CN114903855A - Irinotecan hydrochloride and floxuridine compound liposome pharmaceutical composition and preparation method thereof - Google Patents

Abstract

The invention provides a irinotecan hydrochloride and fluorouracil compound liposome pharmaceutical composition and a preparation method thereof. The invention provides a irinotecan hydrochloride fluorouracil liposome pharmaceutical composition, which comprises irinotecan hydrochloride, fluorouracil, hydrogenated soybean phosphatidylcholine, distearoyl phosphatidylcholine, cholesterol and pelatidylethanolamine. The irinotecan hydrochloride fluorouracil liposome pharmaceutical composition provided by the invention has the advantages that irinotecan hydrochloride has higher encapsulation efficiency, and fluorouracil also has higher encapsulation efficiency, high drug loading, good stability, good drug effect, low toxicity and the like.

Description

Irinotecan hydrochloride and floxuridine compound liposome pharmaceutical composition and preparation method thereof

Technical Field

The invention relates to a liposome pharmaceutical composition and a preparation method thereof, in particular to an irinotecan hydrochloride floxuridine compound liposome pharmaceutical composition and a preparation method thereof.

Background

Irinotecan hydrochloride (Irinotecan, CPT-11) as a water-soluble camptothecin derivative is an effective drug for treating metastatic colorectal cancer, and is still effective in fluorouracil resistant cases.

Fluoropyrimidines have been used for more than forty years for the treatment of advanced colorectal Cancer, 5-fluorouracil (5-FU) being considered as the standard systemic chemotherapy for this indication (Van Laar, J.A.M. et al, Eur.J.cancer (1998) 34: 296-306, Coutinho, A.K., et al, Cancer Control (2003) 10: 224-238). Floxuridine is a derivative of 5-fluorouracil deoxyribonucleoside, has activity proved clinically, is suitable for liver cancer, rectal cancer, esophageal cancer, gastric cancer, breast cancer, lung cancer and the like, and has obvious curative effect on primary liver cancer which can not be resected by operation.

Since the approval of irinotecan in 1996, the combination of irinotecan and 5-FU has become the standard chemotherapeutic approach for first and second line treatment of metastatic colorectal cancer. Although the free drug combination has a good therapeutic effect, since fluoropyrimidines such as 5-FU and floxuridine have the disadvantage of being rapidly eliminated in vivo, in order to improve the efficacy and toxicity profile of the drug, a long infusion time such as a period of up to 24 hours or more for administration is often clinically used, which causes great inconvenience to patients.

CN105796495A discloses an irinotecan hydrochloride liposome pharmaceutical composition and a preparation method thereof, wherein the irinotecan hydrochloride liposome pharmaceutical composition comprises irinotecan hydrochloride, hydrogenated soybean phosphatidylcholine, distearoyl phosphatidylcholine, cholesterol and persevered phosphatidylethanolamine, wherein the weight ratio of the irinotecan hydrochloride, the hydrogenated soybean phosphatidylcholine, the distearoyl phosphatidylcholine, the cholesterol and the persevered phosphatidylethanolamine is 1: 2.1-3.9: 0.5-1.6: 0.75-1.5: 0.25 to 1.25; the preparation method comprises the following steps: (1) dissolving hydrogenated soybean phosphatidylcholine, distearoyl phosphatidylcholine, cholesterol and cultivated phosphatidylethanolamine in an organic solvent, and injecting the solution into an ammonium sulfate solution by a peristaltic pump to form a primary dispersion; (2) homogenizing and finishing the primary dispersion in the step (1), and removing the organic solvent and the non-wrapped ammonium sulfate by using ultrafiltration equipment to prepare a blank liposome; (3) dissolving irinotecan hydrochloride in water, adding an osmotic pressure regulator, a pH regulator and the hollow white liposome in the step (2), fully incubating, filtering for sterilization, and filling to obtain the irinotecan hydrochloride liposome injection.

CN1798544A discloses a liposome composition of water-soluble camptothecin and fluoropyrimidine, specifically comprising irinotecan, floxuridine, DSPC, DSPG, cholesterol in a specific molar ratio, and gradient agent using copper gluconate or copper sulfate, triethanolamine, and emphasizing that the liposome preferably contains metal ion solution, and the metal ion is preferably copper. Wherein the entrapment rate of floxuridine entering the liposome in a passive drug loading mode is basically 3-5%.

Disclosure of Invention

The invention provides a pharmaceutical composition of irinotecan hydrochloride and floxuridine liposome and a preparation method thereof, the pharmaceutical composition plays a synergistic anti-tumor role, and the liposome has higher entrapment rate, particularly higher floxuridine entrapment rate, high drug loading, good stability, good drug effect, light adverse reaction and the like.

The invention provides an irinotecan fluorouridine hydrochloride liposome pharmaceutical composition, which comprises irinotecan hydrochloride, fluorouridine, Hydrogenated Soybean Phosphatidylcholine (HSPC), distearoyl phosphatidylcholine (DSPC), Cholesterol (CHOL) and pelidylated phosphatidylethanolamine.

Wherein the molar ratio of irinotecan hydrochloride (calculated as irinotecan) to floxuridine is 1-5: 5-1, preferably 0.8-1: 1 to 1.25, more preferably 1: 1.

Hydrogenated Soybean Phosphatidylcholine (HSPC), distearoyl phosphatidylcholine (DSPC) and Cholesterol (CHOL) in a molar ratio of 1-9: 0-8: 0.5 to 2. Preferably, the molar ratio of the hydrogenated soybean phosphatidylcholine to the distearoyl phosphatidylcholine to the cholesterol is 1-7: 0-8: 1 to 2. Preferably, the molar ratio of the hydrogenated soybean phosphatidylcholine to the distearoyl phosphatidylcholine to the cholesterol is 1-5: 0-6: 1 to 2. Preferably, the molar ratio of the hydrogenated soybean phosphatidylcholine to the distearoyl phosphatidylcholine to the cholesterol is 1-5: 0-4: 1 to 2. Preferably, the molar ratio of the hydrogenated soybean phosphatidylcholine to the distearoyl phosphatidylcholine to the cholesterol is 1-4: 0-8: 1 to 2. Preferably, the molar ratio of the hydrogenated soybean phosphatidylcholine to the distearoyl phosphatidylcholine to the cholesterol is 1-4: 0-5: 1 to 2.

The molar ratio of the culture phosphatidylethanolamine (DSPE-mPEG) to the sum of the Hydrogenated Soybean Phosphatidylcholine (HSPC), distearoyl phosphatidylcholine (DSPC) and cholesterol is 1: 30-60. The peletized phosphatidylethanolamine is polyethylene glycol 1000 phosphatidylethanolamine, polyethylene glycol 2000 phosphatidylethanolamine or polyethylene glycol 3350 phosphatidylethanolamine, preferably polyethylene glycol 2000 phosphatidylethanolamine.

The mass ratio of the irinotecan hydrochloride to the sum of the hydrogenated soybean phosphatidylcholine and the distearoyl phosphatidylcholine is 1: 4-10.

The irinotecan floxuridine hydrochloride liposome pharmaceutical composition provided by the invention can also comprise an osmotic pressure regulator. The osmotic pressure regulator is one or more selected from glucose, mannitol, sucrose, lactose, trehalose and galactose, and preferably glucose, mannitol or sucrose. Wherein the weight ratio of the osmotic pressure regulator to the phospholipid is 0.25-0.8: 1, preferably, the weight ratio of the osmotic pressure regulator to the phospholipid is 0.25-0.6: 1, wherein the weight of the phospholipid refers to the sum of the weight of hydrogenated soybean phosphatidylcholine, distearoyl phosphatidylcholine and esterified phosphatidylethanolamine.

The irinotecan floxuridine hydrochloride lipidosome pharmaceutical composition provided by the invention is administrated in a parenteral form and can be an injection or a freeze-dried powder injection.

The invention provides a preparation method of irinotecan floxuridine hydrochloride liposome pharmaceutical composition, which comprises the following steps: dissolving hydrogenated soybean phosphatidylcholine, distearoyl phosphatidylcholine, cholesterol and cultured phosphatidylethanolamine in an organic solvent, evaporating the organic solvent under reduced pressure to form a membrane, adding an ammonium sulfate sucrose solution or an ammonium sulfate aqueous solution to hydrate to form a suspension, homogenizing, grading, and removing the unencapsulated ammonium sulfate and sucrose or ammonium sulfate by an ultrafiltration system to obtain an empty liposome; adding irinotecan floxuridine hydrochloride sucrose solution, fully incubating, removing unencapsulated free drug by an ultrafiltration system, sterilizing and filtering to obtain the irinotecan floxuridine hydrochloride liposome injection.

The invention provides a preparation method of irinotecan floxuridine hydrochloride liposome pharmaceutical composition, which comprises the following steps:

(1) dissolving hydrogenated soybean phosphatidylcholine, distearoyl phosphatidylcholine, cholesterol and cultivated phosphatidylethanolamine in a proper amount of organic solvent, evaporating the organic solvent at 50-75 ℃ under reduced pressure, and adding an ammonium sulfate sucrose solution or an ammonium sulfate aqueous solution to form a suspension;

(2) granulating the suspension prepared in the step (1) by a high-pressure homogenizer, and removing unencapsulated ammonium sulfate and sucrose or ammonium sulfate by an ultrafiltration system to prepare blank liposome;

(3) dissolving irinotecan fluorouridine hydrochloride in water, adding an osmotic pressure regulator and the hollow white liposome in the step (2), and fully incubating.

(4) Removing unencapsulated free drugs from the incubated suspension through an ultrafiltration system to prepare the irinotecan floxuridine hydrochloride liposome, and sterilizing and filtering to obtain the irinotecan floxuridine hydrochloride liposome injection.

The invention provides a preparation method of an irinotecan floxuridine hydrochloride lipidosome pharmaceutical composition, which comprises the following steps:

(1) dissolving hydrogenated soybean phosphatidylcholine, distearoyl phosphatidylcholine, cholesterol and cultivated phosphatidylethanolamine in an organic solvent, and injecting the solution into an ammonium sulfate sucrose solution or an ammonium sulfate aqueous solution to form a primary dispersion;

(2) homogenizing and finishing the primary dispersion in the step (1), and removing the organic solvent, the uncoated ammonium sulfate and the sucrose or the ammonium sulfate by an ultrafiltration device to prepare a blank liposome;

(3) adding irinotecan floxuridine hydrochloride into an osmotic pressure regulator for dissolving, and then fully incubating with the hollow white liposome in the step (2);

(4) removing unencapsulated free drugs from the incubated suspension through an ultrafiltration system to prepare the irinotecan floxuridine hydrochloride liposome, and sterilizing and filtering to obtain the irinotecan floxuridine hydrochloride liposome injection.

In the preparation method, the organic solvent in step (1) is selected from one or more of chloroform, methanol, ethanol, diethyl ether, isopropanol, acetone and petroleum ether, preferably one of isopropanol, methanol or ethanol, and more preferably ethanol.

The ammonium sulfate solution in the step (1) may be 0.1mol/L, 0.15mol/L, 0.25mol/L or 0.30mol/L, preferably 0.25mol/L ammonium sulfate aqueous solution.

And (3) homogenizing and finishing the granules in the step (2), and preferably sequentially extruding the granules through 0.2, 0.1 mu m or 0.05 mu m polycarbonate films to obtain the product.

The irinotecan hydrochloride floxuridine liposome pharmaceutical composition provided by the invention has higher entrapment rate of irinotecan hydrochloride and higher entrapment rate of floxuridine, wherein the pre-loading entrapment rate of irinotecan hydrochloride is 80% -95%, and the pre-loading entrapment rate of floxuridine is 5% -8%. The irinotecan fluuridine hydrochloride compound liposome provided by the invention has the advantages of simple formula and process, low cost and contribution to industrial mass production. Meanwhile, the irinotecan fluuridine hydrochloride liposome pharmaceutical composition provided by the invention has higher drug effect and lower toxicity, and is more beneficial to treatment and use of patients.

Drawings

FIG. 1 example 1 formula 1 particle size.

Figure 2 example 1 particle size of formula 2.

FIG. 3 particle size of formula 3 of example 1.

Figure 4 example 2R1 particle size.

Fig. 5 example 2R2 particle size.

Fig. 6 example 2R3 particle size.

Fig. 7 example 2R4 particle size.

Fig. 8 example 2R5 particle size.

Fig. 9 example 2R6 particle size.

Fig. 10 example 2R7 particle size.

The specific implementation mode is as follows:

the following examples are intended to further illustrate the invention, but the invention is not limited thereto.

EXAMPLE 1 Liposomal injection of irinotecan floxuridine hydrochloride prepared with different long circulating phospholipids

TABLE 1 formulation ratio of irinotecan hydrochloride floxuridine liposome injection

The preparation method comprises the following steps:

weighing hydrogenated soybean phosphatidylcholine, distearoyl phosphatidylcholine, cholesterol and cultured phosphatidyl ethanol according to the prescription amount, placing the weighed hydrogenated soybean phosphatidylcholine, distearoyl phosphatidylcholine, cholesterol and cultured phosphatidyl ethanol into a 500ml round-bottom flask, adding a proper amount of absolute ethanol for dissolving, evaporating the ethanol on a rotary evaporator at 50-75 ℃ under reduced pressure to form a uniform lipid membrane at the bottom of the flask, washing the membrane by using 100ml of 0.25mol/L ammonium sulfate sucrose solution or ammonium sulfate aqueous solution for hydration, passing the membrane through a homogenizer for 3 times of 0.2 mu m, extruding and granulating the membrane by 0.1 mu m or 0.05 mu m, removing the unencapsulated ammonium sulfate and sucrose or ammonium sulfate by ultrafiltration, and supplementing water for injection continuously in the ultrafiltration process to obtain blank liposomes.

Dissolving irinotecan fluorouridine hydrochloride in water according to the prescription amount, adding 8-9% of osmotic pressure regulator, adding the mixture into the blank liposome, incubating at 50-60 ℃ for 20 minutes to obtain a drug-loaded liposome, removing unencapsulated drug by ultrafiltration, adjusting the drug concentration by using a sucrose solution, sterilizing and filtering by using a 0.22 mu m filter membrane, and filling into a penicillin bottle to obtain the irinotecan fluorouridine hydrochloride liposome injection.

The irinotecan floxuridine hydrochloride liposome injection prepared according to each formula is respectively subjected to particle size detection and encapsulation efficiency detection, and the results are shown in table 2:

TABLE 2 TABLE 1 indexes of irinotecan floxuridine hydrochloride liposome injection

As is clear from Table 2, there was no significant difference in particle size and appearance between the respective portions. When the molar ratio of the cultured phosphatidylethanolamine to the sum of the mass of the hydrogenated phosphatidylcholine, the distearoyl phosphatidylcholine and the cholesterol is 1:15, the encapsulation efficiency of irinotecan hydrochloride and floxuridine in the obtained compound liposome is very low; when the molar ratio of the cultured phosphatidylethanolamine to the sum of the masses of the hydrogenated phosphatidylcholine, the distearoyl phosphatidylcholine and the cholesterol is 1: when the content is 30-60 hours, the entrapment rate of irinotecan hydrochloride and floxuridine in the compound liposome is higher, so that the molar ratio of the selectively-cultured phosphatidylethanolamine to the sum of the mass of the hydrogenated phosphatidylcholine, the distearoyl phosphatidylcholine and the cholesterol is 1: between 30 and 60.

Example 2 irinotecan floxuridine hydrochloride liposome injection prepared by different phospholipid ratios

TABLE 3 prescription ratio of irinotecan hydrochloride floxuridine liposome injection

The preparation method comprises the following steps:

respectively weighing hydrogenated soybean phosphatidylcholine, distearoyl phosphatidylcholine, cholesterol and cultured phosphatidylethanolamine according to the prescription amount, placing the hydrogenated soybean phosphatidylcholine, distearoyl phosphatidylcholine, cholesterol and cultured phosphatidylethanolamine into a 250ml round-bottom flask, adding a proper amount of absolute ethyl alcohol to dissolve the absolute ethyl alcohol, evaporating the ethyl alcohol on a rotary evaporator at 50-75 ℃ under reduced pressure to form a uniform lipid membrane on the bottom of the flask, washing the membrane with 100ml of 0.25mol/L ammonium sulfate sucrose solution or ammonium sulfate aqueous solution, passing the membrane through a homogenizer for 3 times of 0.2 mu m, extruding and granulating the membrane by 0.1 mu m or 0.05 mu m, removing the unencapsulated ammonium sulfate and sucrose or ammonium sulfate by ultrafiltration, and continuously supplementing injection water in the ultrafiltration process to obtain blank liposomes.

Dissolving irinotecan fluorouridine hydrochloride in water according to the prescription amount, adding 8-9% of osmotic pressure regulator, adding the mixture into the blank liposome, incubating at 50-60 ℃ for 20 minutes to obtain a drug-loaded liposome, removing unencapsulated drug by ultrafiltration, adjusting the drug concentration by using a sucrose solution, sterilizing and filtering by using a 0.22 mu m filter membrane, and filling into a penicillin bottle to obtain the irinotecan fluorouridine hydrochloride liposome injection.

2. The above formulations were tested for encapsulation efficiency and particle size, and the results are shown in table 4:

TABLE 4 encapsulation efficiency and particle size of irinotecan hydrochloride floxuridine liposome injection

From the results in Table 4, it can be seen that: the encapsulation of irinotecan hydrochloride and floxuridine was substantially equivalent between different phospholipid formulations, with no apparent difference in particle size.

Example 3 irinotecan hydrochloride floxuridine liposome injection

TABLE 5 irinotecan hydrochloride floxuridine liposome injection ratio

The preparation method comprises the following steps:

respectively putting hydrogenated soybean phosphatidylcholine, distearoyl phosphatidylcholine, cholesterol and cultured phosphatidylethanolamine with the prescription amount into a 250ml round bottom flask, adding a proper amount of absolute ethyl alcohol to dissolve the absolute ethyl alcohol, evaporating the ethyl alcohol on a rotary evaporator at 50-75 ℃ under reduced pressure to form a uniform lipid membrane on the bottom of the flask, washing the membrane with 100ml of 0.25mol/L ammonium sulfate sucrose solution or ammonium sulfate aqueous solution by hydration, passing through a homogenizer for 3 times with the diameter of 0.2 mu m, extruding with the diameter of 0.1 mu m or 0.05 mu m, removing the unencapsulated ammonium sulfate and sucrose or ammonium sulfate by ultrafiltration, and continuously supplementing water for injection in the ultrafiltration process to complete granules to obtain blank liposomes.

Dissolving irinotecan fluorouridine hydrochloride in water according to the prescription amount, adding 8-9% of osmotic pressure regulator, adding the mixture into the blank liposome, incubating at 50-60 ℃ for 20 minutes to obtain a drug-loaded liposome, removing unencapsulated drug by ultrafiltration, adjusting the drug concentration by using a sucrose solution, sterilizing and filtering by using a 0.22 mu m filter membrane, and filling into a penicillin bottle to obtain the irinotecan fluorouridine hydrochloride liposome injection, wherein the entrapment rate of irinotecan hydrochloride is 82.33%, and the entrapment rate of fluorouridine is 5.04%.

Example 4 irinotecan hydrochloride floxuridine liposome injection

TABLE 6 irinotecan hydrochloride floxuridine liposome injection ratio

Irinotecan hydrochloride	0.073g
		Floxuridine	0.60g
Hydrogenated soybean phosphatidylcholine	0.31g
		Distearoyl phosphatidylcholine	0.42g
Cholesterol	0.051g
		DSPE-mPEG2000	0.096g
Ammonium sulfate	3.3g
		Sucrose	5g
Water for injection	Adding to the desired volume

The preparation method comprises the following steps:

respectively putting hydrogenated soybean phosphatidylcholine, distearoyl phosphatidylcholine, cholesterol and cultivated phosphatidylethanolamine with the prescription amount into a 250ml round bottom flask, adding a proper amount of absolute ethyl alcohol to dissolve the absolute ethyl alcohol, evaporating the ethyl alcohol on a rotary evaporator at 50-75 ℃ under reduced pressure to form a uniform lipid membrane on the bottom of the flask, washing the membrane with 100ml of 0.3mol/L ammonium sulfate sucrose solution or ammonium sulfate aqueous solution by hydration, passing through a homogenizer for 3 times with the diameter of 0.2 mu m, extruding out with the diameter of 0.1 mu m or 0.05 mu m, removing the unencapsulated ammonium sulfate and sucrose or ammonium sulfate by ultrafiltration, and continuously supplementing water for injection in the ultrafiltration process to complete granules to obtain blank liposomes.

Dissolving irinotecan fluorouridine hydrochloride in water according to the prescription amount, adding an osmotic pressure regulator, adding the mixture into the blank liposome, incubating for 20 minutes at 50-60 ℃ to obtain a drug-loaded liposome, removing unencapsulated drugs through ultrafiltration, adjusting the drug concentration through a sucrose solution, sterilizing and filtering through a 0.22-micron filter membrane, and filling into a penicillin bottle to obtain the irinotecan fluorouridine hydrochloride liposome injection, wherein the encapsulation rate of irinotecan hydrochloride is 88.54%, and the encapsulation rate of fluorouridine is 5.73%.

Test example 1 evaluation of drug efficacy of formulations of different phospholipids and compound solutions

1. Test materials

ICR mouse Beijing Weitonghua license number SCXK (Jing) 2012-

2. Test prescription

Prescription 1: r1, irinotecan fluorouridine hydrochloride solution, weighing 37.5mg of irinotecan hydrochloride and 13.63mg of fluorouridine, and dissolving in 150ml of 5% glucose injection to obtain irinotecan fluorouridine hydrochloride solution.

Prescription 1: r1, irinotecan fluorouridine hydrochloride solution, weighing 37.5mg of irinotecan hydrochloride and 13.63mg of fluorouridine, and dissolving in 150ml of 5% glucose injection to obtain irinotecan fluorouridine hydrochloride solution.

Prescription 2-4: R2-R4, irinotecan floxuridine hydrochloride liposome injection prepared according to the preparation method of example 2, wherein: r2: distearoylphosphatidylcholine 736mg, distearoylphosphatidylglycerol 213mg, cholesterol 51mg, ammonium sulfate 3.3g, irinotecan hydrochloride 99.5mg, floxuridine 862.2mg were weighed and prepared according to the preparation method of example 2.

R3: distearoylphosphatidylcholine 736mg, distearoylphosphatidylglycerol 213mg, cholesterol 51mg, phosphatidylethanolamine 120.9mg, ammonium sulfate 3.3g, irinotecan hydrochloride 99.5mg, floxuridine 801.1mg were weighed and prepared according to the preparation method of example 2.

R4: the preparation method of example 2 was followed using 736mg of distearoylphosphatidylcholine, 206mg of hydrogenated soybean phosphatidylcholine, 51mg of cholesterol, 120.9mg of phosphatidylethanolamine, 3.3g of ammonium sulfate, 99.5mg of irinotecan hydrochloride, and 791.88mg of floxuridine.

TABLE 7 pharmacodynamic test prescription and accession number

3. Test methods and results

Inoculating S180 tumor-bearing mice solid tumor model, wherein the administration dose is 5mg/kg, 8 mice are administered for each prescription once every three days, the administration is carried out three times in total, the mice are killed on the fifteenth day after inoculation, the tumors are stripped and weighed, and the tumor inhibition rate is calculated, and the prescription and the tumor inhibition rate are shown in Table 8:

TABLE 8 results of pharmacodynamic tests on tumor inhibition rates of groups of mice

Numbering	Dosage (mg/kg)	Tumor inhibition Rate (%)	Number of animals (d1/d15, only)
				R1	5*3	38.4％	8/8
R2	5*3	85.6％	8/8
				R3	5*3	90.8％	8/8
R4	5*3	98.1％	8/8

The results in Table 8 show that the compound liposome prescription group has an obviously better tumor inhibition rate than the compound solution group, and each compound liposome group has an tumor inhibition rate higher than 85 percent and shows a stronger tumor inhibition effect; as can be seen by comparing the R2 prescription with the R3 prescription, the long circulation added in the prescription can prolong the retention time of the medicine in vivo and improve the tumor inhibition rate, and the tumor inhibition rate of the R4 prescription is obviously higher than that of the R2 prescription and the R3 prescription, thereby proving that the R4 prescription has obvious advantages in the aspect of drug effect compared with other prescriptions.

Test example 2 evaluation of drug efficacy of formulations with different phospholipid ratios

1. Test materials

ICR mouse Beijing Weitonglihua license number SCXK (Jing) 2012-000211400700140517

Positive control drug: cyclophosphamide (CTX) shanxi prader pharmaceutical products company batch No.: 04130902

EXAMPLE 2 irinotecan floxuridine hydrochloride liposome injection prepared by the preparation method of R1-R4

Physiological saline: batch number of pharmaceutical products of open rescue limited: 14112034

2. Test methods and results

Inoculating S180 tumor-bearing mice solid tumor model, wherein the administration dose is 5mg/kg, 10 mice are administered once every three days and three times in total, the death condition of the mice is recorded in the experimental period, the mice are killed on the fifteenth day after inoculation, the tumors are stripped and weighed, and the tumor inhibition rate is calculated, and the prescription and the tumor inhibition rate results are shown in a table 9 and a table 10:

TABLE 9 pharmacodynamic test prescription and numbering

TABLE 10 results of pharmacodynamic tests on tumor inhibition and body weight of various groups of mice

Note: p < 0.05; p is less than 0.01; p < 0.001, compared with physiological saline group

The results in Table 10 show that the mean value of the tumor weight of the Cyclophosphamide (CTX) group is 0.619 +/-0.348 g, the tumor inhibition rate is 36.19 percent, the tumor inhibition rate of each formula is superior to that of a positive control group (cyclophosphamide), the tumor inhibition rate of the R4 formula is more than 80 percent, particularly the tumor inhibition rate of the R2 formula and the R3 formula is more than 85 percent, and the Cyclophosphamide (CTX) group has stronger anti-tumor effect.

Test example 3 comparison of drug efficacy of irinotecan hydrochloride floxuridine liposome injection

1. Test materials

Mice: slek Balb/c license number 20211019Abzz0619000377

2. Sample preparation of irinotecan fluorouridine hydrochloride liposome injection:

preparation of control: accurately weighing DSPC 1017.59mg, DSPG 294.75mg and CHOL 71.13mg, placing the weighed materials into a 1000ml eggplant-shaped bottle, adding a proper amount of chloroform/methanol/water (95:4:1 volume ratio) for dissolution, carrying out water bath at 70 ℃, drying by using nitrogen, transferring the bottle to a rotary evaporator, continuously drying for at least 4 hours in vacuum, preparing a membrane washing solution containing copper gluconate 4.54 percent and triethanolamine 3.28 percent, regulating the pH value of the solution to be 7.4 by using the triethanolamine, taking 150ml, washing the membrane at 70 ℃, carrying out water bath at 70 ℃, finishing the particles to the particle size of 110nm +/-20 nm, and carrying out ultrafiltration to remove the gluconone in an external water phase. A blank liposome suspension was formed.

Weighing raw materials according to a medicine-lipid ratio of 0.12:1, dissolving in SHE solution (weighing 205.38g of sucrose, 9.53g of HEPES, 17.54g of EDTA17, dissolving, adjusting pH to 7.4 by adding triethanolamine, and fixing volume to 2000ml), adding sodium hydroxide to adjust pH to 7.4 for medicine carrying, preheating the medicine solution and blank liposome at 50 ℃ for 5min respectively, carrying medicine according to a volume ratio of 1:1, and carrying out water bath at 50 ℃ for 60 min. And removing the unencapsulated drug by ultrafiltration to obtain the irinotecan floxuridine hydrochloride liposome.

Preparation of the test article:

irinotecan hydrochloride floxuridine liposome injection, lot 21070813, Nanjing green leaf pharmaceutical Co., Ltd, prepared according to the preparation method of example 4.

3. Test methods and results

Inoculating S180 tumor-bearing mice solid tumor models, wherein the administration doses are respectively 2.5mg/kg, 5mg/kg and 10mg/kg, 5 mice are administered twice a week and are continuously administered for three weeks, the death condition of the mice is recorded in the experimental period, the mice are killed on the 25 th day after inoculation, the tumors are stripped and weighed, and the tumor inhibition rate is calculated, and the prescription and the tumor inhibition rate result are shown in a table 11:

TABLE 11 results of pharmacodynamic tests on tumor inhibition and body weight of various groups of mice

From the results in table 11, it can be seen that the low dose group of the test article had comparable efficacy to the control article, and was slightly better than the control article; but the drug effect of the high-dose group of the test product is obviously better than that of the reference product. And under the high dose of the reference substance, all animals die after the administration of the mice shows that the reference substance has higher toxicity; the test product has no animal death at high dose, the tumor inhibition rate is more than 75 percent, and the test product has obvious anti-tumor effect and low toxicity.

Test example 4 irinotecan hydrochloride floxuridine liposome injection Long-term stability

Irinotecan fluorouridine hydrochloride liposome injection prepared according to the formula R4 in example 2 was subjected to accelerated stability test according to the Chinese pharmacopoeia 2020, and the results are shown in Table 12:

TABLE 12 Long term stability study

Long-term stability data shows that all indexes such as encapsulation efficiency and the like are not obviously increased or reduced, and all data show good stability.

Claims (10)

1. An irinotecan hydrochloride fluorouracil liposome pharmaceutical composition comprises irinotecan hydrochloride, fluorouracil, hydrogenated soybean phosphatidylcholine, distearoyl phosphatidylcholine, cholesterol and pelatidylethanolamine, wherein the molar ratio of irinotecan hydrochloride to fluorouracil is 1-5: 5-1 in terms of irinotecan, and is characterized in that: the molar ratio of the hydrogenated soybean phosphatidylcholine to the distearoyl phosphatidylcholine to the cholesterol is 1-9: 0-8: 0.5-2, and the molar ratio of the cultivated phosphatidylethanolamine to the sum of the hydrogenated soybean phosphatidylcholine, the distearoyl phosphatidylcholine and the cholesterol is 1: 30-60.

2. Irinotecan hydrochloride fluorouracil liposome pharmaceutical composition according to claim 1, characterized in that: wherein the molar ratio of irinotecan hydrochloride to fluorouracil calculated from irinotecan is 1: 1.

3. irinotecan hydrochloride fluorouracil liposome pharmaceutical composition according to claim 1, characterized in that: the molar ratio of the hydrogenated soybean phosphatidylcholine to the distearoyl phosphatidylcholine to the cholesterol is 1-4: 0-8: 1 to 2.

4. Irinotecan hydrochloride fluorouracil liposome pharmaceutical composition according to claim 3, characterized in that: hydrogenated soybean phosphatidylcholine, distearoyl phosphatidylcholine and cholesterol in a molar ratio of 1-4: 0-3: 1 to 2.

5. The irinotecan hydrochloride fluorouracil liposome pharmaceutical composition according to any one of claims 1 to 4, characterized in that: the mass ratio of the irinotecan hydrochloride to the sum of the hydrogenated soybean phosphatidylcholine and the distearoyl phosphatidylcholine is 1: 4-10.

6. The irinotecan hydrochloride fluorouracil liposome pharmaceutical composition according to any one of claims 1 to 4, characterized in that: the composition can also comprise an osmotic pressure regulator, wherein the osmotic pressure regulator is selected from one or more of glucose, mannitol, sucrose, lactose, trehalose and galactose.

7. Irinotecan hydrochloride fluorouracil liposome pharmaceutical composition according to claim 6, characterized in that: the weight ratio of the osmotic pressure regulator to the phospholipid is 0.25-0.6: 1, wherein the weight of the phospholipid refers to the sum of the weight of hydrogenated soybean phosphatidylcholine, distearoyl phosphatidylcholine and esterified phosphatidylethanolamine.

8. A preparation method of irinotecan hydrochloride fluorouracil liposome pharmaceutical composition is characterized by comprising the following steps: dissolving hydrogenated soybean phosphatidylcholine, distearoyl phosphatidylcholine, cholesterol and cultured phosphatidylethanolamine in an organic solvent, evaporating the organic solvent under reduced pressure to form a membrane, adding an ammonium sulfate sucrose solution or an ammonium sulfate aqueous solution to hydrate to form a suspension, homogenizing, grading, and removing the unencapsulated ammonium sulfate and sucrose or ammonium sulfate by an ultrafiltration system to obtain an empty liposome; adding irinotecan fluorouracil hydrochloride sucrose solution, fully incubating, removing unencapsulated free drug by an ultrafiltration system, sterilizing and filtering to obtain the irinotecan fluorouracil hydrochloride liposome injection.

9. The production method according to claim 8, characterized in that:

(1) dissolving hydrogenated soybean phosphatidylcholine, distearoyl phosphatidylcholine, cholesterol and cultivated phosphatidylethanolamine in a proper amount of organic solvent, evaporating the organic solvent at 50-75 ℃ under reduced pressure, and adding an ammonium sulfate sucrose solution or an ammonium sulfate aqueous solution to form a suspension;

(2) granulating the suspension prepared in the step (1) by a high-pressure homogenizer, and removing the unencapsulated ammonium sulfate and sucrose or ammonium sulfate by an ultrafiltration system to prepare blank liposome;

(3) dissolving irinotecan hydrochloride fluorouracil in water, adding an osmotic pressure regulator and the hollow white liposome in the step (2), and fully incubating;

(4) and removing unencapsulated free drugs from the incubated suspension through an ultrafiltration system to prepare the irinotecan hydrochloride fluorouracil liposome, and sterilizing and filtering to obtain the irinotecan hydrochloride fluorouracil liposome injection.

10. The production method according to claim 9, characterized in that:

(1) dissolving hydrogenated soybean phosphatidylcholine, distearoyl phosphatidylcholine, cholesterol and cultivated phosphatidylethanolamine in an organic solvent, and injecting into an ammonium sulfate sucrose solution or an ammonium sulfate aqueous solution to form a primary dispersion;

(2) homogenizing and finishing the primary dispersion in the step (1), and removing the organic solvent, the uncoated ammonium sulfate and the sucrose or the ammonium sulfate by an ultrafiltration device to prepare a blank liposome;

(3) adding irinotecan hydrochloride fluorouracil into an osmotic pressure regulator for dissolving, and then fully incubating with the hollow white liposome in the step (2);

(4) and removing unencapsulated free drugs from the incubated suspension through an ultrafiltration system to prepare the irinotecan hydrochloride fluorouracil liposome, and sterilizing and filtering to obtain the irinotecan hydrochloride fluorouracil liposome injection.

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