Preparation method of oxaliplatin for injection

Abstract

The present invention relates to a preparation method of oxaliplatin for injection, including: preparing a liposome solvent by a liposome preparation and an amphiphilic membrane stabilizer, preparing a chitosan solution, mixing the chitosan liposome micelle with the liposome solvent; mixing the chitosan liposome micelle with a free oxaliplatin solution to form an oxaliplatin polymer micelle in which oxaliplatin is encapsulated in the chitosan liposome micelle; freeze-drying to obtain oxaliplatin for injection. The present invention provides a method of making oxaliplatin exist in the form of polymer micelles by selecting a specific liposome preparation and an amphiphilic membrane stabilizer, greatly improving the cancer cell inhibition rate, delaying the drug release time, improving the circulation time of the drug in the blood, and ensuring storage stability.

Description

Technical Field

[0001] The invention relates to the technical field of oxaliplatin drug preparation, and in particular to a method for preparing oxaliplatin for injection. Background Art

[0002] Chemotherapy is an important method for treating cancer. However, most chemotherapeutic drugs lack specific biodistribution in the body, which leads to toxic side effects on normal cells and organs, thus limiting their clinical application. In addition, drug resistance and multidrug resistance of tumor cells have also become a very important factor leading to the failure of tumor chemotherapy.

[0003] Oxaliplatin was first developed by Debiopharm. It is a platinum metal complex and a new generation of platinum anti-tumor drugs after cisplatin and carboplatin. It does not have the renal toxicity of cisplatin, nor the bone marrow suppression of carboplatin. It binds to DNA rapidly, and its presence can be shown by measuring the adducts of white blood cells one hour after administration in the human body. The lyophilized powder injection currently used in clinical practice, however, during the use of oxaliplatin, due to its rapid metabolism in the body, lack of targeting, and instability in the free state of oxaliplatin, the drug degrades rapidly and has limited efficacy.

[0004] With the development of nanotechnology, attempts have been made to apply nanotechnology to pharmaceutical products. Liposomes are closed endoplasmic reticulum, which are formed by bilayer membranes with hydrophobic groups of phospholipids facing each other. Liposomes are composed of the same components as biological membranes and therefore have a high degree of biocompatibility. In addition, liposomes can carry both hydrophilic and hydrophobic drugs, because hydrophilic drugs can be contained in the endoplasmic reticulum, while hydrophobic drugs can be embedded in the liposome membrane. The physical properties of liposomes also determine that they can deliver drugs to tumors through blood circulation. Summary of the invention

[0005] In order to solve the shortcomings of the prior art, the present invention provides a method for preparing oxaliplatin for injection, comprising the following steps:

[0006] Step S1: accurately measure the liposome preparation, dissolve it in a chloroform solvent, stir and heat to 80 degrees Celsius to obtain a solution with a mass concentration of 45-55 g / L;

[0007] Step S2: Take low molecular weight chitosan, add deionized water to dissolve it, and the concentration is between 4-6g / L; and add the liposome solution obtained in step S1 under magnetic stirring, the volume ratio of the liposome solution to the chitosan solution is 1:5, and stir and react at 80 degrees Celsius for 5 hours. After cooling to room temperature, remove the chloroform solvent by vacuum distillation, and dialyze the reaction solution with distilled water for 3 days, freeze-dry the dialyzate, and wash the unreacted liposomes with anhydrous ethanol to obtain chitosan-liposome micelles;

[0008] Step S3: Add 90% of the total amount of water for injection into the preparation tank, then add the prescribed amount of oxaliplatin and lactose, stir until completely dissolved into a clear liquid, add more water for injection until the preparation tank is full, measure the pH value of the liquid to be between 4.5-6.5, in g / mL units, add 0.05% of the total amount of the preparation liquid, stir and adsorb for 30 minutes, and filter and remove carbon with a 3um titanium rod; the liquid is sterilized and filtered through 0.22um;

[0009] Step S4: weighing chitosan-liposome micelles at a ratio of 5 times the content of oxaliplatin in the oxaliplatin solution, adding the chitosan-liposome micelles to the oxaliplatin solution, and subjecting the chitosan-liposome micelles to ultrasonic treatment in a water bath at room temperature for 15 minutes to obtain an emulsion; then vortexing the chitosan-liposome micelles to convert the W / O emulsion into an O / W emulsion, thereby forming oxaliplatin polymer micelles in which oxaliplatin is encapsulated in the chitosan-liposome micelles;

[0010] Step S5: filling the oxaliplatin polymer micelles and freeze-drying them to obtain oxaliplatin for injection.

[0011] Wherein, in step S1, the liposome is selected from soybean phosphatidylcholine, egg yolk phospholipids or soybean lecithin.

[0012] Wherein, in the step S1, an amphiphilic membrane stabilizer is added at the same time, the amphiphilic membrane stabilizer is selected from cholesterol, stigmasterol or yeast sterol, and the concentration of the amphiphilic membrane stabilizer is one half of the liposome concentration.

[0013] Wherein, in the step S3, in the prepared oxaliplatin solution, the mass concentrations of oxaliplatin and lactose are respectively between 4.0-4.5 g / L and 35-40 g / L.

[0014] Among them, in the step S3, the prescription of the prepared oxaliplatin solution involves: dissolving 50g oxaliplatin and 450g lactose in 12000mL water for injection.

[0015] Wherein, in step S5, the freeze-drying step comprises:

[0016] Step S51: After the product enters the box, the shelf temperature is uniformly reduced to -40 degrees Celsius and kept warm for 1 hour, then the shelf temperature is raised to -20 degrees Celsius and kept warm for 2 hours. After the insulation is completed, the shelf temperature is further reduced to -45 degrees Celsius. After the product temperature reaches -35 degrees Celsius, it is kept warm for 6 hours.

[0017] Step S52, primary drying: quickly lower the condenser temperature to below -50 degrees Celsius, evacuate to below 10 Pa, raise the shelf temperature to between -14 and -16 degrees Celsius within 1.5 hours, start timing and keep warm for 17-19 hours, then quickly raise the shelf temperature to -5 to -3 degrees Celsius, keep warm until the ice crystals of the product completely disappear, and continue to keep warm for 4 hours;

[0018] Step S53, secondary drying: raise the shelf temperature to 15 degrees Celsius within 1 hour, keep it warm for 3 hours, then raise the shelf temperature to 40 degrees Celsius, and when the product temperature rises to 35 degrees Celsius, keep it warm for 12 hours and take it out of the box.

[0019] The preparation method of oxaliplatin for injection provided by the present invention enables oxaliplatin to exist in the form of polymer micelles through the selection of specific liposome preparations and amphiphilic membrane stabilizers, thereby greatly improving the cancer cell inhibition rate, delaying the drug release time, increasing the drug circulation time in the blood, and ensuring storage stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 :Effect of oxaliplatin polymer micelles prepared by different liposomes on the inhibition rate of colorectal cancer cells.

[0021] Figure 2 :Effect of oxaliplatin polymer micelles prepared by different liposomes on the inhibition rate of gastric cancer cells.

[0022] Figure 3 : Effects of different amphiphilic membrane stabilizers on the sustained release of prepared oxaliplatin polymer micelles. DETAILED DESCRIPTION

[0023] In order to have a further understanding of the technical solution and beneficial effects of the present invention, the technical solution and beneficial effects produced by the present invention are described in detail below with reference to the accompanying drawings.

[0024] 1. Selection of phospholipid membrane materials

[0025] Liposomes are closed endoplasmic reticulum formed by a bilayer membrane with hydrophobic groups of phospholipids facing each other. They have high biocompatibility and can transport oxaliplatin to specific tumor cells through blood circulation, thereby improving the targeting of the drug.

[0026] The phospholipid membrane materials commonly used in the prior art for preparing cancer drug targeting micelles include egg yolk phosphatidylcholine, soybean phosphatidylcholine, dipalmitoyl phosphatidylcholine, egg yolk lecithin, soybean lecithin, phosphatidylethanolamine and hydrogenated lecithin.

[0027] In the present invention, chitosan-phospholipid micelles coated with oxaliplatin are first prepared based on the above-mentioned phospholipid membrane materials, and the inhibition rate of the drug on several specific cancer cells under different micelles is verified respectively, so as to obtain the phospholipid membrane material most suitable for preparing oxaliplatin micelles.

[0028] The preparation method is as follows:

[0029] 1. Accurately measure 1 g of liposome preparation, dissolve it in 20 mL of chloroform solvent, stir and heat to 80 degrees Celsius;

[0030] 2. Take 0.5 g of low molecular weight chitosan, add 100 mL of deionized water to dissolve; add the liposome solution obtained in step 1 under magnetic stirring, stir and react at 80 degrees Celsius for 5 hours, cool to room temperature, remove the chloroform solvent by vacuum distillation, and dialyze the reaction solution with distilled water for 3 days, freeze-dry the dialyzate, and wash the unreacted liposomes with anhydrous ethanol to obtain chitosan-liposome micelles;

[0031] 3. Add 90% of the total amount of water for injection into the preparation tank, then add the prescribed amount of oxaliplatin and lactose, stir until completely dissolved into a clear liquid, add more water for injection until the preparation is complete, measure the pH value of the solution to be between 4.5-6.5, in g / mL units, add 0.05% of the total amount of the preparation of medicinal activated carbon, stir and adsorb for 30 minutes, filter and remove carbon with a 3um titanium rod; the solution is sterilized and filtered through 0.22um; a mass concentration of 4.2g / L oxaliplatin solution is obtained;

[0032] 4. Weigh chitosan-liposome micelles at a ratio of 5 times the oxaliplatin content in the oxaliplatin solution, add them to the oxaliplatin solution, and ultrasonicate them in a water bath at room temperature for 15 minutes to obtain an emulsion; then vortex treat to convert the W / O emulsion into an O / W emulsion to form oxaliplatin polymer micelles in which oxaliplatin is encapsulated in the chitosan-liposome micelles.

[0033] Investigate the effect of polymer micelles formed under different liposomes on the inhibition rate of specific cancer cells:

[0034] Colorectal cancer and gastric cancer were used as the effector cancer cells: the cells were cultured in RPMI-1640 culture medium containing 10% newborn calf serum in a culture flask, placed in a 37-degree Celsius incubator, and 5% carbon dioxide was passed through the culture medium. The cell growth status was observed. When the cells grew to near fusion, the old culture medium was aspirated, the cells were washed with PBS, and 0.3% trypsin was added for digestion. The cells were seeded at a density of 8000 / well in a 96-well culture plate, the temperature was adjusted to 37 degrees Celsius, 5% carbon dioxide was passed through, and cultured for 24 hours. The culture medium was aspirated, rinsed with PBS, and then fresh culture medium was added. Different concentrations of oxaliplatin polymer micelle solution and free oxaliplatin solution were added respectively, and 5 parallel wells were set for each concentration. A negative control group was also set up. The temperature was continued to be adjusted to 37 degrees Celsius, 5% carbon dioxide was passed through, and after culturing for 48 hours, 5 mg / mL was added to each well. 20uL of MTT aqueous solution was added, and the cells were cultured for another 4 hours. The culture medium in the wells was removed, and 100uL of DMSO was added to each well to dissolve the purple crystalline product. The cells were shaken at 37 degrees Celsius for half an hour, and the absorbance at 570nm was measured using an ELISA reader. Cell inhibition rate = 1-(absorbance of the experimental group / absorbance of the negative control group). The results are shown in Table 1. Figure 1 and Figure 2 The data involved are shown in Table 1 and Table 2.

[0035] Table 1: Effects of oxaliplatin polymer micelles prepared by different liposomes on the inhibition rate of colorectal cancer cells

[0036]

[0037] Table 2: Effects of oxaliplatin polymer micelles prepared by different liposomes on the inhibition rate of gastric cancer cells

[0038]

[0039] The results show that when egg yolk phosphatidylcholine, egg yolk phospholipids and soybean lecithin are used as liposomes, the prepared oxaliplatin polymer micelles have an excellent inhibition rate on colorectal cancer cells and gastric cancer cells.

[0040] 2. Amphiphilic membrane stabilizer

[0041] In order to stabilize and regulate the membrane fluidity of liposomes, in the above method for preparing oxaliplatin polymer micelles, an amphiphilic membrane stabilizer can be added simultaneously to combine with the liposomes to prevent them from agglomerating into a crystal structure, so that the chitosan-liposome micelles form a vesicle-type gel, encapsulate oxaliplatin therein, control the release rate of the drug, and prolong the systemic circulation time.

[0042] Common amphiphilic membrane stabilizers for regulating liposome membrane stability include cholesterol, sodium cholesterol phosphate, sodium cholesterol sulfonate, lanosterol, stigmasterol, brassicasterol, yeast sterol, etc. Soy lecithin is used as liposomes, and chitosan-liposome micelles are modified with the above stabilizers respectively; the method is as follows:

[0043] In the above method for preparing oxaliplatin polymer micelles, an amphiphilic membrane stabilizer is added simultaneously in step 1, and its concentration is half of that of soybean lecithin, and the remaining steps are the same.

[0044] The effects of different amphiphilic membrane stabilizers on the sustained release of oxaliplatin polymer micelles were verified by the following methods:

[0045] Thirty-six rats were randomly divided into 9 groups, with 4 rats in each group as parallel controls. They were injected with the above-mentioned oxaliplatin polymer micelles with different amphiphilic membrane stabilizers added, oxaliplatin polymer micelles without amphiphilic membrane stabilizers added, and free oxaliplatin solution, with a mass concentration of 1% and an injection volume of 50 mL. Blood was collected at 0.5h, 1h, 2h, 4h, 8h, 15h, and 20h after administration, and the platinum concentration in plasma was determined by atomic absorption spectrometry. The results are shown in Table 1. Figure 3 The data involved are shown in Table 3.

[0046] Table 3: Effects of different amphiphilic membrane stabilizers on the sustained release of prepared oxaliplatin polymer micelles

[0047]

[0048] The experimental results show that cholesterol, stigmasterol and yeast sterol, when used as amphiphilic membrane stabilizers, can have a good drug sustained-release effect on oxaliplatin and prolong the blood circulation time in the body.

[0049] 3. Long-term stability

[0050] Based on the liposomes and amphiphilic membrane stabilizers selected above, the final oxaliplatin lyophilized powder for injection was prepared, and the same method was used to verify its inhibition rate on cancer cells and its long-term stability under storage conditions.

[0051] Embodiment 1:

[0052] 1. Accurately measure 1g of soybean phosphatidylcholine and 0.5g of cholesterol, dissolve them in 20mL of chloroform solvent, stir and heat to 80 degrees Celsius;

[0053] 2. Take 0.5 g of low molecular weight chitosan, add 100 mL of deionized water to dissolve; add the liposome solution obtained in step 1 under magnetic stirring, stir and react at 80 degrees Celsius for 5 hours, cool to room temperature, remove the chloroform solvent by vacuum distillation, and dialyze the reaction solution with distilled water for 3 days, freeze-dry the dialyzate, and wash the unreacted liposomes with anhydrous ethanol to obtain chitosan-soybean phosphatidylcholine micelles;

[0054] 3. Add 90% of the total amount of water for injection into the preparation tank, then add the prescribed amount of oxaliplatin and lactose, stir until completely dissolved into a clear liquid, add more water for injection until the preparation is complete, measure the pH value of the solution to be between 4.5-6.5, in g / mL units, add 0.05% of the total amount of the preparation of medicinal activated carbon, stir and adsorb for 30 minutes, filter and remove carbon with a 3um titanium rod; the solution is sterilized and filtered through 0.22um; a mass concentration of 4.2g / L oxaliplatin solution is obtained;

[0055] 4. Weigh chitosan-liposome micelles at a ratio of 5 times the oxaliplatin content in the oxaliplatin solution, add them to the oxaliplatin solution, and ultrasonicate them in a water bath at room temperature for 15 minutes to obtain an emulsion; then vortex treat to convert the W / O emulsion into an O / W emulsion to form oxaliplatin polymer micelles in which oxaliplatin is encapsulated in the chitosan-liposome micelles.

[0056] 5. Freeze drying:

[0057] Pre-freezing: After the product enters the box, the shelf temperature is uniformly reduced to -40 degrees Celsius and kept warm for 1 hour. The shelf temperature is then raised to -20 degrees Celsius and kept warm for 2 hours. After the insulation is completed, the shelf temperature is further reduced to -45 degrees Celsius. After the product temperature reaches -35 degrees Celsius, it is kept warm for another 6 hours.

[0058] Primary drying: quickly lower the condenser temperature to below -50 degrees Celsius, evacuate to below 10 Pa, raise the shelf temperature to between -14 and -16 degrees Celsius within 1.5 hours, start timing and keep warm for 17-19 hours, then quickly raise the shelf temperature to -5 to -3 degrees Celsius, keep warm until the ice crystals of the product completely disappear, and continue to keep warm for 4 hours;

[0059] Secondary drying: Raise the shelf temperature to 15 degrees Celsius within 1 hour, keep it warm for 3 hours, then raise the shelf temperature to 40 degrees Celsius. When the product temperature rises to 35 degrees Celsius, keep it warm for 12 hours and take it out of the box.

[0060] Embodiment 2:

[0061] The liposome and the amphiphilic membrane stabilizer are selected from egg yolk phospholipids and stigmasterol respectively, and the remaining steps are the same as those in Example 1.

[0062] Embodiment 3:

[0063] The liposome and the amphiphilic membrane stabilizer are selected from soybean lecithin and yeast sterol respectively, and the remaining steps are the same as those in Example 1.

[0064] Embodiment 4:

[0065] In step 1, the added amounts of liposome and amphiphilic membrane stabilizer are 0.9 g and 0.45 g respectively, and the remaining steps are the same as in Example 1.

[0066] Embodiment 5:

[0067] In step 2, the amount of chitosan added is 0.4 g. In step 3, the mass concentration of the prepared oxaliplatin solution is 4.5 g / L. The remaining steps are the same as in Example 2.

[0068] Embodiment 6:

[0069] In step 1, the added amounts of liposome and amphiphilic membrane stabilizer are 1.1 g and 0.55 g respectively, and the remaining steps are the same as in Example 1.

[0070] At the same time, we purchased two reagents that were already on the market. We placed them both in a high temperature environment of 50 degrees Celsius and observed the accelerated test results within 6 months. The results are as follows:

[0071] Table 4: Accelerated test results of oxaliplatin provided by the present invention and commercially available oxaliplatin

[0072]

[0073]

[0074] As can be seen from the above table, the oxaliplatin for injection provided by the present invention, through the selection of specific liposomes and amphiphilic membrane stabilizers, not only improves the cancer cell inhibition rate and improves the drug sustained-release effect, but also has a high degree of stability compared to existing commercially available products. After being stored at high temperature for 6 months, the effective ingredients and impurities are almost unchanged.

[0075] The preparation method of oxaliplatin for injection provided by the present invention enables oxaliplatin to exist in the form of polymer micelles through the selection of specific liposome preparations and amphiphilic membrane stabilizers, greatly improves the cancer cell inhibition rate of colorectal cancer cells and gastric cancer cells, delays the drug release time, increases the circulation time of the drug in the blood, and ensures storage stability.

[0076] Although the present invention has been described using the above preferred embodiments, they are not intended to limit the scope of protection of the present invention. Any person skilled in the art may make various changes and modifications to the above embodiments without departing from the spirit and scope of the present invention. The scope of protection of the present invention shall be based on the definition of the claims.

Claims

1. A method for preparing oxaliplatin for injection, characterized in that: The steps include: Step S1: accurately measure the liposome preparation, dissolve it in chloroform solvent, stir and heat to 80 degrees Celsius to prepare a liposome solution with a mass concentration of 45-55 g / L; Step S2: Take low molecular weight chitosan, add deionized water to dissolve it, and the concentration is between 4-6g / L; and add the liposome solution obtained in step S1 under magnetic stirring, the volume ratio of the liposome solution to the chitosan solution is 1:5, and stir and react at 80 degrees Celsius for 5 hours. After cooling to room temperature, remove the chloroform solvent by vacuum distillation, and dialyze the reaction solution with distilled water for 3 days, freeze-dry the dialyzate, and wash the unreacted liposomes with anhydrous ethanol to obtain chitosan-liposome micelles; Step S3: Add 90% of the total amount of water for injection into the preparation tank, then add the prescribed amount of oxaliplatin and lactose, stir until completely dissolved into a clear liquid, add water for injection to the total amount of the preparation, measure the pH value of the liquid to be between 4.5-6.5, in g / mL units, add 0.05% of the total amount of the preparation of medicinal activated carbon, stir and adsorb for 30 minutes, and filter with a 3um titanium rod to remove carbon; the liquid is sterilized and filtered through 0.22um; Step S4: weighing chitosan-liposome micelles at a ratio of 5 times the content of oxaliplatin in the oxaliplatin solution, adding the chitosan-liposome micelles to the oxaliplatin solution, and subjecting the chitosan-liposome micelles to ultrasonic treatment in a water bath at room temperature for 15 minutes to obtain an emulsion; then vortexing the chitosan-liposome micelles to convert the W / O emulsion into an O / W emulsion, thereby forming oxaliplatin polymer micelles in which oxaliplatin is encapsulated in the chitosan-liposome micelles; Step S5: filling the oxaliplatin polymer micelles, and freeze-drying them to obtain oxaliplatin for injection; In step S1, the liposome is selected from egg yolk phosphatidylcholine, egg yolk phospholipids or soybean lecithin; In the step S1, an amphiphilic membrane stabilizer is added at the same time. The amphiphilic membrane stabilizer is selected from cholesterol, stigmasterol or yeast sterol, and the concentration of the amphiphilic membrane stabilizer is half of the liposome concentration.

2. The method for preparing oxaliplatin for injection according to claim 1, characterized in that: In the step S3, in the prepared oxaliplatin solution, the mass concentrations of oxaliplatin and lactose are respectively between 4.0-4.5 g / L and 35-40 g / L.

3. The method for preparing oxaliplatin for injection according to claim 1, characterized in that: In the step S3, the oxaliplatin solution prepared has a prescription involving: dissolving 50 g of oxaliplatin and 450 g of lactose in 12000 mL of water for injection.

4. The method for preparing oxaliplatin for injection according to claim 1, characterized in that: In step S5, the freeze-drying step includes: Step S51: After the product enters the box, the shelf temperature is uniformly reduced to -40 degrees Celsius and kept warm for 1 hour, then the shelf temperature is raised to -20 degrees Celsius and kept warm for 2 hours. After the insulation is completed, the shelf temperature is further reduced to -45 degrees Celsius. After the product temperature reaches -35 degrees Celsius, it is kept warm for 6 hours; Step S52, primary drying: quickly lower the condenser temperature to below -50 degrees Celsius, evacuate to below 10 Pa, raise the shelf temperature to between -14 and -16 degrees Celsius within 1.5 hours, start timing and keep warm for 17-19 hours, then quickly raise the shelf temperature to -5 to -3 degrees Celsius, keep warm until the ice crystals of the product completely disappear, and continue to keep warm for 4 hours; Step S53, secondary drying: raise the shelf temperature to 15 degrees Celsius within 1 hour, keep it warm for 3 hours, then raise the shelf temperature to 40 degrees Celsius, and when the product temperature rises to 35 degrees Celsius, keep it warm for 12 hours and take it out of the box.

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