CN101810577B - Gossypol intravenous injection fatty emulsion for curing tumors - Google Patents

Abstract

The invention discloses a fat emulsion for intravenous injection of gossypol for treating tumors, which consists of the following components per 1000ml: 40-80g of medicinal salt of gossypol, 200-300g of ethyl oleate or medium-chain fatty acid triglyceride ( As an oil phase), 30-50 g of soybean lecithin (as a surfactant), 10-40 g of absolute ethanol or PEG400 (as a co-surfactant), and the balance is water. Studies have shown that the gossypol fat emulsion of the present invention, when the drug carrier is used according to the stated dosage, the drug-loading amount of gossypol is significantly higher than that of oral preparations and common injection preparations, and is stable and uniform, and drug crystallization or layering is not easy to occur. Compared with oral preparations, the bioavailability is significantly improved. When the average particle size of the milk droplets is 70-150nm, the stability is the best, and it has a sustained release effect, and has a significant targeting effect on tumor tissues, which can avoid the drug’s effect on the gastrointestinal tract. Stimulate.

Description

Gossypol Intravenous Injection of Fat Emulsion for Tumor Treatment

technical field

The invention relates to an antitumor pharmaceutical composition, in particular to an intravenous injection preparation containing gossypol.

Background technique

Gossypol, also known as gossypol, is a yellow phenolic compound that naturally exists in the seeds, leaves, stems and roots of cotton, a plant of the Malvaceae family. At room temperature, gossypol is yellow crystal, soluble in ethanol, ether, acetone, chloroform and other organic solvents, also soluble in oil, but insoluble in water, hexane and petroleum ether with low boiling point, most of the organic solvents are It is highly toxic to the human body and is not suitable as a drug carrier.

Gossypol is well known as a male birth control drug. In recent years, its anti-tumor effect has been paid more and more attention. It has a wide range of killing effects on tumor cells, such as uterine fibroids, prostate cancer, colon cancer, lung cancer, liver cancer, laryngeal cancer, head and neck tumors, glioma, etc.

As a broad-spectrum antitumor drug, gossypol has the advantages of abundant sources and low cost, and can play a synergistic therapeutic effect with chemotherapy and radiotherapy. my country is a large cotton-producing country with rich sources of gossypol. Strengthening the research and development of gossypol tumor preparations will have good economic and social benefits.

The gossypol preparations currently on the market are only gossypol acetate oral preparations, which are used as oral male contraceptives or for the treatment of gynecological diseases, including menorrhagia or disorders, endometriosis and the like. However, because gossypol and gossypol acetate are insoluble in water, their oral preparations have low bioavailability, and have gastrointestinal reactions such as gastrointestinal irritation, loss of appetite, nausea, and vomiting, and "peak-valley" phenomenon.

Chinese patent 200510022777.9 (applicant: Shenyang Pharmaceutical University, publication number CN1827117, publication date: September 06, 2006, date of authorization announcement: December 16, 2009) discloses a gossypol acetate slow-release tablet that combines cotton acetate Phenol is added to slow-release materials to form sustained-release preparations to avoid the phenomenon of "peaks and valleys", and is used to solve the phenomenon of "peaks and valleys". However, the drug loading of sustained-release preparations is limited after adding many sustained-release excipients, and the irritating effect on the gastrointestinal tract cannot be avoided. And gossypol acetate is used for the relatively high dose of treating tumor, and the disclosed gossypol acetate of U.S. Patent No. 5,385,936 (date of application: July 12, 1990, publication date: January 31, 1995) shows that gossypol acetate is used for the treatment of tumors. , the human dose of gossypol acetate for the treatment of tumors is 40-100mg/day. In its Phase I clinical trial, it still has no effect on some tumors when the dosage reaches 70mg/day. It can be seen that the unit dose of gossypol acetate should be at least 40-60mg, in order to reduce the number of administrations to patients and obtain a better curative effect. And above-mentioned preparation can't realize targeted delivery.

Fat emulsion is a pharmaceutical dosage form that can significantly improve the solubility of drugs. It is of great help to improve the drug loading and pharmacological characteristics of poorly water-soluble drugs. At present, fat emulsions of several drugs have been studied in China. However, in order to obtain a stable fat emulsion for different drugs, there are large differences in the requirements for the type and dosage of excipients, that is, drug carriers, and a large number of screening experiments are required to obtain the best drug carrier and ratio. At present, there is no report about the fat emulsion of gossypol in the prior art.

Contents of the invention

Aiming at the above prior art, the present invention provides a gossypol intravenous injection preparation for treating tumors, specifically a gossypol intravenous fat emulsion.

The present invention is achieved through the following technical solutions:

A fat emulsion for intravenous injection of gossypol for treating tumors, which consists of the following components per 1000ml: 40-80g of medicinal salt of gossypol, 200-300g of ethyl oleate or medium-chain fatty acid triglyceride (as the oil phase) , 30-50g of soybean lecithin (as a surfactant), 10-40g of absolute ethanol or PEG400 (as a co-surfactant), and the balance is water.

The average droplet diameter of the fat emulsion for intravenous injection of gossypol is 70-150 nm.

The medicinal salt of gossypol is gossypol acetate or gossypol formate.

The medium-chain fatty acid refers to C8-C10 fatty acid.

Gossypol intravenous fat emulsion of the present invention can be prepared in the following manner:

(1) After the medicinal salt of gossypol is dissolved in ether, it is stirred and mixed with the oil phase, surfactant and co-surfactant under heating, and evaporated under reduced pressure to remove the ether as the oil phase medicinal liquid, and heated at a constant temperature for subsequent use;

(2) Heat an appropriate amount of sterilized water for injection to 60-70°C as the aqueous phase liquid;

(3) Slowly drop the oil phase medicinal solution into the aqueous phase liquid at a constant temperature, and stir while adding until a transparent and uniform solution is formed, then use sterilized water for injection to make up the volume, and then repeatedly homogenize with a homogenizer to obtain the product.

In addition, during preparation, part of the co-surfactant can also be mixed with sterilized water for injection as an aqueous phase liquid; the medicinal salt of gossypol can also be directly mixed with the oil phase, surfactant and co-surfactant as an aqueous phase liquid. oily phase without ether.

The gossypol fat emulsion of the present invention has been studied and shown that when the drug carrier is used according to the dosage, the drug loading of gossypol is significantly higher than that of oral preparations and common injection preparations. The drug loading amount of the salt can reach up to 90 mg, and it is stable and uniform, and drug crystallization or layering is not easy to occur. Compared with the oral preparation, the gossypol intravenous injection preparation of the present invention has significantly improved bioavailability, and the stability is the best when the average particle size of emulsion droplets is 70-150nm, has a slow-release effect, and has significant targeting to tumor tissue, It can avoid the irritation of the drug to the gastrointestinal tract. The preparation process of the preparation is simple and beneficial to industrial production. In summary, the present invention has outstanding substantive features and remarkable progress.

Description of drawings

Fig. 1 is the medicine-time curve after the single tail vein injection of gossypol acetate intravenous injection fat emulsion rat;

Fig. 2 is the tissue content after a single tail vein injection of gossypol acetate intravenously injected fat emulsion tumor-bearing mice.

Detailed ways

The present invention will be further described below in conjunction with embodiment:

The preparation of embodiment 1 gossypol acetate intravenous injection fat emulsion

Party:

Gossypol Acetate 40g

Ethyl oleate 207g

Soy Lecithin 35g

Absolute ethanol 18g

Sterile water for injection to 1000ml

preparation:

Dissolve the prescribed amount of gossypol acetate in ether, then add ethyl oleate, soybean lecithin, and absolute ethanol in a constant temperature water bath at 60°C, and stir while adding. The stirring speed is 1600rpm, stir for 2 hours, and evaporate under reduced pressure to remove the ether , as an oil phase medicinal solution, stored in a constant temperature water bath at 60°C for later use;

Preserve an appropriate amount of sterilized water for injection in a constant temperature water bath at 60°C as the aqueous phase liquid;

Slowly drop the oil phase liquid into the water phase liquid at a constant temperature of 60°C, and stir while adding. The stirring speed is 2200rpm. Stir for 1 hour until a transparent and uniform solution is formed. Dilute to 1000ml with sterilized water for injection, and then use homogeneous The homogenizer (NS10012k high-pressure homogenizer, Niro Soavi S.p.A., Italy) was repeatedly homogenized 5 times at 60Mpa, and filtered with a 0.45 μm microporous membrane. Packed into 1000 parts, sterilized and stored.

In the obtained sample, the average diameter of emulsion droplets is 104nm, and the encapsulation efficiency is 87.2%.

The preparation of embodiment 2 gossypol acetate intravenous injection fat emulsion

Party:

Gossypol Acetate 60g

Ethyl oleate 220g

Soy Lecithin 43g

PEG400 35g

Sterile water for injection to 1000ml

preparation:

Dissolve the prescribed amount of gossypol acetate in ether, then add ethyl oleate, soybean lecithin, and PEG400 in a constant temperature water bath at 70°C, and stir while adding, at a stirring speed of 1700rpm, stir for 1.5 hours, and evaporate under reduced pressure to remove the ether, as Oil phase liquid, stored in a constant temperature water bath at 70°C for later use;

Store an appropriate amount of sterile water for injection in a constant temperature water bath at 70°C as the aqueous phase liquid;

Slowly drop the oil phase liquid into the water phase liquid at a constant temperature of 70°C, stir while adding, the stirring speed is 2000rpm, stir for 1 hour until a transparent and uniform solution is formed, dilute to 1000ml with sterilized water for injection, and then use homogeneous The homogenizer (NS10012k high-pressure homogenizer, Niro Soavi S.p.A., Italy) was repeatedly homogenized 6 times at 60Mpa, and filtered with a 0.45 μm microporous membrane. Packed into 1000 parts, sterilized and stored.

In the obtained sample, the average diameter of emulsion droplets is 142nm, and the encapsulation efficiency is 93.6%.

The preparation of embodiment 3 gossypol acetate intravenous injection fat emulsion

Party:

Gossypol Acetate 80g

Medium-chain fatty acid (C8-C10) triglycerides 297g

Soy Lecithin 36g

PEG400 29g

Sterile water for injection to 1000ml

preparation:

Dissolve the prescribed amount of gossypol acetate in ether, then add medium-chain fatty acid (C8-C10) triglycerides, soybean lecithin, and PEG400 in a constant temperature water bath at 70°C, and stir while adding. The stirring speed is 1800rpm, and stir for 2 hours , evaporated under reduced pressure to remove ether, used as an oil phase liquid, and stored in a constant temperature water bath at 70°C for later use;

Store an appropriate amount of sterile water for injection in a constant temperature water bath at 70°C as the aqueous phase liquid;

Slowly drop the oil phase liquid into the water phase liquid at a constant temperature of 70°C, stir while adding, the stirring speed is 2400rpm, stir for 1 hour until a transparent and uniform solution is formed, dilute to 1000ml with sterile water for injection, and then Machine (NS10012k high-pressure homogenizer, Niro Soavi S.p.A., Italy) repeated homogenization 6 times at 40Mpa, and filtered with 0.45 μm microporous membrane. Packed into 1000 parts, sterilized and stored.

In the obtained sample, the average diameter of emulsion droplets is 93nm, and the encapsulation efficiency is 97.4%.

The preparation of embodiment 4 gossypol acetate intravenous injection fat emulsion

Party:

Gossypol Acetate 50g

Medium-chain fatty acid (C8-C10) triglycerides 242g

Soy Lecithin 47g

Anhydrous ethanol 24g

Sterile water for injection to 1000ml

preparation:

Mix the prescribed amount of gossypol acetate with medium-chain fatty acid (C8-C10) triglycerides, soybean lecithin, and absolute ethanol (15g) in a constant temperature water bath at 70°C, and stir while adding. The stirring speed is 1800rpm, and stir for 1 hour , as an oil phase liquid, stored in a constant temperature water bath at 70°C for later use;

Add an appropriate amount of sterilized water for injection, add the remaining amount of absolute ethanol, and store in a constant temperature water bath at 70°C as the aqueous phase liquid;

Slowly drop the oil phase liquid into the water phase liquid at a constant temperature of 70°C, stir while adding, the stirring speed is 2000rpm, stir for 1 hour until a transparent and uniform solution is formed, dilute to 1000ml with sterilized water for injection, and then use homogeneous The homogenizer (NS10012k high-pressure homogenizer, Niro Soavi S.p.A., Italy) was repeatedly homogenized 6 times at 50Mpa, and filtered with a 0.45 μm microporous membrane. Packed into 1000 parts, sterilized and stored.

In the obtained sample, the average diameter of emulsion droplets is 129nm, and the encapsulation efficiency is 80.1%.

The preparation of embodiment 5 gossypol formic acid intravenous injection fat emulsion

Party:

Gossypol formate 60g

Medium-chain fatty acid (C8-C10) triglycerides 263g

Soy Lecithin 35g

Anhydrous ethanol 20g

Sterile water for injection to 1000ml

preparation:

Mix the prescribed amount of gossypol formic acid with medium-chain fatty acid (C8-C10) triglycerides, soybean lecithin, and absolute ethanol in a constant temperature water bath at 70°C, and stir while adding at a stirring speed of 1600rpm for 2 hours. Phase liquid, stored in a constant temperature water bath at 70°C for later use;

Preserve an appropriate amount of sterilized water for injection in a constant temperature water bath at 70°C as the aqueous phase liquid;

Slowly drop the oil phase liquid into the water phase liquid at a constant temperature of 70°C, stir while adding, the stirring speed is 2400rpm, stir for 1 hour until a transparent and uniform solution is formed, dilute to 1000ml with sterile water for injection, and then The homogenizer (NS10012k high-pressure homogenizer, Niro Soavi S.p.A., Italy) was repeatedly homogenized 5 times at 40Mpa, and filtered with a 0.45 μm microporous membrane. Packed into 1000 parts, sterilized and stored.

In the obtained sample, the average particle diameter of emulsion droplets is 87nm, and the encapsulation efficiency is 79.4%.

The pharmacokinetic experiment of embodiment 6 gossypol acetate intravenous injection fat emulsion in rat body

12 Wister rats, 180-240g, half male and half male were fasted one night before the experiment, were randomly divided into 2 groups, 6 rats in each group, and the experimental group was given the intravenous fat emulsion prepared in Example 1 by single tail vein injection (with acetic acid Gossypol 40mg/kg body weight administration), the control group made a suspension of gossypol acetate suspension and 0.25% carboxymethyl cellulose for oral gavage administration (administration with gossypol acetate 40mg/kg body weight) .

Blood samples were taken at 0, 30, 60 minutes and 2, 8, 16, and 24 hours after administration to determine the content of gossypol acetate in plasma, and the drug-time curve was drawn. The results are shown in Figure 1. It can be seen from the drug-time curve that intravenous injection of fat emulsion has significant sustained-release characteristics, and its bioavailability is significantly higher than that of oral administration.

Example 7 Tissue Distribution Experiment of Gossypol Acetate Intravenous Injection of Fat Emulsion in Tumor-bearing Mice

The C-57 model mice of Dutch Lewis lung cancer were purchased from the Experimental Animal Center of Shaanxi Academy of Traditional Chinese Medicine. The C-57 mice were provided by the Experimental Animal Center of Fourth Military Medical University. They were 4-6 weeks old and weighed 15-25g. There were 60 mice in total. male. The model tumor was excised and ground to make a cell homogenate, which was inoculated subcutaneously in the ventral side of the root of the right forelimb of C-57 experimental mice. Each mouse was inoculated with 2.0×10 ⁷ tumor cells, with a total volume of 0.20 ml. After 2 weeks, the tumor grew and was used in the experiment.

The tumor-bearing mice were randomly divided into 2 groups, 30 in each group. The experimental group was given the intravenous fat emulsion prepared in Example 2 by single tail vein injection (administered with gossypol acetate 40 mg/kg body weight), and the control group was given acetic acid Gossypol was suspended in 0.25% carboxymethyl cellulose to make a suspension orally administered by oral gavage (administered with gossypol acetate 40 mg/kg body weight). 1, 2, 8, 12, 16, and 24 hours after administration, 5 mice in each group were sacrificed, and the tumor tissue, heart, liver, spleen, lung, kidney, and brain were taken out to determine the tissue content. The relative content was calculated according to the concentration of the drug in the tissue and the weight of the tissue, and the results are shown in Figure 2.

It can be seen from the tissue distribution diagram that the content of gossypol acetate in the tumor tissue after the intravenous injection of gossypol acetate is significantly higher than that of the oral administration preparation. Intravenous injection of gossypol acetate fat emulsion has a certain targeting effect on tumor tissue.

Example 8 Toxicity Test of Gossypol Acetate Intravenous Injection Fat Emulsion

100 SPF grade KM mice, with a body weight of 30-40g, were randomly divided into 5 groups of 20 in each group of male and female, and three groups of experimental groups were injected into the intravenous injections prepared in Examples 1, 3 and 4 respectively (with acetic acid Gossypol 80mg/kg body weight administration), positive control group, gossypol acetate suspended in 0.25% carboxymethyl cellulose to make suspension oral gavage administration (gossypol acetate 80mg/kg body weight administration), the blank control group was given physiological saline by tail vein injection, 2ml/only, and administered once a day for 7 consecutive days. General observation, blood On the first, third, fifth, and seventh day after administration, 5 animals in each group were sacrificed for pathological anatomy, and pathological examination was carried out under an optical microscope.

General observation finds that the incidence rate of vomiting after the administration of the positive control group is 65%, and the incidence rate of vomiting in the experimental group is 5% (intravenous injection prepared in embodiment 1), 0% (intravenous injection prepared in embodiment 3), and 0% (intravenous injection prepared in implementation example 3). Intravenous injection prepared in Example 4), each experimental group was significantly different from the control group (p<0.05). And the vomiting symptoms of the experimental group were relatively mild. The hypokalemia symptom incidence rate of experimental group and positive control group is respectively 35% (intravenous injection made in embodiment 1), 20% (intravenous injection made in embodiment 3), 40% (intravenous injection made in embodiment 4), 80% %, each experimental group was significantly different from the control group (p<0.05). . Pathological anatomy showed no significant tissue damage. The blank control group had no abnormal symptoms.

The results showed that the incidence of adverse reactions of gossypol intravenous injection of lipid emulsion was significantly lower than that of oral administration. Its lower gastrointestinal irritation is related to the route of administration, and the lower incidence of hypokalemia may be related to the slower release rate of the drug.

Stability experiment of embodiment 9 gossypol intravenous injection fat emulsion

Stability test was carried out to the intravenous injection fat emulsion obtained in Examples 1-5.

(1) Strong light irradiation test: The gossypol intravenous fat emulsion was placed under 4000lx light for 5 days and 10 days, and then observed. The results found that compared with before placing, the injection remained clear and transparent, and no oil-water layer was seen.

(2) High humidity test: put gossypol intravenously injected fat emulsion in airtight containers under the conditions of 25%, 75% and 92.5% relative humidity, respectively, and investigate the stability after 5 days and 10 days. Compared with before placement, the intravenous injection remained clear and transparent, with no oil-water layering.

(3) High temperature test: put gossypol intravenously injected fat emulsion in a closed container and place it under the conditions of 40, 60, and 80°C respectively, and investigate the stability after 5 days and 10 days. Compared with before being placed, it still remains clear and transparent, and no oil-water layering is seen.

(4) Accelerated test: The gossypol intravenously injected fat emulsion was centrifuged at 4000rpm for 10min, and it remained clear and transparent without oil-water separation.

(5) Inspection of retained samples at room temperature: The gossypol intravenously injected fat emulsion was placed under the conditions of temperature 25°C and relative humidity 75% for 1, 2, 3, 6, 12, and 24 months, and it remained clear and transparent, with no oil or moisture Layer and drug precipitation.

Claims (2)

1. gossypol intravenous injection fatty emulsion for the treatment of tumor, it is characterized in that: every 1000ml is composed of the following components for described gossypol intravenous injection fatty emulsion: the pharmaceutical salts 40～80g of gossypol, ethyl oleate or medium-chain fatty acid triglyceride 200～300g, soybean lecithin 30～50g, dehydrated alcohol or PEG400 10～40g, surplus is a water; Described medium-chain fatty acid is meant the fatty acid of C8～C10.

2. the gossypol intravenous injection fatty emulsion of the described treatment tumor of claim 1 is characterized in that, the pharmaceutical salts of described gossypol is gossypol acetate or formic acid gossypol.

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