulsifier, and glycerol as an isotonic agent. Introlipid was officially approved for clinical use in Sweden in 1962. In 1967, the article "Fat Emulsion Applied to Complete Parenteral Nutrition" published by Hallberg et al. comprehensively summarized the experimental and clinical research of Introlipid, proved that its clinical application is safe and reliable, and proposed the guidelines for the clinical application of Introlipid. This article and the article "Growth, development and positive nitrogen balance under long-term complete parenteral nutrition" published by Dudrick et al. in 1968 are recognized as two classic works that have a great impact on the development of parenteral nutrition into routine clinical application. After more than 20 years, Introlipid has been widely used all over the world, and developed countries such as Germany, France, Japan, and the United States have successively developed their own fat emulsions. In 1976, the relevant authorities of the United States approved the routine application of fat emulsion in clinical practice. At present, there are at least 20 kinds of fat emulsions available in the world. my country also entered into a joint venture with Sweden's Kabi Company in the late 1980s to mass-produce Introlipid for clinical application. In 1973, Solassol et al. first introduced the concept of three-in-one (All-in-one, AIO) solution, which proved that fat emulsion and all other nutrient solutions could be mixed in a bottle or bag, and could be maintained under certain conditions and time. Stabilizing and nutritional support effects. Since then, AIO infusion plastic bags have been popularized. This simple but significant improvement greatly simplifies the preparation and infusion of parenteral nutrition solutions, and broadens the way for the widespread clinical application of fat emulsions. So far, common fat emulsion refers to the oil-in-water (oil in water, o/w) submicroemulsion with fatty acid glyceride as dispersed phase and egg yolk lecithin as emulsifier. Since the composition and particle size of fat emulsion droplets are very similar to those of chylomicrons in the blood after oral intake of fatty foods, it is generally believed that the behavior of fat emulsion droplets in the human body is also similar to that of chylomicrons. With the in-depth research on fat emulsion, it is also playing an increasingly important role in the pharmaceutical field.

The special physical and chemical properties and low toxicity of fat emulsion determine that it can be used as a good carrier of fat-soluble drugs, especially anticancer drugs, anesthetic drugs and anti-inflammatory drugs. The commonly used method is to wrap the drug in the lipid core part of the fat emulsion. Since this structure is also similar to microspheres, the name of lipid microspheres (Lipid Microsphere, LM) also came into being. It is generally believed that lipid microspheres are made by dissolving drugs in fatty oil and emulsifying and dispersing them in the water phase through phospholipids. The diameter is about 200nm.

LM has many physical chemical and biological advantages:

1. It is a good carrier for fat-soluble drugs. Many clinical drugs have poor water solubility and must rely on organic solvents to function. Organic solvents not only have certain toxicity themselves, but may also interfere with the efficacy of drugs.

2. It can effectively increase the stability of the drug. In the drug-containing lipid microspheres, a considerable part of the drug is distributed in the oil phase or the oil-water interface membrane, avoiding direct contact with water. For drugs that are prone to hydrolysis or are sensitive to pH changes, this "segregation" acts to increase stability.

3. The drug in LM is partly wrapped in the oil phase or interfacial membrane, which avoids direct contact with body fluids, thus reducing the local and vascular irritation that the drug itself may produce. In addition, the drug is slowly released from the oil phase in the body, which can avoid adverse reactions caused by the initial concentration of the drug when it is injected.

4. Small particles with a particle size of about 200nm can be swallowed by the phagocytes of the reticular tissue system of the body and stay in the reticular tissue system (such as liver, lung, etc.), which is targeted. This feature is useful for anti-tumor It is particularly important to improve drug efficacy and reduce toxic and side effects of drugs.

Takahashi et al's relevant literature reports: LM was prepared as a trial with bleomycin, and water was used as a control, and tumor-bearing rats were used as a test. Intravenous or intratumoral administration was carried out, and then the tumor size and drug concentration in the tumor were measured respectively. The results showed that: the tumor in the LM group shrank by more than 50%, and the drug could still be detected 10 hours after administration; while the tumor in the water group had no obvious Shrunken, and the drug was undetectable after 30 minutes to 1 hour. The above shows that different dosage forms of drugs have different effects, which shows that LM has the characteristics of selective distribution and slow release, and is a suitable carrier dosage form for diseases of the liver and lymphoid tissue system and cancer chemotherapy. Studies have proved that this feature is particularly important for anticancer drugs to improve efficacy and reduce side effects.

In recent years, the targeted transfer of LM as a drug carrier has gradually attracted people's attention. Once the drug lipid emulsion was developed, it was widely used clinically and received good benefits. The development and marketing of a series of drug-loaded fat emulsion products such as Lipo PGE1 and Lipo PGI2 developed by Yu Mizushima in Japan indicates the broad prospects of such preparations. In particular, the mature emulsion production process and the application of synthetic or natural surfactants and the improvement of emulsion production technology have promoted the development of emulsion products with commercial value and stable shape (stable period is about 2 years), for The development and production of medicated fat emulsion has created better conditions. Such as etomidate, propofol, dexamethasone palmitate lipid emulsion, flurbiprofen ethoxylated α-ethyl ester lipid emulsion, prostaglandin E1 lipid emulsion, etc.

Theoretically, to dissolve insoluble drugs, two conditions are necessary: 1. Effective shaking or stirring process; 2. Undissolved particles have a large specific surface area. In our experiment, these two principles are used. The basic theory of our experiment is as follows: First, adding the drug powder into the emulsion under high-speed stirring can obtain a kind of ultrafine oil droplets and fine particles of the drug dispersed in water at the same time coarse dispersion system. In the second step, a high-pressure homogenization process is carried out on the dispersion system, and the high-speed airflow impacts to form a supersonic stirring, so that the drug particles are rapidly dissolved and penetrate into the oil-water interface film in molecular form. The drug can be added in two forms, one is ultrafine powder, and the other is nanoscale dispersed suspension. There are also two methods for adding the drug, that is, adding the drug directly to the prepared emulsion for injection or adding the drug to the colostrum and then diluting. At present, this technology has been successfully applied to amphotericin B (Amphotericin B), and R.H.Müller et al. prepared a film-loaded drug nanoemulsion for amphotericin B injection in 2001.

The demethylcantharidin lipid microsphere injection (oil-in-water emulsion) prepared by the present invention reduces the vascular irritation of sodium demethylcantharidinate, improves the curative effect and reduces toxic and side effects, so it has certain innovation and comparative advantages. Strong practicality.

Contents of the invention

The object of the present invention is to provide a kind of lipid microsphere injection (oil-in-water emulsion) containing sodium demethylcantharidinate and preparation method.

The injection contains sodium demethylcantharidinate, a fat-soluble medium, water and a surfactant. Wherein, sodium demethylcantharidinate can be demethylcantharidin, or demethylcantharidin can be reacted to generate sodium demethylcantharidin during preparation preparation process.

Fat-soluble media indicate a broad class of physiologically acceptable substances, whether mineral, vegetable, animal, essential or synthetic, or mixtures thereof. Thus, fat-soluble media is used to refer to a wide range of substances with quite different chemical properties. When classifying oils by type or function, eg mineral oil is derived from petroleum and contains aliphatic or waxy hydrocarbons, aromatic hydrocarbons or mixed aliphatic and aromatic based hydrocarbons. Also included in the class of mineral oils are petroleum derived oils such as refined paraffinic oils and the like. In the vegetable oil category, oils are mainly derived from seeds or nuts and include drying oils such as linseed and tung oil; semi-drying oils such as safflower and soybean oil; non-drying oils such as castor oil, cottonseed oil and coconut oil and Can be used as soap stock such as palm oil and coconut oil. In the animal oil category, oils are usually derived from fats that are tallow, lard, and stearic acid. Liquid animal oils include fish oil, oleic acid, spermaceti, etc. They usually contain high amounts of fatty acids. Contains some vegetable oils, such as olive oil, cottonseed oil, corn oil and peanut oil, and also contains some special fish oils, which are widely used as medicine due to their rich vitamins, such as cod liver, shark liver oil, etc. Liquid fatty oils such as mono-, di-, tri-glycerides, or mixtures thereof are preferred oils. Medium chain length triglycerides are also useful oils according to the invention. Preferred are long-chain fatty acid glycerides, medium-chain fatty acid glycerides, and mixtures thereof.

The surfactant used may be any surfactant, typically phospholipids, Tween, poloxamer, sodium oleate, oleic acid, cholic acid, deoxycholic acid and mixtures thereof. The phospholipids are selected from lecithin, soy lecithin, and mixtures thereof. The Tween is selected from Tween 20, Tween 40, Tween 60, Tween 80, Tween 85, and mixtures thereof. Preferred are lecithin, soy lecithin, sodium oleate, oleic acid, Tween 80, Pluronic F68, and mixtures thereof.

Typically, the formula of the present invention consists of:

Oil phase 5%～30%

Sodium demethylcantharidinate 0.01%～0.5%

Surfactant 0.5%～5%

Pure glycerin 0.5%～5%

The rest is water for injection

Various additives can also be added to the composition, if desired. Contains metal chelating agents if possible. Common metal chelating agents are edetate disodium (edetate disodium salt), edetate sodium calcium (edetate calcium disodium salt), and mixtures thereof. Metal chelating agents are used in amounts of about 0% to about 1% of the formulation.

The invention also relates to the preparation method of lipid microsphere injection and oil-in-water emulsion containing sodium demethylcantharidinate. Contains one or more of the following steps:

1. Add sodium demethylcantharidate ultrafine particle suspension or ultrafine powder to a blank emulsion that does not contain sodium demethylcantharidate and mix to prepare sodium demethylcantharidate lipid microsphere injection.

2. Add sodium demethylcantharidate ultrafine particle suspension or ultrafine powder to the emulsion containing part of sodium demethylcantharidate and mix to obtain sodium demethylcantharidate lipid microsphere injection .

3. adding sodium demethylcantharidinate into a water-soluble medium containing a surfactant, then mixing with the oil phase, and stirring at a high speed to prepare colostrum.

4. The preparation method of the preparation mainly includes a high-speed stirring process and a high-pressure homogenization process.

5. The preparation is sterilized by high-pressure rotary sterilization.

The present invention relates to a method of mixing sodium demethylcantharidate into oil, comprising the steps of: dissolving sodium demethylcantharidate in a solution of said oil and said sodium demethylcantharidate co-solvent; and Removal of the co-solvent has constituted the oil solution of the sodium demethylcantharidate. Typically the co-solvent is a short chain alcohol. Especially methanol, ethanol and isopropanol. These co-solvents are finally removed by evaporation.

The preparation of the invention uses the principle of drug loading on the interface film, which improves the drug loading capacity of sodium demethylcantharidinate, which is insoluble in both oil and water, in the emulsion, reduces its toxicity and enables targeted drug release in vivo. Wherein, sodium demethylcantharidate has a certain distribution (about 20-80%) in the interfacial film of the oil-water two phases, and the remainder of sodium demethylcantharidate is distributed in the water phase and the oil phase. This preparation is a preparation of antineoplastic drugs, and the main characteristics in the body after injection are low toxicity and liver targeting.

Description of drawings:

Figure 1 is a schematic diagram of the structure of lipid microspheres.

A-2表示制剂以氯化钠注射液(0.9％)稀释20-100倍后平均粒径，

B-1表示制剂以葡萄糖注射液(5.4％)稀释1-10倍后平均粒径，B-1表示制剂以葡萄糖注射液(5.4％)稀释20-100倍后平均粒径，SD-A-1表示以氯化钠注射液(0.9％)稀释1-10倍后粒度分布值，

SD-A-2表示以氯化钠注射液(0.9％)稀释20-100倍后粒度分布值，SD-B-1表示以葡萄糖注射液(5.4％)稀释1-10倍后粒度分布值，SD-B-2表示以葡萄糖注射液(5.4％)稀释20-100倍后粒度分布值。)Fig. 2 is the result figure of particle size determination after dilution of sodium demethylcantharidinate lipid microspheres. (in, A-1 represents the average particle diameter after the preparation is diluted 1-10 times with sodium chloride injection (0.9%),

A-2 represents the average particle diameter after the preparation is diluted 20-100 times with sodium chloride injection (0.9%),

B-1 represents the average particle diameter after the preparation is diluted 1-10 times with glucose injection (5.4%), B-1 represents the average particle diameter after the preparation is diluted 20-100 times with glucose injection (5.4%), SD-A-1 represents the particle size distribution value after diluting 1-10 times with sodium chloride injection (0.9%),

SD-A-2 represents the particle size distribution value after diluting 20-100 times with sodium chloride injection (0.9%), SD-B-1 represents the particle size distribution value after diluting 1-10 times with glucose injection (5.4%), SD-B-2 represents the particle size distribution value after diluting 20-100 times with glucose injection (5.4%). )

Fig. 3 is a graph showing the measurement results of the ζ-potential of the diluted sample.

Figure 4 is a distribution diagram of the diluted sample in the aqueous phase.

Figure 5 is the NCTD plasma concentration-time curve.

(in, Demethylcantharidinate sodium lipid microsphere injection, Demethylcantharidinate sodium ordinary injection)

Fig. 6 is the drug concentration-time curve in the heart of NCTD. (in, Demethylcantharidinate sodium lipid microsphere injection, Demethylcantharidinate sodium ordinary injection)

Fig. 7 is the drug concentration-time curve in liver of NCTD. (in, Demethylcantharidinate sodium lipid microsphere injection, Demethylcantharidinate sodium ordinary injection)

表示去甲基斑蝥酸钠脂质微球注射液，

表示去甲基斑蝥酸钠普通注射液)Fig. 8 is the drug concentration-time curve in NCTD kidney. (in,

Demethylcantharidinate sodium lipid microsphere injection,

Demethylcantharidinate sodium ordinary injection)

Fig. 9 is a graph showing drug distribution in various tissues in rats after administration for 0.5 hours. (in, Indicates Sodium Demethylcantharidate Lipid Microsphere Injection, ■Indicates Sodium Demethylcantharidate Ordinary Injection)

表示去甲基斑蝥酸钠脂质微球注射液，■表示去甲基斑蝥酸钠普通注射液)Fig. 10 is a graph showing drug distribution in various tissues in rats after administration for 6 hours. (in,

Indicates Sodium Demethylcantharidate Lipid Microsphere Injection, ■Indicates Sodium Demethylcantharidate Ordinary Injection)

Fig. 11 is a graph showing drug distribution in various tissues of rats administered for 12 hours. (in, Indicates Sodium Demethylcantharidate Lipid Microsphere Injection, ■Indicates Sodium Demethylcantharidate Ordinary Injection)

Figure 12 is a graph showing the distribution of AUC values in various tissues in rats after 24 hours of administration. (in, Indicates Sodium Demethylcantharidate Lipid Microsphere Injection, ■Indicates Sodium Demethylcantharidate Ordinary Injection)

Detailed ways

Example 1

Preparation of Sodium Demethylcantharidinate Raw Material

Although sodium demethylcantharidinate has an injection, it is all obtained from the direct hydrolysis of norcantharidin to obtain a sodium saline solution. Its raw materials are not produced and sold. And preparing norcantharidin sodium from norcantharidin is the most basic organic reaction, therefore, we refer to bibliographical reports, take norcantharidin as raw material, through hydrolysis into salt, dehydration, washing, drying, make norcantharidin Sodium Cantharidinate. The reaction formula is as follows:

The general synthesis method is as follows:

Sodium hydroxide 10-30g, add water 100mL, let it cool after dissolving, add 10-30g of norcantharidin while stirring, after almost all dissolved, filter out a small amount of insoluble matter. Concentrate the solution under reduced pressure to nearly dryness to obtain a large amount of white powdery solid, add 5-50mL of ethanol, filter with suction to obtain white crystals, wash with ethanol several times, check the pH value of the eluate after each washing, and wait for the eluate to When the pH value is 4-9, filter with suction and dry in vacuum to obtain a white crystalline powder.

Refining: Dissolve 5-50g of the above white powder in 5-50mL of water, filter with a G3 vertical melting funnel, add 100-1000mL of ethanol to the filtrate, leave it for 2-20 hours, suction filter, and vacuum dry to obtain a white powdery solid.

Example 2

Prescription preparation process of sodium demethylcantharidinate preparation

Prescription 1:

Coix seed oil for injection 5-30g

Sodium demethylcantharidate 0.01g-5g

Lecithin 0.5-5g

Glycerin 0-5g

Add water for injection to 100ml

Preparation method 1:

(1) Mix the prescribed amount of lecithin, glycerin and sodium demethylcantharidinate with an appropriate amount of water for injection preheated to 40-100°C, transfer to a tissue masher, stir for several minutes, 1-5 times, Until the ingredients are evenly dispersed; (2) Add the prescribed amount of oil phase preheated to 40-100°C to the above dispersion liquid, transfer it to a tissue grinder, stir for several minutes, 1-5 times, until the oil phase is uniform Disperse; (3) Take the above-mentioned colostrum and add water for injection preheated to 40-100°C to the full amount, transfer it to a high-pressure homogenizer, homogenize it for 1-10 times, and take a sample for microscopic inspection until the oil droplet is below 0.5 microns; (4) The submicron emulsion of demethylcantharidinate sodium was taken and sealed in an infusion bottle, filled with nitrogen, and sterilized in a high-pressure rotary sterilizer.

Prescription 2:

Soybean oil for injection 5-30g

Sodium demethylcantharidate 0.01g-5g

Soy lecithin 0.5-5g

Glycerin 0-5g

Tween-80 0-1g

Add water for injection to 100ml

Preparation method 2:

(1) Disperse the prescribed amount of sodium demethylcantharidinate in 0%-5% Tween-80 solution, stir with a high-speed mixer for several minutes, 1-5 times, and then homogenize 1-10 times with a high-pressure homogenizer (50-100MP, 20-100 DEG C) to get final product, the demethylcantharidate sodium content of the prepared suspension is 10 times of nanoemulsion; (2) lecithin, glycerin and preheating to 40- Mix with an appropriate amount of water for injection at 100°C, transfer to a tissue masher, stir for a few minutes, 1-5 times, until the ingredients are evenly dispersed; (3) Add the prescribed amount of preheated to 40-100 Transfer the soybean oil at ℃ into the tissue masher, stir for several minutes, 1-5 times, until the oil phase is evenly dispersed, add water for injection at 20-100℃ to the full amount; (4) supercharge sodium demethylcantharidinate Add the microparticle suspension into the blank colostrum, homogenize under high pressure for 1-10 times, take a sample for microscopic inspection, until the oil droplet is below 0.5 micron; (5) Take demethylcantharidinate sodium submicroemulsion and pot it into an infusion bottle , filled with nitrogen, and sterilized in a high-pressure rotary sterilizer.

Prescription 3:

Brucea javanica oil for injection 5-30g

Sodium demethylcantharidate 0.01g-5g

Lecithin 0.5-5g

Glycerin 0-5g

Tween-80 0-1g

Add water for injection to 100ml

Preparation method 3:

(1)～(3) The steps are the same as the preparation method 2; (4) add the blank colostrum into the high-pressure homogenizer and homogenize it for 1-10 times, take a sample for microscopic examination, until the oil droplet is below 0.5 microns; (5) remove the Add the sodium methylcantharidinate ultrafine particle suspension into the blank emulsion and stir at a high speed; (6) get the submicron emulsion of demethylcantharidinate and fill it in an infusion bottle, fill it with nitrogen, place it in a high-pressure rotary sterilizer, Sterilize.

Prescription 4:

Oil phase (soybean oil/MCT) 5-30g

Sodium demethylcantharidate 0.01-5g

Lecithin 0.5-5g

Glycerin 0-5g

Sodium oleate 0-1g

EDTA 0-1g

Add water for injection to 100mL

Preparation method 4:

(1) Disperse glycerin for injection, sodium oleate, EDTA, F68 in an appropriate amount of water for injection, heat to 40-100°C in a magnetic stirrer and stir until completely dissolved; (2) Lecithin for injection, Tween-80, Add it to the mixed oil phase composed of MCT for injection and soybean oil for injection, heat to 40-100°C and stir until the lecithin is completely dissolved, add sodium demethylcantharidinate, and perform high-pressure homogenization. (3) Add the oil phase to the water phase, place it in a high-speed tissue grinder and stir for several minutes, 1-5 times. (4) Adjust the pH value to 4-9, dilute with water for injection to the prescribed volume, transfer to a high-pressure homogenizer for homogenization 3-10 times. (5) Bottling, sealing with nitrogen, placing in a high-pressure rotary sterilizer, and sterilizing.

In addition to the composition in the prescription listed above, prepare 5 preparations in addition by the above method, and their composition is as follows: Sodium desmethylcantharidinate, desmethylcantharidin, and mixtures thereof 0.2g Soybean oil, safflower oil, coix seed oil, javanica oil, medium chain fatty acid glycerides 5-30g lecithin, soybean lecithin 0-5g Oleic Acid, Sodium Oleate 0-1g Cholic Acid, Sodium Deoxycholate 0-1g Glycerin, Sorbitol, Mannitol, Glucose 0-5g EDTA 0-1g Poloxamer F68 0-5g Tween 80 0-1g

Example 3

Screening of Dilution Medium of Sodium Demethylcantharidinate Lipid Microspheres

The present invention studies the effects of different dilution media and different time after dilution on the properties of sodium demethylcantharidinate lipid microspheres to screen out more suitable clinical dilution media and determine the safe use time after dilution.

Experimental method: Dilute sodium demethylcantharidinate lipid microsphere sample with sodium chloride injection (0.9%) respectively 1-10 times (code name A-1) and 20-100 times (code name A-2), with Glucose injection (5.4%) was diluted 1-10 times (code B-1) and 20-100 times (code B-2), and the particle size, ζ-potential and Drug package rate. See attached drawing 2 for the particle size measurement results of the diluted sample, see attached drawing 3 for the ζ-potential change of the diluted sample, see attached drawing 4 for the drug distribution over time after dilution of the sample, and see attached drawing for the distribution diagram in the water phase of the diluted sample 5,

Based on the above experimental results, it is safe and reasonable to use sodium demethylcantharidinate lipid microspheres within 2 hours when diluted 1-10 times with grape injection in clinical use.

Example 4

Irritation Test of Sodium Demethylcantharidinate Lipid Microspheres Injection

(1) Vascular stimulation test

6 New Zealand white rabbits were selected, 3 white rabbits were injected with demethylcantharidate sodium injection Qs in the ear vein of the right ear, and the left ear was injected with the same dose of sterile 5% glucose injection as a control; the other 3 white rabbits were injected with Demethylcantharidinate sodium lipid microsphere injection Qz was injected into the auricular vein of the right ear, and the same dose of sterile 5% glucose injection was injected into the left ear as a control. Once a day, for three consecutive days, 24 hours after the last administration, the white rabbits were killed, and the reaction at the injection site was observed with the naked eye, and the blood vessels and surrounding tissues of the rabbit ears were dissected for paraffin sections, stained, and examined with a light microscope.

The results showed that the vascular irritation test of the two formulations of sodium demethylcantharidinate injection was visually observed: the vascular irritation of Qz was weaker than that of Qs; the microscopic examination report showed that: the Qs group had a certain degree of vascular irritation to the ear blood vessels of New Zealand white rabbits. Irritation, the Qz group had no obvious irritation to the ear blood vessels of New Zealand white rabbits.

(2) Muscle stimulation test

Select 4 New Zealand white rabbits, 2 of each dosage form, inject 1ml of Qs and Qz injections into the right quadriceps muscle respectively, and inject the same amount of sterile 5% glucose injection into the left quadriceps muscle as a control, inject After 48 hours, the white rabbit was executed, and the quadriceps muscle was dissected and taken out, cut longitudinally, and the reaction of the muscle tissue at the injection site was observed to determine the degree of reaction.

Level 0: No change.

Grade 1: Mild hyperemia, the scope of which is below 0.5cm×1.0cm.

Grade 2: Moderate hyperemia, the scope of which is above 0.5cm×1.0cm.

Grade 3: Severe congestion with muscle degeneration.

Grade 4: necrosis with brown degeneration.

Grade 5: Extensive necrosis occurs.

Then calculate the sum of the response series of the four quadriceps femoris muscles. If the difference between the highest value and the lowest value of the quadriceps muscle response series is greater than 2, another 2 healthy white rabbits should be taken for a new experiment. After the result is obtained, if the sum of the response series of the four quadriceps muscles is less than 10, it is considered that the local irritation test of the test product is in compliance with the regulations.

The results showed that the muscle stimulation of Qz was weaker than that of Qs.

Example 5

Hemolytic test

Take 20ml of blood from the common carotid artery of New Zealand white rabbits, put it in a flask, and gently stir it with a glass rod. After a few minutes, remove the fibrin, take out the blood, add an equal amount of 5% glucose injection, centrifuge, and remove the supernatant ; The precipitated red blood cells were washed with 5% glucose injection and centrifuged. Repeat this until the supernatant is transparent, and make a 2% suspension with 5% glucose injection according to the volume of red blood cells.

Take 7 clean test tubes, number them respectively, add each solution in the table below in turn, the 6th tube does not add the test solution as a blank control tube, and the 7th tube replaces 5% glucose injection with distilled water, shake well, and place in a 37°C water bath , observe whether there is hemolysis at 0.5 hour, 1 hour, 2 hours, and 3 hours.

The results showed that no hemolysis occurred in both Qs and Qz, and the hemolysis tests of the two dosage forms were all qualified.

Example 6

Allergy test

Take 8 healthy guinea pigs, 4 Qs and 4 Qz dosage forms respectively, and inject 0.5ml of the corresponding test solution intraperitoneally into each guinea pig, once every other day, a total of three times. On the 14th and 21st days after the first injection, 1 ml of the corresponding test drug solution was injected into the lateral vein of the hind paw of each guinea pig (dose conversion is the same as before), and the challenge was carried out. Observe 2 hours for each intravenous administration, if there are two or more of nose scratching, piloerection, coughing, and dyspnea, it will be judged as positive; if there is one of convulsions, incontinence, collapse, shock, and death judged as positive. Everything normal is negative.

The results showed that both formulations of Qs and Qz met the standard of allergic experiment.

Example 7

Rat plasma pharmacokinetic study dosing regimen

Twelve rats were randomly divided into two groups, 6 rats in each group, and fasted overnight before the experiment. Group A was the control group, injected demethylcantharidate sodium chloride injection (2mg·mL ^-1 ) into the right posterior femoral vein, and group B was the test group, injected demethylcantharidate sodium into the right posterior femoral vein For lipid microspheres, take 0.5mL of blood from the orbit at 10min, 20min, 30min, 45min, 1h, 2h, 4h, 6h, 8h, 12h, and 24h, put them in pre-heparinized conical centrifuge tubes, centrifuge, and accurately draw the upper layer Add 20 μL of water for injection to 200 μL of plasma, and after treatment according to a certain method, take 20 μL of the sample for injection, and calculate the concentration of sodium demethylcantharidate in the sample at each time point using the standard curve of the day.

The experimental results are shown in Figure 6

Compartment model pharmacokinetic parameters

The mean blood concentration data of sodium desmethylcantharidinate lipid microspheres and injection were processed with the 3P97 pharmacokinetic program. According to the AIC and the degree of fitting, the model attributes of the two were judged. The results showed that the blood concentration data of sodium demethylcantharidinate lipid microspheres and injection were in line with the two-compartment model.

Non-compartmental Model Pharmacokinetic Parameters

The in vivo process is described by the blood drug concentration-time measured data, and the pharmacokinetic parameters are calculated. The results showed that the main pharmacokinetic parameters AUC _0-∞ , AUMC _0-∞ , AUC _0-t , AUMC _0-t , S ₂ , MRT, VRT had no significant difference.

Example 8

Kinetic study of tissue distribution in rats

Dosing regimen

60 rats were randomly divided into two groups, 30 rats in each group. Group A was the control group, injected NCTD sodium chloride injection into the right posterior femoral vein, and group B was the test group, injected NCTD lipid microspheres into the right posterior femoral vein at 0.5h, 2h, 6h, 12h, and 24h respectively (6 rats per point, 3 each in Group A and Group B) the animals were executed by decapitation, the heart, liver, spleen, lung, kidney, and brain were taken out, rinsed with normal saline, blotted dry with filter paper, weighed 0.5g, Add 1mL normal saline to homogenate, directly homogenate the tissue less than 0.5g, centrifuge, take the supernatant, after processing according to the item "Tissue Sample Processing and Measurement", take 20μL sample, calculate each time point according to the standard curve of the day Concentration of NCTD in the samples.

Experimental results

See Tables 10-12 for the drug concentration measurement results at each time point after the intravenous injection of NCTD lipid microspheres and NCTD injection in experimental animals, and the relationship between the average value and time of each tissue drug concentration at each time point in experimental animals is shown in Figures 6-12 .

Under the administration conditions of this test, the blood drug concentrations in rat spleen, lung, and brain could not be detected within 24 hours after administration of the test preparation and the reference preparation, so the results were not listed.

In addition, two animals died after 12h-24h in the rat group given the test preparation, and they still died after the supplementary experiment, so only one group of rat tissue distribution data was obtained in the test preparation group at 24h.

The plasma pharmacokinetic test results in rats showed that sodium demethylcantharidinate lipid microsphere injection had no obvious sustained-release effect in rats. The experimental results of tissue distribution kinetics showed that sodium demethylcantharidinate was more distributed in kidney, liver, heart and plasma, but hardly distributed in lung, spleen and brain. After changing the dosage form, except for a slight change in the overall distribution in the liver, the distribution characteristics of other tissues in the body basically did not change.

Taking demethylcantharidinate sodium solution as a reference preparation, the tissue distribution dynamics characteristics in rats were measured, and the results showed that after intravenous administration of demethylcantharidinate sodium into lipid microspheres, the drug did not change. The residence time and drug distribution in spleen, lung, kidney, brain, and plasma of rats were slightly increased in liver tissue.

Claims (10)

1, a kind of lipide microsphere injection that contains demethylate disodium cantharidinate, it is characterized in that: it comprises demethylate disodium cantharidinate, fat-soluble medium, water, surfactant and isoosmotic adjusting agent, wherein said fat-soluble medium is 5% to about 30% in the preparation consumption, demethylate disodium cantharidinate is 0.01% to 5% in the preparation consumption, surfactant is 0.5% to 5% in the preparation consumption, isoosmotic adjusting agent is 0.5% to 5% in the preparation consumption, and surplus is a water.

2, a kind of lipide microsphere injection that contains demethylate disodium cantharidinate according to claim 1 is characterized in that: also contain metal-chelator in the preparation; Isoosmotic adjusting agent glycerol, sorbitol, mannitol, glucose, and composition thereof; Also contain sodium hydroxide, hydrochloric acid, and composition thereof, wherein chelating agen is 0% to 1% in the preparation consumption, isoosmotic adjusting agent is 0.5% to 5% in the preparation consumption.

3, a kind of lipide microsphere injection that contains demethylate disodium cantharidinate according to claim 1, it is characterized in that: described injection comprises the oil in water emulsion that contains demethylate disodium cantharidinate that intravenous transfusion is used, and the compositions that constitutes lipide microsphere injection is identical with the compositions of oil in water emulsion.

4, according to claim 1 or 3 described a kind of lipide microsphere injections that contain demethylate disodium cantharidinate, it is characterized in that: the principle of described injection application interface film medicine carrying, demethylate disodium cantharidinate has distribution to account for 20～80% in the biphase interfacial film of profit in the injection, and the remainder demethylate disodium cantharidinate is distributed in water and the oil phase.

5, according to claim 1 or 3 described a kind of lipide microsphere injections that contain demethylate disodium cantharidinate, it is characterized in that: demethylate disodium cantharidinate can be a cantharidin in the described injection, also can be to generate demethylate disodium cantharidinate through reaction by cantharidin in the formulation preparation process.

6, a kind of lipide microsphere injection that contains demethylate disodium cantharidinate according to claim 1, it is characterized in that: described fat-soluble medium is selected from mineral oil, vegetable oil, animal oil, quintessence oil and artificial oil, or its mixture, described surfactant is selected from phospholipid, tween, pluronic, enuatrol, oleic acid, cholic acid, deoxycholic acid and composition thereof.

7, a kind of lipide microsphere injection that contains demethylate disodium cantharidinate according to claim 1 is characterized in that: described grease separation is from safflower oil, soybean oil, and Semen Maydis oil, MCT Oil, Semen Coicis oil, Oleum Fructus Bruceae, and composition thereof; Wherein said phospholipid is selected from lecithin, fabaceous lecithin, or the two mixture; Described tween is selected from polysorbas20, polysorbate40, polysorbate60, Tween 80, polysorbate85, or the arbitrary proportion mixture of above different size.

8, a kind of preparation method that contains the lipide microsphere injection of demethylate disodium cantharidinate comprises following steps: the powder of demethylate disodium cantharidinate ultramicro powder suspension or ultra micro is added to the blank Emulsion that does not contain demethylate disodium cantharidinate or contains in the Emulsion of part demethylate disodium cantharidinate through mixed demethylate disodium cantharidinate lipide microsphere injection.

9, a kind of lipide microsphere injection that contains demethylate disodium cantharidinate, it is characterized in that: comprise following processing step: demethylate disodium cantharidinate is added in the water-soluble medium that contains surfactant, mix with oil phase then, after the process high-speed stirred prepares colostrum, carry out the high pressure homogenize process, the high-pressure rotary sterilization is adopted in the sterilization of preparation.

10, a kind of lipide microsphere injection that contains demethylate disodium cantharidinate, it is characterized in that: comprise following processing step: with demethylate disodium cantharidinate and fat-soluble medium mixed after, redispersion is at aqueous phase, after the process high-speed stirred prepares colostrum, carry out the high pressure homogenize process, the high-pressure rotary sterilization is adopted in the sterilization of preparation.

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