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    • 专利摘要:
    LIPOSOME AND IRINOTECAN OR ITS CHLORIDATE AND ITS METHOD OF PREPARATION. The present invention relates to an irinotecan liposome or its hydrochloride and its method of preparation. The liposome comprises irinotecan or its hydrochloride, neutral phoslolipid and cholesterol, where the weight ratio of cholesterol to neutral phospholipid is 1: 3-5. Said liposome is prepared by the ion gradient method.
    公开号:BR112012012151B1
    申请号:R112012012151-0
    申请日:2009-12-03
    公开日:2021-01-19
    发明作者:Xinyong Tong；Guofeng Lei；Chengxia Yu；Liang Chen
    申请人:Jiangsu Hengrui Medicine Co., Ltd.；Shanghai Hengrui Pharmaceutical Co., Ltd.；
    IPC主号:
    专利说明:

    FIELD OF THE INVENTION
    [0001] The present invention relates to an irinotecan liposome or its hydrochloride and its method of preparation and an injection comprising said liposome and its method of preparation. BACKGROUND OF THE INVENTION
    [0002] Irinotecan is a semi-synthetic derivative of camptothecin. Camptothecin can specifically bind topoisomerase I, which can induce reversible ruptures of single strands of DNA and then unwind the double stranded structure of DNA. Irinotecan and its active metabolite SN-38 can bind to the topoisomerase I-DNA complex, thus preventing the reconnection of the simple strand fracture. It has been proved that the cytotoxicity of irinotecan can be attributed to the interaction of replicase and triple topoisomerase I-DNA-irinotecan (or SN-38) complexes, which break the double strand of DNA in DNA synthesis.
    [0003] Irinotecan hydrochloride is widely used in the treatment of malignant tumors, with the advantages of obvious pharmacological effects and clinical efficacy. However, it has the same problem as other camptothecin derivatives: the saturated lactone ring in the irinotecan structure is pH dependent and can be reversibly transformed into its carboxylated form under physiological conditions, with which the anti-tumor activity is weakened. The existing commercial formulations of irinotecan hydrochloride are liquid injection and lyophilized powder for injection. After intravenous administration at the clinic, the free drug will lose its activity, because the lactone ring in its structure is susceptible to being hydrolyzed in the carboxylate form in the alkaline physiological environment, thereby indirectly reducing efficacy, and these formulations have serious side effects. , which are mainly neutropenia and delayed diarrhea.
    [0004] The liposome has been widely studied as a vehicle for drugs in recent years. The main characteristics of the liposome include the protection of the encapsulated drug, increasing the stability of the drug, the alteration of the in vivo distribution behavior of the drug and the transport of the drug to the diseased region by passive or active targeting. As a good anti-cancer drug carrier, the liposome can improve the effectiveness of the drug and reduce the toxicity of the drug.
    [0005] International application WO2005 / 117878 has presented a liposome formulation of irinotecan and its method of preparation. This formulation comprises irinotecan or irinotecan hydrochloride, a phospholipid selected from the group consisting of hydrogenated soy phosphatidylcholine, phosphatidylethanolamine, lecithin and cardiolipin, and cholesterol. Likewise, Chinese patent application CN1994279A also disclosed an irinotecan liposome formulation and its method of preparation, in which phosphatidylcholine is used as a phospholipid to prepare a liposome in Example 5.
    [0006] The formulations mentioned in the patent literature above can have good effects. However, when these formulations are used in humans, the stability, particle size and others are still not satisfactory. DESCRIPTION OF THE INVENTION
    [0007] The present invention features an irinotecan or irinotecan hydrochloride liposome, which has greater drug loading capacity, high encapsulation efficiency, good stability and is suitable to be prepared in a formulation.
    [0008] Until now, some literature (for example, international application WO2005 / 117878 and CN1994279A) has described the composition and methods of preparing the irinotecan liposome. In some formulations, some indexes have had good results. However, there is no information about the stability and control of particle size. After further study of the liposome, we were surprised to find that the amount of cholesterol in particular had an impact on the particle size and stability of the liposome when the selected type of inactive ingredient and the amount used in the formulation met some conditions. We have successfully prepared a liposome with a small and uniform particle size distribution and improved its stability by controlling the ratio between neutral phospholipid and cholesterol. Compared to other formulations, the liposome of the present application has greater storage stability, and other indicators have also improved significantly. In addition, compared to the technologies described in international application WO2005 / 117878 and CN1994279A, these products do not comprise a compound with a basic functional group and a cationic lipid. And the liposome of the present invention has a good anti-tumor effect and some advantages of simple formulation, high drug loading capacity and good storage stability.
    [0009] The liposome of the present invention comprises irinotecan or irinotecan hydrochloride, a neutral phospholipid and cholesterol, and the weight ratio of cholesterol to neutral phospholipid is 1: 3 ~ 5, preferably 1: 3.5 ~ 4.5 , most preferably 1: 4.
    [00010] The neutral phospholipid used in the present invention is selected from the group consisting of hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), soy phosphatidylcholine (SPC) and others. The effect becomes the best when using hydrogenated soy phosphatidylcholine as a neutral phospholipid. The drug loading capacity of the liposome can be greatly improved when the weight ratio of the drug to the phospholipid is further adjusted as follows: Irinotecan or irinotecan hydrochloride 1 neutral phospholipid 2 ~ 5, preferably 2.5-4.
    [00011] The liposome of the present invention can be prepared by liposome preparation methods conventional in the art, preferably by an ion gradient method. When using the ionic gradient method, there is an ionic gradient formed by a buffer between the internal aqueous phase and the external aqueous phase of said liposome. Preferably, the internal aqueous phase of said liposome has a higher concentration of ions than the external aqueous phase, which can improve the stability of the liposome particle size during the storage period, maintain a better efficacy of the drug and allow to control a size of small and uniform medium particle of the liposome, allows the change in the particle size of the liposome to be reduced to a minimum during the storage period.
    [00012] In the present invention, the change in particle size of the liposome during the storage period can be reduced to a minimum by adding a hydrophilic polymer lipid derivative to the formulation. And the liposome cycle time in vivo can be extended by adding a polyethylene glycol derivative to the formulation. The polyethylene glycol derivative is selected from the group consisting of polyethylene glycol 2000-diestearoyl phosphatidyl ethanolamine (DSPE-PEG2000), polyethylene glycol 5000-diestearoyl phosphatidyl ethanolamine, polyethylene glycol 2000-dipalmitoyl phosphatidyl ethanolamine, polyethylene glycol 5000-dipal. To improve the long-term efficacy of the drug, a hydrophilic polymer lipid derivative is preferably added to the liposome in the present invention. Based on this formulation ratio, DSPE-PEG2000 has the most obvious effect. The preferred weight ratio of the lipid derivative to irinotecan or irinotecan hydrochloride is 0.2 ~ 0.4.
    [00013] The liposome can also comprise a charged phospholipid selected from the group consisting of dilauroyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, distearoyl phosphatidyl glycerol, phosphatidylglycerol acid, dimiristate phosphatidyl acid, dioleic acid, dioleyl acid, dioleyl acid a mixture of these, and the weight ratio of the charged phospholipid to the neutral phospholipid is 1: 5 ~ 1: 100.
    [00014] Preferably, the liposome of the present invention comprises the following ratios by weight of ingredients: irinotecan hydrochloride 1 hydrogenated soy phosphatidylcholine 3.4-3.8 polyethylene glycol 2000-distearoyl phosphatidyl ethanolamine 0.34-0.38 cholesterol 0.8-0.95 and the cholesterol to hydrogenated soy phosphatidylcholine ratio is 1: 4.
    [00015] The present invention also provides a method of preparing the irinotecan liposome or irinotecan hydrochloride. The liposome of the present invention can be prepared by a conventional method of preparing liposomes. Those skilled in the art can choose various methods to prepare the liposome according to the formulation presented by the present invention. For the formulation of the liposome in the present invention, the method of preparation by an ionic gradient is preferably selected. The preparation method comprises the following steps: 1) preparation of an empty liposome by any of the following methods A to D: A. dissolution of a neutral phospholipid and cholesterol in anhydrous ethanol or a mixed solvent of anhydrous ethanol and tertiary alcohol butyl according to the desired formulation, mix the mixture with a buffer to obtain a crude empty liposome after removal of ethanol by distillation under reduced pressure and then prepare an empty liposome with the desired particle size using a homogenizer high pressure and / or extrusion equipment; B. dissolution of a neutral phospholipid and cholesterol in chloroform or a mixed solvent of chloroform-methanol according to the desired formulation, formation of a lipid film by means of a rotary evaporator, addition of a hydration buffer to obtain a crude and empty liposome , then, preparation of an empty liposome with the desired particle size using a high pressure homogenizer and / or extrusion equipment; C. mixing a neutral phospholipid, cholesterol and a buffer according to the desired formulation, then preparing an empty liposome with the desired particle size using a high pressure homogenizer and / or extrusion equipment; D. dissolution of a neutral phospholipid and cholesterol in anhydrous ethanol or a mixed solvent of anhydrous ethanol and tert-butyl alcohol according to the desired formulation, mix the mixture with a buffer and then prepare an empty liposome the size of desired particle using a high pressure homogenizer and / or extrusion equipment; 2) formation of an ionic gradient between the internal aqueous phase and the external aqueous phase of the empty liposome: replacement of the external aqueous phase of the empty liposome to form an ionic gradient between the internal aqueous phase and the external aqueous phase of the empty liposome; 3) preparation of a drug-loaded liposome: preparation of an aqueous solution of irinotecan hydrochloride, adding it to the empty liposome dispersion with an ionic gradient and then incubating the dispersion to obtain the drug-loaded liposome under heating and agitation.
    [00016] After said step 3) of preparing a drug-loaded liposome, said method can also comprise the following step of: 4) removing the free drug and concentrating the sample: adding a buffer medium to the hydrochloride liposome irinotecan, removal of the non-encapsulated drug using a tangential flow device and concentration of the sample in the appropriate volume.
    [00017] The present invention also features a liposome injection comprising the above liposome. When preparing a liposome in an injection suitable for human use, it is useful to add a stabilizer. The stabilizer used in the present invention can be a conventional stabilizer, such as vitamin E, ethylene diamine tetraacetic acid and the like. The stabilizer is useful for the stability of the formulation. For the formulation described above, the study of the stabilizer shows that ethylene diamine tetraacetic acid or its salt has the best effect compared to other stabilizers. They are useful for improving the stability of the liposome. Thus, the stabilizer may be ethylene diamine tetraacetic acid, ethylene diamine tetraacetic acid disodium and ethylene diamine tetraacetic acid dicalcium or a mixture thereof. The ratio of the added stabilizer is 0 ~ 0.5% (w / v), and the minimum is not 0%.
    [00018] The composition of the present invention preferably comprises an antioxidant selected from the group consisting of water-soluble antioxidant and oil-soluble antioxidant, wherein said oil-soluble antioxidant is selected from the group consisting of α-tocopherol, α-tocopherol succinate, α-tocopherol acetate and a mixture of these, in which said water-soluble antioxidant is selected from the group consisting of ascorbic acid, sodium bisulfite, sodium sulfite, sodium pyrosulfite, L-cysteine and a mixture of these. The ratio of the added antioxidant is 0 ~ 0.5% (w / v), and the minimum is not 0%.
    [00019] The injection may be in the form of a lyophilized liquid or powder for injection. The formulation may comprise an osmotic pressure regulator selected from the group consisting of glucose, sucrose, sorbitol, mannitol, sodium chloride, glycerin, histidine and its hydrochloride, glycine and its hydrochloride, lysine, serine, glutamic acid, arginine, valine and a mixture of these. The ratio of the added osmotic pressure regulator is 0 ~ 5% (w / v), and the minimum is not 0%.
    [00020] In a formulation in the form of lyophilized powder for injection, the injection also comprises a lyoprotectant and then the injection is prepared with the lyophilized powder for injection after lyophilization. The lyoprotectant is selected from the group consisting of glucose, sucrose, trehalose, mannitol, dextran, lactose and a mixture of these.
    [00021] The preferred liposome injection of the present invention comprises the following weight ratio of ingredients: irinotecan hydrochloride 1 hydrogenated soy phosphatidylcholine 3.4-3.8 polyethylene glycol 2000 - distearoyl phosphatidyl ethanolamine 0.34-0.38 cholesterol 0.8-0.95 ethylene diamine tetraacetic disodium acid 0.05-0.09 and the ratio of cholesterol to hydrogenated soy phosphatidylcholine is 1: 4.
    [00022] The injection preparation method described above comprises the following steps: 1) preparation of an empty liposome by any of the following methods A to D: A. dissolving a neutral phospholipid and cholesterol in anhydrous ethanol or a mixed solvent of anhydrous ethanol and tert-butyl alcohol according to the desired formulation, mix the mixture with a buffer to obtain a crude empty liposome after removing the ethanol by distillation under reduced pressure and then preparing an empty liposome the size of desired particle using a high pressure homogenizer and / or extrusion equipment; B. dissolution of a neutral phospholipid and cholesterol in chloroform or a mixed chloroform-methanol solvent according to the desired formulation, formation of a lipid film by means of a rotary evaporator, addition of a hydration buffer to obtain a crude empty liposome and , then, preparation of an empty liposome with the desired particle size using a high pressure homogenizer and / or extrusion equipment; C. mixing a neutral phospholipid, cholesterol and a buffer according to the desired formulation, then preparing an empty liposome with the desired particle size using a high pressure homogenizer and / or extrusion equipment; D. dissolution of a neutral phospholipid and cholesterol in anhydrous ethanol or a mixed solvent of anhydrous ethanol and tert-butyl alcohol according to the desired formulation, mix the mixture with a buffer and then prepare an empty liposome the size of desired particle using a high pressure homogenizer and / or extrusion equipment; 2) formation of an ionic gradient between the internal aqueous phase and the external aqueous phase of the empty liposome: replacement of the external aqueous phase of the empty liposome to form an ionic gradient between the internal aqueous phase and the external aqueous phase of the empty liposome; 3) preparation of a drug-loaded liposome: preparation of an aqueous solution of irinotecan hydrochloride, adding it to the empty liposome dispersion with an ionic gradient and then incubating the dispersion to obtain the drug-loaded liposome under heating and agitation.
    [00023] After said step 3) of preparing a drug-loaded liposome, said method may also comprise the following step of: 4) removing the free drug and concentrating the sample: adding a buffer medium to the hydrochloride liposome irinotecan, removal of the non-encapsulated drug using a tangential flow device and concentration of the sample in the appropriate volume.
    [00024] After obtaining the liposome, the drug concentration is adjusted by diluting the measured volume; the liposome is sterilized by filtration, then filled and closed to obtain the liposome injection. Or, after adding a lyoprotectant to the drug sample in liposome, the drug concentration is adjusted by diluting the measured volume, the liposome is sterilized by filtration, then filled, closed and lyophilized to obtain the freeze-dried liposome powder for injection.
    [00025] Beneficial effects of the present invention:
    [00026] The liposome formulation of irinotecan or irinotecan hydrochloride has overcome many deficiencies in existing products and technologies. Drug stability can be improved by encapsulating drugs in the internal aqueous phase of the liposome. Since the drug is in the form of a lactone ring in vivo, the concentration of the active metabolite SN-38 is maintained for a long time in the plasma. Generically, the irinotecan liposome formulation or irinotecan hydrochloride can increase the effectiveness of the formulation and reduce the side effects of the drugs.
    [00027] The irinotecan or irinotecan hydrochloride liposome formulation of the present invention solved the problem of low drug loading capacity in the liposome by controlling the specific ratio between the drug, phospholipid and cholesterol. The drug to lipid ratio in the liposome injection is above 0.25 (w / w), and the encapsulation efficiency is above 90%, preferably above 95%. The liposome prepared by the present invention has a smaller particle size and improves stability by optimizing the dosage of cholesterol and phospholipid. By screening the stabilizer, a certain percentage of salts of ethylene diamine tetra-acetic acid is preferably added to the formulation to significantly improve the stability of the liposome, and the particle size distribution of the liposome is uniformly in the range of 10 nm ~ 220 nm. The results of the influencing factor experiment of irinotecan or irinotecan hydrochloride injection show that the particle size and encapsulation efficiency of the sample have no significant change when placed at 40 ° C for 10 days, and all indices meet the requirements. Compared to commercially available formulations, the injection of irinotecan liposome or irinotecan hydrochloride has a significant increase in the rate of tumor inhibition and a significant reduction in its toxicity. BRIEF DESCRIPTION OF THE DRAWINGS
    [00028] Figure 1 shows the particle size distribution of the irinotecan liposome injection or irinotecan hydrochloride according to the present invention.
    [00029] Figure 2 shows the morphology of the injection of irinotecan liposome or irinotecan hydrochloride according to the present invention.
    [00030] Figure 3 shows the results of the in vivo anticancer effect test of the injection of irinotecan liposome or irinotecan hydrochloride according to the present invention. PREFERRED EMBODIMENTS
    [00031] The following examples are intended to illustrate the invention in more detail, but are in no way intended to limit its scope. EXAMPLE 1 Formulation:
    Preparation method:
    [00032] Hydrogenated soy phosphatidylcholine (HSPC) and cholesterol (CHOL) of the formulation amount were dissolved in an appropriate amount of anhydrous ethanol, the resulting lipid solution was mixed with an ammonium sulfate solution (100 mL), the ethanol was removed by distillation with reduced pressure, and then the crude empty liposome is obtained. After 5 homogenization cycles in the high pressure homogenizer (100 MPa (1,000 bar)), the particle size of the liposome was controlled by extrusion of the liposome in the extrusion equipment (two extrusion membranes of 0.1 μm in the extrusion equipment, extrusion five times) and then the aqueous solution of DSPE-PEG2000 was added. With stirring, the mixture was incubated for 20 minutes. The empty liposome was dialyzed using an ultrafiltration device by tangential flow with continuous supplementation of water for injection in progress; then, the empty liposome was finally obtained.
    [00033] The aqueous solution of irinotecan hydrochloride was prepared with water for injection and was added to the dispersion of empty liposomes with the ionic gradient above according to the weight ratio of irinotecan hydrochloride to HSPC of 1: 3.5. With stirring, the mixture was heated to 60 ° C and incubated for 20 minutes and then the drug loaded liposome was obtained. The non-encapsulated drug was removed using a tangential flow ultrafiltration device. 0.45 g of sodium chloride was added to adjust the osmotic pressure after the sample was concentrated to about 50 mL. After adjusting the drug concentration by diluting the measured volume, the liposome was sterilized by filtration with a 0.22 μm filter, filled under nitrogen protection and sealed in a small bottle. The liposome injection of irinotecan hydrochloride was finally obtained.
    [00034] The change in the particle size of each formulation was shown in the table below. The results indicated that the particle size of the sample was the smallest when the weight ratio of phospholipid to cholesterol was 4: 1.

    [00035] The stability of the prepared sample was investigated at 25 ° C at various weight ratios of phospholipid to cholesterol. The results were shown in the table below. After storage at 25 ° C for 60 days, the particle size and encapsulation efficiency of the sample did not change significantly when the weight ratio of phospholipid to cholesterol was 4: 1. However, for samples with other weight-to-cholesterol phospholipid ratios, the sample size increased, and the encapsulation efficiency declined. Consequently, the stability of the sample was best when the weight ratio of phospholipid to cholesterol was 4: 1.

    [00036] Conclusions:
    [00037] Taking all indices into account, better results can be obtained when the cholesterol to phospholipid ratio is 1: 3 ~ 5, more preferably 1: 4. EXAMPLE 2 Formulation:

    [00038] Preparation method:
    [00039] Hydrogenated soy phosphatidylcholine and cholesterol of the formulation amount were dissolved in an appropriate amount of anhydrous ethanol, the resulting lipid solution was mixed with an ammonium sulfate solution (100 mL), the anhydrous ethanol was removed by pressure distillation reduced and then the crude empty liposome is obtained. After 5 homogenization cycles in the high pressure homogenizer (100 MPa (1,000 bar)), the particle size of the liposome was controlled by extrusion of the liposome in the extrusion equipment (two extrusion membranes of 0.1 μm in the extrusion equipment, extrusion five times) and then the aqueous solution of DSPE-PEG2000 was added. With stirring, the mixture was incubated for 20 minutes. The empty liposome was dialyzed using an ultrafiltration device by tangential flow with continuous supplementation of water for injection in progress; then, the empty liposome was finally obtained.
    [00040] The aqueous solution of irinotecan hydrochloride was prepared with water for injection and was added to the dispersion of empty liposomes with the ionic gradient above according to the weight ratio of irinotecan hydrochloride to HSPC of 1: 3.5. With stirring, the mixture was heated to 60 ° C and incubated for 20 minutes and then the drug loaded liposome was obtained. The non-encapsulated drug was removed using a tangential flow ultrafiltration device. 0.45 g of sodium chloride was added to adjust the osmotic pressure after the sample was concentrated to about 50 mL. After adjusting the drug concentration by dilution to a constant volume, the liposome was sterilized by filtration with a 0.22 μm filter, filled under nitrogen protection and sealed in a small bottle. The liposome injection of irinotecan hydrochloride was finally obtained. EXAMPLE 3
    [00041] The formulation and method of preparing the empty liposome were the same as in Example 2, except that the weight ratio of irinotecan hydrochloride to HSPC was 1: 1.5, 1: 2, 1: 3.5, 1: 4 and 1: 5 in the liposome preparation process. The encapsulation efficiency and particle size of the irinotecan hydrochloride liposome sample were shown in the table below:

    [00042] Encapsulation efficiency has been shown to decrease significantly when the weight ratio of irinotecan hydrochloride to HSPC was 1: 1.5, and the drug loading content decreased markedly when the ratio was 1: 5. Both conditions are unsuitable for the preparation of formulations used in clinical application. The encapsulation efficiency and drug loading content were higher when the ratio was 1: 2 ~ 1: 4. EXAMPLE 4
    [00043] The formulation and method of preparing empty liposome and drug loaded liposome were the same as in Example 2, except that the HSPC in the formulation was replaced by high-purity egg phosphatidylcholine (EPC) and high-grade soy phosphatidylcholine purity (SPC), respectively. The stability of the resulting liposome sample was investigated at 25 ° C, and the results were shown in the table below. The test results showed that the stability of the liposome sample prepared with HSPC was the best, and that the main indices had no marked change when stored at 25 ° C for 2 months.
    EXAMPLE 5 Formulation:

    [00044] Preparation method 1:
    [00045] Ethanol injection method: hydrogenated soy phosphatidylcholine, DSPE-PEG2000 and cholesterol from the formulation quantity were dissolved in an appropriate amount of anhydrous ethanol, the resulting lipid solution was injected in a saline solution of irinotecan hydrochloride. Ethanol was removed by distillation under reduced pressure, and then the crude empty liposome was obtained. The particle size of the liposome was controlled by extrusion of the liposome in the extrusion equipment (two extrusion membranes of 0.1 μm in the extrusion equipment, extrusion five times) after 5 homogenization cycles in the high pressure homogenizer (100 MPa (1,000 Pub)). The drug concentration was adjusted by diluting the measured volume, the liposome was sterilized by filtration with a 0.22 μm filter, filled under nitrogen protection and sealed in a small bottle. The liposome injection of irinotecan hydrochloride was finally obtained.
    [00046] Preparation method 2:
    [00047] Film dispersion method: hydrogenated soy phosphatidylcholine, DSPE-PEG2000 and cholesterol from the formulation amount were dissolved in an appropriate amount of chloroform, and the resulting lipid solution was prepared as a film by the rotary evaporator, then the chloroform was removed. Irinotecan hydrochloride saline was added, and the mixture was incubated for 1h. The particle size of the liposome was controlled by extrusion of the liposome in the extrusion equipment (two extrusion membranes of 0.1 μm in the extrusion equipment, extrusion five times) after 5 homogenization cycles in the high pressure homogenizer (100 MPa (1,000 Pub)). The drug concentration was adjusted by diluting the measured volume, the liposome was sterilized by filtration with a 0.22 μm filter, filled under nitrogen protection and sealed in a small bottle. The liposome injection of irinotecan hydrochloride was finally obtained.
    [00048] The encapsulation efficiency and particle size of the irinotecan hydrochloride liposomes prepared by Preparation method 1, 2 and Example 2 were determined.

    [00049] It has been demonstrated that a target product could be prepared by passive drug loading methods, such as an ethanol injection method and a film dispersion method, when preparing the irinotecan hydrochloride liposome. However, the liposome prepared by these methods had low encapsulation efficiency, and only a small amount of the drug can be loaded into the liposome. In contrast, the sample prepared by the active drug loading method (Example 2) had high encapsulation efficiency and drug loading content. In addition, the sample prepared by an active drug loading method had a small, uniform particle size. Thus, in the present invention, the active drug loading method was used to prepare the liposome. Extremely good results were obtained with the preparation of the irinotecan hydrochloride liposome by the ion gradient method. EXAMPLE 6

    [00050] Preparation method:
    [00051] Empty liposome: the lipid ethanol solution was injected, and the solution was homogenized under 100 MPa (1,000 bar), 6 times; extruded 3 times at 200 nm, 5 times at 100 nm; PEG2000-DSPE was added, and the mixture was incubated for 30 min at 60 ° C. Then, the mixture was dialyzed 3 times with a tangential flow device, 50 mL each time, in which Vitamin E (VE) was added to the organic phospholipid solvent, and EDTA was added to the ammonium sulfate solution.
    [00052] Drug loaded liposome: about 10 mg / ml of aqueous irinotecan hydrochloride solution was prepared and added to the empty liposome, then the mixture was incubated at 60 ° C for 15 min. The sample was concentrated to approximately 50 mL using a tangential flow device, and 5 mg / mL of sample was obtained.
    [00053] The stability results were shown in the table below. None of the indexes in the sample had a marked change when only EDTA was added. Significantly improved the stability of the liposome. However, other stabilizers did not significantly improve the stability of the liposome.
    EXAMPLE 7 Formulation (1):


    [00054] Preparation method:
    [00055] Hydrogenated soy phosphatidylcholine and cholesterol of the formulation amount were dissolved in an appropriate amount of anhydrous ethanol, and the resulting lipid solution was mixed with a manganese sulfate solution (100 mL). After anhydrous ethanol is removed by distillation under reduced pressure, the crude empty liposome is obtained. The particle size of the liposome was controlled by extruding the liposome in the extrusion equipment (two 0.1 μm extrusion membranes in the extrusion equipment, extruding five times). The empty liposome was dialyzed using an ultrafiltration device by tangential flow with continuous supplementation of water for injection in progress; then, the empty liposome was obtained. The aqueous solution of irinotecan hydrochloride was prepared with water for injection and was added to the empty liposome dispersion with ionic gradient. With stirring, the mixture was heated to 50 ° C and incubated for 20 minutes and then the drug loaded liposome was obtained. The non-encapsulated drug was removed using a tangential flow ultrafiltration device, and then 2.5 g of mannitol was added to adjust the osmotic pressure. After adjusting the drug concentration by dilution to a constant volume, the liposome was sterilized by filtration with a 0.22 μm filter and then filled under nitrogen protection and sealed in a small bottle. The liposome injection of irinotecan hydrochloride was finally obtained. The particle size of the liposome was measured by the nanoparticle size analyzer (89.3 nm), and the encapsulation efficiency was 97.5%. Formulation (2):

    [00056] Preparation method:
    [00057] Hydrogenated egg lecithin and cholesterol of the formulation amount were dissolved in an appropriate amount of anhydrous ethanol, and the resulting lipid solution was mixed with a solution of manganese sulfate (100 mL). The particle size of the liposome was controlled by extruding the liposome in the extrusion equipment (two 0.1 μm extrusion membranes in the extrusion equipment, extruding five times). The empty liposome was dialyzed using an ultrafiltration device by tangential flow with continuous supplementation of water for injection in progress; then, the empty liposome was obtained. The aqueous solution of irinotecan hydrochloride was prepared with water for injection and was added to the empty liposome dispersion with ionic gradient. With stirring, the mixture was heated to 50 ° C and incubated for 20 minutes and then the drug loaded liposome was obtained. The non-encapsulated drug was removed using a tangential flow ultrafiltration device, and the sample was concentrated to approximately 50 mL. Then, 2.5 g of histidine was added to adjust the osmotic pressure. After adjusting the drug concentration by diluting the measured volume, the liposome was sterilized by filtration with a 0.22 μm filter and then filled under nitrogen protection and sealed in a small bottle. The liposome injection of irinotecan hydrochloride was finally obtained. The particle size of the liposome was measured by the nanoparticle size analyzer (87.6 nm), and the encapsulation efficiency was 98.1%. Formulation (3):

    [00058] Preparation method:
    [00059] Hydrogenated soy phosphatidylcholine and cholesterol of the formulation amount were dissolved in an appropriate amount of anhydrous ethanol, and the resulting lipid solution was mixed with an ammonium sulfate solution (100 mL). After anhydrous ethanol is removed by distillation under reduced pressure, the crude empty liposome is obtained. After 5 cycles of homogenization in the high pressure homogenizer (100 MPa (1,000 bar)), the aqueous solution of DSPE-PEG2000 was added. With stirring, the mixture was incubated for 20 minutes. The empty liposome was dialyzed using an ultrafiltration device by tangential flow with continuous supplementation of water for injection in progress; then, the empty liposome was obtained. The aqueous solution of irinotecan hydrochloride was prepared with water for injection and was added to the empty liposome dispersion with ionic gradient. With stirring, the mixture was heated to 60 ° C and incubated for 20 minutes and then the drug loaded liposome was obtained. The non-encapsulated drug was removed using a tangential flow ultrafiltration device, and the sample was concentrated to approximately 50 mL. Then, 0.45 g of sodium chloride was added to adjust the osmotic pressure. After adjusting the drug concentration by diluting the measured volume, the liposome was sterilized by filtration with a 0.22 μm filter and then filled under nitrogen protection and sealed in a small bottle. The liposome injection of irinotecan hydrochloride was finally obtained. The particle size of the liposome was measured by the nanoparticle size analyzer (87.3 nm), and the encapsulation efficiency was 99.2%. EXAMPLE 8 Formulation:

    [00060] Preparation method:
    [00061] Hydrogenated soy phosphatidylcholine, myocardial phospholipid, DSPE-PEG5000, cholesterol and α-tocopherol of the formulation amount were dissolved in an appropriate amount of anhydrous ethanol, and the resulting lipid solution was mixed with a citric acid solution (100 mL ). After anhydrous ethanol is removed by distillation under reduced pressure, the crude empty liposome is obtained. After 5 homogenization cycles in the high pressure homogenizer (100 MPa (1,000 bar)), the empty liposome was dialyzed using a tangential flow ultrafiltration device with continuous supplementation of sodium chloride solution (0.9%, 400 mL) in the course; then, the empty liposome was obtained. The aqueous solution of irinotecan hydrochloride was prepared with water for injection and was added to the empty liposome dispersion with ionic gradient. With stirring, the mixture was heated to 60 ° C and incubated for 20 minutes and then the drug loaded liposome was obtained. The non-encapsulated drug was removed using a tangential flow ultrafiltration device, and the sample was concentrated to approximately 50 mL. After adjusting the drug concentration by dilution to a constant volume, the liposome was sterilized by filtration with a 0.22 μm filter and then filled under nitrogen protection and sealed in a small bottle. The liposome injection of irinotecan hydrochloride was finally obtained. The particle size of the liposome was measured by the nanoparticle size analyzer (85.8 nm), and the encapsulation efficiency was 98.6%. EXAMPLE 9 Formulation:


    [00062] Method of preparation:
    [00063] DPPC, DPPG and cholesterol of the formulation amount were dissolved in an appropriate amount of anhydrous ethanol, and the resulting lipid solution was mixed with an ammonium sulfate solution (100 mL, containing disodium ethylene diamine tetraacetic acid). After ethanol is removed by distillation under reduced pressure, the crude empty liposome is obtained. After 5 homogenization cycles in the high pressure homogenizer (100 MPa (1,000 bar)), the empty liposome was dialyzed using a tangential flow ultrafiltration device with continuous supplementation of sodium chloride solution (0.9%, 400 mL) in the course; then, the empty liposome was obtained. The aqueous solution of irinotecan hydrochloride was prepared with water for injection and was added to the empty liposome dispersion with ionic gradient. With stirring, the mixture was heated to 60 ° C and incubated for 20 minutes and then the drug loaded liposome was obtained. The non-encapsulated drug was removed using a tangential flow ultrafiltration device, and the sample was concentrated to approximately 50 mL. After adjusting the drug concentration by dilution to a constant volume, the liposome was sterilized by filtration with a 0.22 μm filter and then filled under nitrogen protection and sealed in a small bottle. The liposome injection of irinotecan hydrochloride was finally obtained. The particle size of the liposome was measured by the nanoparticle size analyzer (89.4 nm), and the encapsulation efficiency was 97.2%. EXAMPLE 10 Formulation:


    [00064] Preparation method:
    [00065] Hydrogenated soy phosphatidylcholine, cholesterol and α-tocopherol from the formulation amount were dissolved in an appropriate amount of anhydrous ethanol, and the resulting lipid solution was mixed with an ammonium sulfate solution (100 mL). After ethanol is removed by distillation under reduced pressure, the crude empty liposome is obtained. After 5 homogenization cycles in the high pressure homogenizer (100 MPa (1,000 bar)), the liposome was extruded in the extrusion equipment (five 10 nm extrusion membranes in the extrusion equipment, extruded five times). Then, aqueous solution of DSPE-PEG5000 was added, and the mixture was incubated with shaking for 20 minutes. The empty liposome was dialyzed using a tangential flow ultrafiltration device with continuous supplementation of sodium chloride solution (0.9%, 400 mL) in the course; then, the empty liposome was obtained. The aqueous solution of irinotecan hydrochloride was prepared with water for injection and was added to the empty liposome dispersion with ionic gradient. With stirring, the mixture was heated to 60 ° C and incubated for 20 minutes and then the drug loaded liposome was obtained. The non-encapsulated drug was removed using a tangential flow ultrafiltration device, and the sample was concentrated to approximately 50 mL. Then, sucrose and mannitol were added to the mixture and mixed homogeneously. After adjusting the drug concentration by dilution to a constant volume, the liposome was sterilized by filtration with a 0.22 μm filter and then filled into a penicillin bottle and lyophilized. The lyophilized liposome powder for injection of irinotecan hydrochloride was finally obtained. The particle size of the liposome was measured (90.8 nm) after hydration of the lyophilized powder for injection, and the encapsulation efficiency was 97.5%. Experiment 1
    [00066] The product of example 2 was taken as an example to study the physicochemical characteristics of the product obtained according to the present invention:
    [00067] [Particle size distribution]: An appropriate amount of the sample was diluted with water, then measured by the Dynamic Light Scattering (DLS) method. Detection wavelength: À = 633 nm; detection angle: 173 °; detection temperature: 25 ° C. The particle size was represented by the intensity. The particle size distribution was shown in figure 1. The average particle size was 85.9 nm.
    [00068] [Morphology]: An appropriate amount of the diluted sample was extracted, a copper mesh was placed on a clean filter paper, the sample was dripped on the copper mesh, dyed with phosphotungstic acid and observed with transmission microscopy of electrons (TEM, JEM2010, Japan Electronics Co., Ltd.) after drying. The morphology was shown in figure 2. The appearance of the irinotecan hydrochloride liposome was of a typical bilayer structure, and most particle sizes were below 200 nm. It is consistent with the result measured by Dynamic Light Scattering.
    [00069] [Encapsulation efficiency]: Method for determining the drug content: Column: Agilent ZORBAX Eclipse XDB-C18 (4.6 x 150 mm, 5 μm); mobile phase: acetonitrile buffer solution - 0.05M KH2PO4 (the pH value was adjusted to 4, containing 1% triethylamine) = 20:80; column temperature: 40 ° C; injection volume: 20 μL; flow rate: 1.0 mL / min.
    [00070] Method for the detection of encapsulation efficiency:
    [00071] 1 ml of goi sample solution was pipetted into a 10 ml volumetric flask and diluted to the mark with water. Then, it was stirred homogeneously and ultrafiltered with ultrafilter 8010 (MILLIPORE company). The initial filtrate was discarded, and the subsequent filtrate was reserved as the sample solution. 20 μL of the sample solution and the control were pipetted in liquid chromatography, and the chromatogram was recorded. The free drug content of the formulation was calculated by an external standardization method, recorded as W. The total amount of drug in this product was calculated by a content determination method, recorded as W0. The encapsulation efficiency was calculated using the following equation:

    [00072] Results of the determination: The encapsulation efficiency of the product was 99.4%.
    [00073] [Impact Factor Test]: The impact factors were investigated by placing the product under different conditions. The results were shown in the table below:

    [00074] The result showed that the sample was sensitive to light. In bright light, the sample's appearance turned yellow, the content decreased, and related substances were significantly increased. The encapsulation efficiency and particle size of the sample had no marked change at 40 ° C, while related substances were slightly increased. Large size particles were generated in the sample under low temperature or freeze-thaw conditions. Considering the instability of phospholipid under high temperature and the test results of the impact factor test, the product should be stored under conditions of low temperature and darkness. [In vivo antitumor therapeutic efficacy test]
    [00075] Drug name: The irinotecan hydrochloride liposome (CPT-11 liposome) (prepared according to Example 2) was supplied by Shanghai Hengrui Pharmaceutical Co., LTD .. The injection of irinotecan hydrochloride (CPT- 11) was provided by Jiangsu Hengrui Medicine Co., LTD.
    [00076] Preparation methods: The drug was diluted with saline to the required concentration.
    [00077] Experimental animals: BALB / ca-nude mice, 6 - 7 weeks, Ç, purchased from Shanghai Slac Laboratory Animal Co., LTD. Certificate No.: SCXK (Shanghai) 2007-0005. Environment: SPF level.
    [00078] Experimental protocol:
    [00079] The nude mice were inoculated subcutaneously with Ls-174t human colon cancer cells. After the tumors had grown to 150 - 300 mm3, the mice were randomly divided into teams (d0). The dosage and dosing regimens were shown in the table below. The volume of the tumors and the weight of the mice were measured and recorded 2-3 times a week. The tumor volume (V) was calculated using the following equation: V = 1 / 2xaxb2, where a and b represent the length and width, respectively.

    [00080] Results:
    [00081] Both the CPT-11 and CPT-11 liposomes significantly inhibited the growth of human colon cancer Ls-174t in nude mice. The CPT-11 liposome was dose dependent in inhibiting the growth of Ls-174t. The 4/14 tumor partially regressed when the CPT-11 liposome was administered at a high dose (3 mg / kg). The therapeutic efficacy of the CPT-11 liposome was equivalent to CPT-11 (10 mg / kg) when the CPT-11 liposome was administered at a low dose (1 mg / kg). It has been indicated that the therapeutic efficacy of the CPT-11 liposome can be amplified at least 10 times with respect to the CPT-11 injection. The detailed results were shown in Figure 3.
    权利要求:
    Claims (17)
    [0001]
    1. Irinotecan liposome or irinotecan hydrochloride, characterized by the fact that said liposome comprises irinotecan or irinotecan hydrochloride, a neutral phospholipid and cholesterol, and the weight ratio of cholesterol to neutral phospholipid is 1: 3.5 ~ 4,5, said neutral phospholipid comprises hydrogenated soy phosphatidylcholine; said liposome further comprises a hydrophilic polymer lipid derivative; the weight ratio of the lipid derivative of the hydrophilic polymer to irinotecan or irinotecan hydrochloride is 0.2 ~ 0.4.
    [0002]
    2. Liposome according to claim 1, characterized in that said weight ratio of neutral phospholipid to irinotecan or irinotecan hydrochloride is given as follows: irinotecan or irinotecan hydrochloride 1 neutral phospholipid 2 ~ 5.
    [0003]
    3. Liposome according to claim 2, characterized in that said weight ratio of neutral phospholipid to irinotecan or irinotecan hydrochloride is given as follows: irinotecan or irinotecan hydrochloride 1 neutral phospholipid 2.5 ~ 4.
    [0004]
    4. Liposome, according to claim 1, characterized by the fact that said neutral phospholipid is hydrogenated soy phosphatidylcholine.
    [0005]
    5. Liposome, according to claim 1, characterized by the fact that the said cholesterol to neutral phospholipid ratio is 1: 4.
    [0006]
    6. Liposome according to any one of claims 1 to 5, characterized in that said liposome is prepared by the ion gradient method.
    [0007]
    7. Liposome, according to claim 6, characterized by the fact that said liposome has an ionic gradient formed by a buffer between the internal aqueous phase and the external aqueous phase of the liposome.
    [0008]
    8. Liposome, according to claim 6, characterized by the fact that the internal aqueous phase of said liposome has an ionic concentration greater than the external aqueous phase.
    [0009]
    9. Liposome according to claim 1, characterized by the fact that the lipid derivative of the hydrophilic polymer is DSPE-PEG2000.
    [0010]
    10. Liposome according to claim 1, characterized by the fact that said liposome also comprises a charged phospholipid, wherein said charged phospholipid is selected from the group consisting of dilauroyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, diestearoyl phosphatidyl glycerol, dimiristate phosphatidylglycerol, dioleic acid phosphatidylserine, dioleoyl phosphatidylglycerol, dilauroyl phosphatidic acid, dimiristate phosphatidic acid, diestearoyl phosphatidic acid and a mixture of these, and the weight ratio of the phospholipid with a charge of 1: 5 is: 1.
    [0011]
    11. Liposome according to claim 1, characterized in that said liposome comprises the following weight ratios of ingredients: irinotecan hydrochloride 1, hydrogenated soy phosphatidylcholine 3,4-3,8, polyethylene glycol 2000-diestearoil phosphatidyl ethanolamine 0.34-0.38, cholesterol 0.8-0.95, and the ratio of cholesterol to hydrogenated soy phosphatidylcholine is 1: 4.
    [0012]
    12. Method of preparing said liposome as defined in any one of claims 1 to 11, characterized in that said method of preparation comprises the following steps of: 1) preparation of an empty liposome by any of the following methods A to D: A. dissolution of a neutral phospholipid and cholesterol in anhydrous ethanol or a mixed solvent of anhydrous ethanol and tert-butyl alcohol according to the desired formulation, mix the mixture with a buffer to obtain a crude empty liposome after ethanol removal by distillation with reduced pressure and then preparing an empty liposome with the desired particle size using a high pressure homogenizer and / or extrusion equipment; B. dissolution of a neutral phospholipid and cholesterol in chloroform or a mixed chloroform-methanol solvent according to the desired formulation, formation of a lipid film by means of a rotary evaporator, addition of a hydration buffer to obtain a crude empty liposome and , then, preparation of an empty liposome with the desired particle size using a high pressure homogenizer and / or extrusion equipment; C. mixing a neutral phospholipid, cholesterol and a buffer according to the desired formulation, then preparing an empty liposome with the desired particle size using a high pressure homogenizer and / or extrusion equipment; D. dissolution of a neutral phospholipid and cholesterol in anhydrous ethanol or a mixed solvent of anhydrous ethanol and tert-butyl alcohol according to the desired formulation, mix the mixture with a buffer and then prepare an empty liposome the size of desired particle using a high pressure homogenizer and / or extrusion equipment; 2) formation of an ionic gradient between the internal aqueous phase and the external aqueous phase of the empty liposome: replacement of the external aqueous phase of the empty liposome to form an ionic gradient between the internal aqueous phase and the external aqueous phase of the empty liposome; 3) preparation of a drug-loaded liposome: preparation of an aqueous solution of irinotecan hydrochloride, adding it to the empty liposome dispersion with an ionic gradient and then incubating the dispersion to obtain the drug-loaded liposome under heating and agitation; 4) removal of the free drug and concentration of the sample: addition of a buffer medium to the irinotecan hydrochloride liposome, removal of the non-encapsulated drug using a tangential flow device, and concentration of the sample in the appropriate volume.
    [0013]
    13. Preparation method according to claim 12, characterized in that said buffer is selected from the group consisting of a buffer comprising salts of the Na +, K +, Fe2 +, Ca 2 +, Ba2 + ions, Mn2 +, Mg2 +, Li +, NH4 +, H + and a mixture of these.
    [0014]
    14. Liposome injection, characterized by the fact that it comprises the irinotecan liposome or irinotecan hydrochloride as defined in any one of claims 1 to 11; said injection further comprises a stabilizer, wherein said stabilizer is selected from the group consisting of ethylene diamine tetraacetic acid, ethylene diamine tetraacetic acid disodium, ethylene diamine tetraacetic acid dicalcium and a mixture thereof; the ratio of the added stabilizer is 0 ~ 0.5% (w / v), and the minimum is not 0%; an osmotic pressure regulator, in which said osmotic pressure regulator is selected from the group consisting of glucose, sucrose, sorbitol, mannitol, sodium chloride, glycerin, histidine and its hydrochloride, glycine and its hydrochloride, lysine, serine, acid glutamic, arginine, valine and a mixture of these; the ratio of said osmotic pressure regulator added is 0 ~ 5% (w / v), and the minimum is not 0%; an antioxidant, wherein said antioxidant is selected from the group consisting of a water-soluble antioxidant and an oil-soluble antioxidant; wherein said oil-soluble antioxidant is selected from the group consisting of α-tocopherol, α-tocopherol succinate, α-tocopherol acetate and a mixture thereof; wherein said water-soluble antioxidant is selected from the group consisting of ascorbic acid, sodium bisulfite, sodium sulfite, sodium pyrosulfite, L-cysteine and a mixture thereof; the ratio of the added antioxidant is 0 ~ 0.5% (w / v), and the minimum is not 0%; said injection is a liquid injection or lyophilized powder for injection.
    [0015]
    15. Liposome injection, according to claim 14, characterized by the fact that said injection is lyophilized powder for injection that comprises the lyoprotectant, and is prepared by lyophilization.
    [0016]
    16. Liposome injection according to claim 14, characterized in that said injection comprises the following weight ratios of ingredients: irinotecan hydrochloride 1, hydrogenated soy phosphatidylcholine 3,4-3,8, polyethylene glycol 2000 - distearoyl phosphatidyl ethanolamine 0.34-0.38, cholesterol 0.8-0.95, disodium ethylene diamine tetraacetic acid 0.05-0.09, and the cholesterol to hydrogenated soy phosphatidylcholine ratio is 1: 4.
    [0017]
    17. Process for preparing said liposome injection as defined in any of claims 14 to 16, characterized in that said process comprises the method of preparation as defined in any of claims 12 to 13; said preparation process also comprises the following steps: volume measurement, sterilization and subpackaging: adjustment of the drug concentration in the liposome, volume measurement, filtration sterilization, filling of the vials and closing to obtain the liposome injection; or adding a lyoprotectant to the drug sample in liposome, adjusting the drug concentration, measuring the volume, filter sterilization, filling the vials, closing, then lyophilizing to obtain the lyophilized powder for injection.
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    同族专利:
    公开号 | 公开日
    SI2508170T1|2015-12-31|
    CY1116811T1|2017-03-15|
    EP2508170B1|2015-07-29|
    BR112012012151A2|2016-04-12|
    PT2508170E|2015-10-16|
    KR101780915B1|2017-09-21|
    CA2782911C|2016-08-23|
    WO2011066684A1|2011-06-09|
    RU2526114C2|2014-08-20|
    MX2012005775A|2012-06-13|
    JP5645954B2|2014-12-24|
    US10022365B2|2018-07-17|
    SMT201500245B|2015-10-30|
    DK2508170T3|2015-09-21|
    KR20120089754A|2012-08-13|
    US20120282325A1|2012-11-08|
    BR112012012151B8|2021-05-25|
    EP2508170A4|2014-01-15|
    EP2508170A1|2012-10-10|
    ZA201203316B|2013-11-27|
    AU2009356132B2|2015-01-22|
    HUE027467T2|2016-10-28|
    ES2547698T3|2015-10-08|
    HK1159482A1|2012-08-03|
    CN102271659B|2013-09-18|
    KR20160140992A|2016-12-07|
    JP2013512262A|2013-04-11|
    HRP20150911T1|2015-10-23|
    US20170189392A1|2017-07-06|
    CN102271659A|2011-12-07|
    PL2508170T3|2015-12-31|
    CA2782911A1|2011-06-09|
    AU2009356132A1|2012-06-21|
    RU2012123875A|2014-01-20|
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    法律状态:
    2018-01-16| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|
    2018-04-17| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|
    2020-08-25| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application [chapter 6.1 patent gazette]|
    2020-12-01| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|
    2021-01-19| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 10 (DEZ) ANOS CONTADOS A PARTIR DE 19/01/2021, OBSERVADAS AS CONDICOES LEGAIS. |
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    优先权:
    申请号 | 申请日 | 专利标题
    PCT/CN2009/075298|WO2011066684A1|2009-12-03|2009-12-03|Liposome of irinotecan or its hydrochloride and preparation method thereof|
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