CN104306336A - Technology for preparing citric acid daunorubicin lipidosome injection liquid - Google Patents

Abstract

The invention discloses a preparation process of daunorubicin citrate liposome injection, which improves the encapsulation rate of the final product through multiple extrusion preparation of blank liposomes, gradient adjustment of pH value and other operating steps To more than 97%, and the release rate and stability are good, which effectively improves the safety and effectiveness of the product, enhances the safety of patients' medication, thereby improves the compliance of patients with medication, and significantly enhances the therapeutic effect.

Description

Preparation process of daunorubicin citrate liposome injection

technical field

The invention belongs to the technical field of medicine, and in particular relates to a preparation process of daunorubicin citrate liposome injection.

Background technique

Daunorubicin is the first generation of anthracycline antitumor antibiotics. It is mainly used for various types of acute leukemia. It is not absorbed orally and can only be administered intravenously. It is commonly used in the form of hydrochloride and citrate.

Liposomes are lipid microcapsules with an aqueous inner core composed of phospholipid bilayers. Due to its characteristics of slow release, low toxicity and tissue targeting, it is often used in many aspects such as entrapped drugs and gene transfer.

Daunorubicin citrate liposome injection is a product developed by Gilead Company, which encapsulates daunorubicin citrate in liposomes before injection. After daunorubicin is encapsulated in liposomes, it can not only avoid the phagocytosis of phagocytic cells, but also change the distribution of daunorubicin in tissues, increase its orientation to cancerous cells, and enable daunorubicin to select Sexually kill cancer cells or inhibit the proliferation of cancer cells without damaging or inhibiting normal cells and tissues. Therefore, the curative effect of daunorubicin with liposome as carrier has been improved, the toxicity has been reduced, and the immune response has been alleviated.

Daunorubicin liposome injection can have these advantages due to the fact that daunorubicin can be well encapsulated in liposomes, and the encapsulation rate is an intuitive performance of the encapsulation degree. At present, the encapsulation rate of daunorubicin citrate liposome injection in the market is 90%. From the perspective of drug safety and effectiveness, the encapsulation rate still has a large room for improvement.

Contents of the invention

The object of the present invention is to provide a kind of preparation technology of daunorubicin citrate liposome injection, the encapsulation efficiency of the daunorubicin citrate liposome injection that this technology prepares is more than 97%, And the stability is good.

In order to realize the object of the present invention, the inventor provides the following technical solutions.

The preparation technology of daunorubicin citrate liposome injection comprises the following steps:

A. Prepare citric acid solution

Prepare a citric acid solution with a concentration of 8.64% and a pH value of 5.0 for subsequent use;

B. Preparation of Blank Liposomes

At 68-72°C, mix 37.65 parts by weight of distearoylphosphatidylcholine, 9 parts by weight of cholesterol, 42 parts by volume of absolute ethanol with 258 parts by volume of the citric acid solution obtained in step A, and stir for 1 hour to obtain Lipid dissolving solution, spare;

Under the condition of 68-72°C, put the lipid solution in a nano-extruder to extrude to prepare blank liposomes, first use a membrane with a pore size of 0.2 μm, and repeatedly extrude under the condition of an extrusion pressure of 450-600Psi The average particle diameter of the blank liposome is 200-220nm, and then the membrane with a pore diameter of 0.1 μm is repeatedly extruded to the average particle diameter of the blank liposome under the condition of extrusion pressure 200-300Psi is 120-130nm, and then Use a membrane with a pore size of 0.05 μm to repeatedly squeeze the blank liposomes at an extrusion pressure of 120-150 Psi until the average particle size of the blank liposomes is 55-62 nm, and store the blank liposomes at 4°C for future use. ;

C. Prepare sucrose solution

Prepare a sucrose solution with a concentration of 8.5% and a pH value of 5.0 for subsequent use;

D. Blank liposome dialysis

At 4°C, seal the blank liposomes prepared in step B in a dialysis bag, place them in the sucrose solution obtained in step C and dialyze for 4 hours, repeat this, and dialyze 3 times in total, replacing the sucrose solution each time;

After the dialysis is completed, adjust the pH value of the blank liposome to 8.0 and set aside;

E. Preparation of daunorubicin hydrochloride solution

Weigh 2.4 parts by weight of daunorubicin hydrochloride, dissolve it in 90 parts by volume of the sucrose solution obtained in step C, then adjust the pH value to 8.0, and finally add the sucrose solution obtained in step C to settle to 100 parts by volume to obtain daunorubicin hydrochloride solution, spare;

F. Preparation of sucrose solution

Prepare a sucrose solution with a concentration of 8.5% and a pH value of 8.0 for subsequent use;

G. Preparation of daunorubicin citrate liposome solution

At 68-72°C, mix the dialyzed blank liposome obtained in step D, the daunorubicin hydrochloride solution obtained in step E, and 200 parts by volume of the sucrose solution obtained in step F, and stir until the pH value is 5.6-6.0 , stop stirring to obtain daunorubicin citrate liposome solution, cool to room temperature, and set aside;

H. Preparation of Glycine Sucrose Calcium Chloride Buffer

Each 100ml contains 7.6g sucrose, 0.376g glycine, 0.028 calcium chloride dihydrate, pH value is 5.8;

I. prepare daunorubicin citrate liposome injection

In step G gained daunorubicin citrate liposome solution, adding glycine sucrose calcium chloride buffer solution and diluting to the concentration of daunorubicin is 2mg/ml, gained daunorubicin citrate liposome dilution solution Sterile filtration is carried out through a 0.2 μm membrane to obtain daunorubicin citrate liposome injection.

For the preparation process of the above-mentioned daunorubicin citrate liposome injection, the citric acid solution is prepared as described in step A, and the pH value is adjusted to 5.0 by using 1mol/L NaOH solution.

In the preparation process of the above-mentioned daunorubicin citrate liposome injection, the sucrose solution is prepared as described in step C, and the pH value is adjusted to 5.0 by using 1mol/L HCl solution.

In the preparation process of the above-mentioned daunorubicin citrate liposome injection, the blank liposomes described in step D were dialyzed, and the pH value of the blank liposomes was adjusted to 8.0 using 1mol/L NaOH solution.

For the preparation process of the above-mentioned daunorubicin citrate liposome injection, the daunorubicin hydrochloride solution is prepared as described in step E, and the pH value is adjusted to 8.0 by using 0.1mol/L NaOH solution.

In the preparation process of the above-mentioned daunorubicin citrate liposome injection, the sucrose solution is prepared as described in step F, and the pH value is adjusted to 8.0 by using 2mol/L NaOH solution.

The above preparation process of daunorubicin citrate liposome injection, the preparation of glycine sucrose calcium chloride buffer solution described in step H, using 2mol/L hydrochloric acid solution to adjust the pH value to 5.8.

In the preparation process of the present invention, parts by weight correspond to parts by volume, and the corresponding relationship is gram to milliliter, and so on.

The process of blank liposome dialysis described in step D is static dialysis, reaches saturation in 4 hours, and can be dialyzed completely after 3 times. During dialysis, vertically suspending the dialysis bag containing blank liposomes in sucrose solution can make dialysis more sufficient.

In the process of preparing the daunorubicin citrate liposome solution described in step G, the solution is mixed and stirred to a pH value of 5.6-6.0. Under this condition, the encapsulation efficiency and release degree of the final product are better, and the pH value is too high. High or too low will affect the encapsulation efficiency and release degree of the final product.

The glycine sucrose calcium chloride buffer solution prepared as described in step H, the buffer solution under this ratio condition can dilute the concentrated liposome solution on the one hand, and can make the final product in a more stable environment on the other hand. to the buffering effect.

The water used in the preparation process of the present invention is sterile water for injection.

The daunorubicin citrate liposome injection obtained by the preparation process of the present invention contains daunorubicin 50mg, distearoylphosphatidylcholine 376.5mg, cholesterol 90mg, sucrose 2125mg, glycine 94mg in every 25ml , calcium chloride 7mg, wherein, the molar ratio of daunorubicin, distearoylphosphatidylcholine, and cholesterol is 1:10:5. Under this ratio condition, the best encapsulation rate and release degree and the most stable state can be obtained.

The preparation process of the present invention is through the gradient adjustment of the pH value of each raw material solution, and finally the materials with a pH value of 8.0 are mixed to prepare daunorubicin citrate liposome injection. The sealing rate reached over 97%.

According to the daunorubicin citrate liposome injection obtained by the preparation process of the present invention, the encapsulation rate is above 97%, the release degree and stability are good, the safety and effectiveness of the product are significantly improved, and it can Improve patient compliance, thereby improving the effectiveness of treatment.

Detailed ways

The content of the present invention will be further described in detail below in conjunction with specific embodiments.

Example 1

A. Prepare citric acid solution

Accurately weigh 43.2g of citric acid, pour it into 450ml of water and dissolve it. After the dissolution is complete, adjust the pH value of the solution to 5.0 with 1mol/L NaOH, add water to 500ml to obtain a concentration of 8.64%, and a pH value of 5.0. Citric acid solution, for later use;

B. Preparation of Blank Liposomes

Accurately weigh 37.65g of distearoylphosphatidylcholine and 9g of cholesterol, mix distearoylphosphatidylcholine and cholesterol with 42ml of absolute ethanol and 258ml of citric acid solution at 68-72°C, and stir After 1 hour, the lipid solution was obtained and set aside;

Under the condition of 68-72°C, the lipid solution was placed in a nano-extruder to extrude to prepare blank liposomes. First, a polycarbonate membrane with a pore size of 0.2 μm was used to repeatedly extrude under the condition of an extrusion pressure of 450Psi. Press 5 times, the average particle diameter of detection blank liposome is 220nm, then use the polycarbonate membrane that aperture is 0.1 μ m, squeeze repeatedly 5 times under the condition of extrusion pressure 200Psi, detect the average particle diameter of blank liposome. Diameter is 130nm, then use the polycarbonate membrane that pore diameter is 0.05 μ m, squeeze repeatedly 5 times under the condition of extrusion pressure 120Psi, the average particle diameter of detection blank liposome is 62nm, and gained blank liposome is placed in 4 Store under the condition of ℃, spare;

C. Prepare sucrose solution

Weigh 0.85 kg of sucrose, set the volume to 10 liters, adjust the pH value to 5.0 with a 1mol/L HCl solution, and obtain a sucrose solution with a concentration of 8.5% and a pH value of 5.0 for subsequent use;

D. Blank liposome dialysis

Put the blank liposome obtained in step B into a dialysis bag, pour 3L of the sucrose solution obtained in step C into a 5L beaker in a constant temperature environment at 4°C, and then vertically suspend the dialysis bag containing the blank liposome solution in the In the beaker, start magnetic stirring at the same time, and dialyze for 4 hours. Repeat this, a total of 3 times of dialysis, each time replaced with new sucrose solution;

After the dialysis is completed, adjust the pH value of the blank liposome to 8.0 with 1mol/L NaOH solution, and set aside;

E. Preparation of daunorubicin hydrochloride solution

Accurately weigh 2.4g of daunorubicin hydrochloride in a safe operation cabinet, dissolve it in 90ml of the sucrose solution obtained in step C, after completely dissolving, adjust the pH value to 8.0 with 0.1mol/L NaOH solution, and finally add the obtained product in step C Dilute the sucrose solution to 100ml to obtain a daunorubicin hydrochloride solution for subsequent use;

F. Preparation of sucrose solution

Weigh 25.5g of sucrose, add 250ml of water to dissolve, adjust the pH value to 8.0 with 2mol/L NaOH solution, add water to make the volume to 300ml, and obtain a sucrose solution with a concentration of 8.5% and a pH value of 8.0, and set aside;

G. Preparation of daunorubicin citrate liposome solution

At 68-72°C, mix the dialyzed blank liposomes obtained in step D, the daunorubicin hydrochloride solution obtained in step E, and the sucrose solution obtained in 200ml of step F, and stir until the pH value is 5.6, then stop stirring. Obtain daunorubicin citrate liposome solution, cool to room temperature, and set aside;

H. Preparation of Glycine Sucrose Calcium Chloride Buffer

Weigh 76 g of sucrose, 3.76 g of glycine, and 0.28 g of calcium chloride dihydrate, set the volume to 1000 ml, adjust the pH value to 5.8 with 2 mol/L HCl solution, and obtain glycine sucrose calcium chloride buffer solution, and set aside;

I. prepare daunorubicin citrate liposome injection

Carry out the content determination of daunorubicin with high performance liquid phase, adopt the external standard method to calculate the content of daunorubicin in the liposome solution, be 4.56mg/ml, step G gained daunorubicin citrate liposome The volume of solution is 600ml, adds glycine sucrose calcium chloride buffer solution 640ml wherein, obtains the liposome solution that daunorubicin concentration is 2mg/ml, carries out aseptic filtration through the PES sterile filter membrane of 0.2 μm, promptly obtains Daunorubicin citrate liposome injection.

Nitrogen filled and divided into vials, capped, packaged, labeled, and put into storage.

Example 2

A. Prepare citric acid solution

Accurately weigh 43.2g of citric acid, pour it into 450ml of water and dissolve it. After the dissolution is complete, adjust the pH value of the solution to 5.0 with 1mol/L NaOH, add water to 500ml to obtain a concentration of 8.64%, and a pH value of 5.0. Citric acid solution, for later use;

B. Preparation of Blank Liposomes

Accurately weigh 37.65g of distearoylphosphatidylcholine and 9g of cholesterol, mix distearoylphosphatidylcholine and cholesterol with 42ml of absolute ethanol and 258ml of citric acid solution at 68-72°C, and stir After 1 hour, the lipid solution was obtained and set aside;

Under the condition of 68-72°C, put the lipid solution in a nano-extruder to extrude to prepare blank liposomes, first use a polycarbonate membrane with a pore size of 0.2 μm, and repeatedly extrude under the condition of an extrusion pressure of 600Psi Press 5 times, the average particle diameter of detection blank liposome is 200nm, then use the polycarbonate membrane that aperture is 0.1 μ m, squeeze repeatedly 5 times under the condition of extrusion pressure 300Psi, detect the average particle diameter of blank liposome. Diameter is 120nm, then use the polycarbonate membrane that aperture is 0.05 μ m, squeeze repeatedly 5 times under the condition of extrusion pressure 150Psi, the average particle diameter of detection blank liposome is 55nm, gained blank liposome is placed in 4 Store under the condition of ℃, spare;

C. Prepare sucrose solution

Weigh 0.85 kg of sucrose, add water to make the volume up to 10 liters, adjust the pH value to 5.0 with 1mol/L HCl solution, and obtain a sucrose solution with a concentration of 8.5% and a pH value of 5.0 for subsequent use;

D. Blank liposome dialysis

Put the blank liposome obtained in step B into a dialysis bag, pour 3L of the sucrose solution obtained in step C into a 5L beaker in a constant temperature environment at 4°C, and then vertically suspend the dialysis bag containing the blank liposome solution in the In the beaker, start magnetic stirring at the same time, and dialyze for 4 hours. Repeat this, a total of 3 times of dialysis, each time replaced with new sucrose solution;

After the dialysis is completed, adjust the pH value of the blank liposome to 8.0 with 1mol/L NaOH solution, and set aside;

E. Preparation of daunorubicin hydrochloride solution

Accurately weigh 2.4g of daunorubicin hydrochloride in a safe operation cabinet, dissolve it in 90ml of the sucrose solution obtained in step C, after completely dissolving, adjust the pH value to 8.0 with 0.1mol/L NaOH solution, and finally add the obtained product in step C Dilute the sucrose solution to 100ml to obtain a daunorubicin hydrochloride solution for subsequent use;

F. Preparation of sucrose solution

Weigh 25.5g of sucrose, add 250ml of water to dissolve, adjust the pH value to 8.0 with 2mol/L NaOH solution, add water to make the volume to 300ml, and obtain a sucrose solution with a concentration of 8.5% and a pH value of 8.0, and set aside;

G. Preparation of daunorubicin citrate liposome solution

At 68-72°C, mix the dialyzed blank liposomes obtained in step D, the daunorubicin hydrochloride solution obtained in step E, and the sucrose solution obtained in 200ml of step F, and stir until the pH value is 6.0, then stop stirring. Obtain daunorubicin citrate liposome solution, cool to room temperature, and set aside;

H. Preparation of Glycine Sucrose Calcium Chloride Buffer

Weigh 76 g of sucrose, 3.76 g of glycine, and 0.28 g of calcium chloride dihydrate, set the volume to 1000 ml, adjust the pH value to 5.8 with 2 mol/L HCl solution, and obtain glycine sucrose calcium chloride buffer solution, and set aside;

I. prepare daunorubicin citrate liposome injection

Carry out the content determination of daunorubicin with high performance liquid phase, adopt the external standard method to calculate the content of daunorubicin in the liposome solution, be 4.68mg/ml, step G gained daunorubicin citrate liposome The volume of solution is 600ml, adds glycine sucrose calcium chloride buffer solution 804ml wherein, obtains the liposome solution that daunorubicin concentration is 2mg/ml, carries out aseptic filtration through the PES sterile filter membrane of 0.2 μm, promptly obtains Daunorubicin citrate liposome injection.

Nitrogen filled and divided into vials, capped, packaged, labeled, and put into storage.

Example 3

The inventor has carried out quality inspection to daunorubicin citrate liposome injection obtained according to the preparation process described in the present invention, and its stability has been investigated, and the specific results are shown in Table 1.

Accelerated 6-month stability test conditions: T = 25 ° C, RH = 60%

Table 1

As can be seen from Table 1, the encapsulation efficiency of daunorubicin citrate liposome injection obtained according to the preparation process of the present invention is more than 97%, and the encapsulation efficiency does not significantly change after 6 months of accelerated test , more than 95%, other detection indicators are stable, indicating good product stability. Compared with the 90% encapsulation rate of commercially available products, the quality has been significantly improved.

Claims (7)

1. the preparation technology of daunorubicin citrate liposome injection is characterized in that, comprises the following operation steps: A. Prepare citric acid solution Prepare a citric acid solution with a concentration of 8.64% and a pH value of 5.0 for subsequent use; B. Preparation of Blank Liposomes At 68-72°C, mix 37.65 parts by weight of distearoylphosphatidylcholine, 9 parts by weight of cholesterol, 42 parts by volume of absolute ethanol with 258 parts by volume of the citric acid solution obtained in step A, and stir for 1 hour to obtain Lipid dissolving solution, spare; Under the condition of 68-72°C, put the lipid solution in a nano-extruder to extrude to prepare blank liposomes, first use a membrane with a pore size of 0.2 μm, and repeatedly extrude under the condition of an extrusion pressure of 450-600Psi The average particle diameter of the blank liposome is 200-220nm, and then the membrane with a pore diameter of 0.1 μm is repeatedly extruded to the average particle diameter of the blank liposome under the condition of extrusion pressure 200-300Psi is 120-130nm, and then Use a membrane with a pore size of 0.05 μm to repeatedly squeeze the blank liposomes at an extrusion pressure of 120-150 Psi until the average particle size of the blank liposomes is 55-62 nm, and store the blank liposomes at 4°C for future use. ; C. Prepare sucrose solution Prepare a sucrose solution with a concentration of 8.5% and a pH value of 5.0 for subsequent use; D. Blank liposome dialysis At 4°C, seal the blank liposomes prepared in step B in a dialysis bag, place them in the sucrose solution obtained in step C and dialyze for 4 hours, repeat this, and dialyze 3 times in total, replacing the sucrose solution each time; After the dialysis is completed, adjust the pH value of the blank liposome to 8.0 and set aside; E. Preparation of daunorubicin hydrochloride solution Weigh 2.4 parts by weight of daunorubicin hydrochloride, dissolve it in 90 parts by volume of the sucrose solution obtained in step C, then adjust the pH value to 8.0, and finally add the sucrose solution obtained in step C to settle to 100 parts by volume to obtain daunorubicin hydrochloride solution, spare; F. Preparation of sucrose solution Prepare a sucrose solution with a concentration of 8.5% and a pH value of 8.0 for subsequent use; G. Preparation of daunorubicin citrate liposome solution At 68-72°C, mix the dialyzed blank liposome obtained in step D, the daunorubicin hydrochloride solution obtained in step E, and 200 parts by volume of the sucrose solution obtained in step F, and stir until the pH value is 5.6-6.0 , stop stirring to obtain daunorubicin citrate liposome solution, cool to room temperature, and set aside; H. Preparation of Glycine Sucrose Calcium Chloride Buffer Each 100ml contains 7.6g sucrose, 0.376g glycine, 0.028 calcium chloride dihydrate, pH value is 5.8; I. prepare daunorubicin citrate liposome injection In step G gained daunorubicin citrate liposome solution, adding glycine sucrose calcium chloride buffer solution and diluting to the concentration of daunorubicin is 2mg/ml, gained daunorubicin citrate liposome dilution solution Sterile filtration is carried out through a 0.2 μm membrane to obtain daunorubicin citrate liposome injection. 2. the preparation technology of daunorubicin citrate liposome injection according to claim 1, is characterized in that, the described preparation citric acid solution of step A, uses the NaOH solution of 1mol/L to regulate pH value 5.0. 3. the preparation technology of daunorubicin citrate liposome injection according to claim 1 is characterized in that, the described preparation sucrose solution of step C, uses the HCl solution of 1mol/L to regulate pH value to be 5.0. 4. the preparation technology of daunorubicin citrate liposome injection according to claim 1 is characterized in that, the blank liposome dialysis described in step D uses the NaOH solution of 1mol/L to regulate blank lipid body pH to 8.0. 5. the preparation technology of daunorubicin citrate liposome injection according to claim 1 is characterized in that, the preparation daunorubicin hydrochloride solution described in step E, uses the NaOH solution adjustment of 0.1mol/L pH to 8.0. 6. the preparation technology of daunorubicin citrate liposome injection according to claim 1 is characterized in that, the described preparation sucrose solution of step F, uses the NaOH solution of 2mol/L to regulate pH value to be 8.0. 7. the preparation technology of daunorubicin citrate liposome injection according to claim 1 is characterized in that, the described preparation glycine sucrose calcium chloride buffer solution of step H, uses the HCl solution adjustment of 2mol/L The pH value is 5.8.