WO2011066684A1 - 伊立替康或盐酸伊立替康脂质体及其制备方法 - Google Patents

伊立替康或盐酸伊立替康脂质体及其制备方法 Download PDF

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WO2011066684A1
WO2011066684A1 PCT/CN2009/075298 CN2009075298W WO2011066684A1 WO 2011066684 A1 WO2011066684 A1 WO 2011066684A1 CN 2009075298 W CN2009075298 W CN 2009075298W WO 2011066684 A1 WO2011066684 A1 WO 2011066684A1
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Prior art keywords
liposome
injection
irinotecan
cholesterol
drug
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PCT/CN2009/075298
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English (en)
French (fr)
Inventor
仝新勇
雷国锋
余成霞
陈亮
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江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
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Priority to CA2782911A priority Critical patent/CA2782911C/en
Priority to KR1020167033432A priority patent/KR101780915B1/ko
Priority to PL09851784T priority patent/PL2508170T3/pl
Priority to US13/512,048 priority patent/US20120282325A1/en
Priority to PCT/CN2009/075298 priority patent/WO2011066684A1/zh
Priority to ES09851784.0T priority patent/ES2547698T3/es
Priority to SI200931279T priority patent/SI2508170T1/sl
Priority to HUE09851784A priority patent/HUE027467T2/en
Priority to RU2012123875/15A priority patent/RU2526114C2/ru
Priority to PT98517840T priority patent/PT2508170E/pt
Priority to EP09851784.0A priority patent/EP2508170B1/en
Priority to DK09851784.0T priority patent/DK2508170T3/en
Application filed by 江苏恒瑞医药股份有限公司, 上海恒瑞医药有限公司 filed Critical 江苏恒瑞医药股份有限公司
Priority to CN2009801540269A priority patent/CN102271659B/zh
Priority to AU2009356132A priority patent/AU2009356132B2/en
Priority to MX2012005775A priority patent/MX2012005775A/es
Priority to JP2012541296A priority patent/JP5645954B2/ja
Priority to BR112012012151A priority patent/BR112012012151B8/pt
Priority to KR1020127015958A priority patent/KR20120089754A/ko
Publication of WO2011066684A1 publication Critical patent/WO2011066684A1/zh
Priority to HK11113962.4A priority patent/HK1159482A1/xx
Priority to ZA2012/03316A priority patent/ZA201203316B/en
Priority to HRP20150911TT priority patent/HRP20150911T1/hr
Priority to SM201500245T priority patent/SMT201500245B/xx
Priority to CY20151100943T priority patent/CY1116811T1/el
Priority to US15/464,922 priority patent/US10022365B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1277—Processes for preparing; Proliposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1277—Processes for preparing; Proliposomes
    • A61K9/1278—Post-loading, e.g. by ion or pH gradient
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to an irinotecan or irinotecan hydrochloride liposome, a preparation method thereof, and an injection containing the liposome and a preparation method thereof.
  • Irinotecan is a semisynthetic derivative of camptothecin. Camptothecin specifically binds to topoisomerase I, which induces reversible single-strand breaks, thereby unwinding the DNA double-stranded structure; irinotecan and its active metabolite SN-38 and topoisomerase The I-DNA complex binds, thereby preventing re-ligation of the cleavage single strand.
  • Existing studies suggest that the cytotoxic effect of irinotecan is attributed to the interaction between the replicase and the topoisomerase I-DNA-irinotecan (or SN-38) triple complex during DNA synthesis, resulting in DNA double Chain breakage.
  • Irinotecan hydrochloride has obvious pharmacological effects and is clinically effective. It is widely used in the treatment of malignant tumors. However, like other camptothecin drugs, the same problem exists in that the saturated lactone ring in its structure has pH dependence. sexually, under physiological conditions, it can be reversibly converted into its carboxylate form, which reduces the antitumor activity.
  • the existing commercial preparation of irinotecan hydrochloride is irinotecan hydrochloride injection and its freeze-dried powder preparation. After intravenous administration, the free drug is directly in the alkaline environment, and its structure is The lactone ring is susceptible to hydrolysis to a carboxylate form, thereby losing activity and indirectly reducing the efficacy of the drug. Moreover, the toxic side effects of the preparation are large, mainly manifested as neutropenia and delayed diarrhea.
  • liposome As a drug carrier widely studied in recent years, liposome is mainly used to protect encapsulated drugs, increase drug stability, change the distribution behavior of drugs in vivo, and carry drugs passively or actively to the lesions. . As a good carrier of antitumor drugs, liposomes can effectively improve the efficacy of drugs and reduce toxic side effects.
  • irinotecan liposome containing irinotecan or irinotecan hydrochloride, selected from hydrogenated soybean phosphatidylcholine, phosphatidylethanolamine, lecithin, cardiolipin Such as phospholipids, cholesterol.
  • Chinese patent application CN1994279A also discloses a prescription and preparation method of irinotecan liposome, which Liposomes were prepared using the phosphatidylcholine as a phospholipid in Example 5.
  • the object of the present invention is to provide a irinotecan or irinotecan hydrochloride liposome which has a high drug loading amount and can simultaneously satisfy a high encapsulation efficiency and a good stability and is suitable for preparation into a preparation.
  • liposome compositions and preparation methods of irinotecan are described in some documents (e.g., international applications WO2005/117878 and CN1994279A), some of the indicators of the individual schemes are better. However, no information is provided on the control of stability, particle size, etc.
  • the applicant's research on liposomes has surprisingly found that when the choice of excipients and dosages meets certain conditions, the amount of special cholesterol has a certain effect on the size and stability of the liposomes.
  • the ratio between the two is controlled based on neutral phosphorus and cholesterol, which makes the particle size of the liposome small and uniform, and improves the stability of the liposome. Compared with other prescriptions, the stability and the like of the liposome of the present application are markedly improved.
  • the present invention does not contain a basic functional group compound and a cationic lipid in comparison with the technologies of the international applications WO2005/117878 and CN1994279A, and has a simple prescription composition, a high drug loading amount, and good stability, and the liposome of the present invention has a very high Good anti-tumor effect.
  • the liposome of the present invention contains irinotecan or irinotecan hydrochloride, neutral phospholipid and cholesterol, wherein the weight ratio of the cholesterol to the neutral phospholipid is 1:3 to 5, and the preferred ratio is 1:3.5-4.5, the most It is preferably 1:4.
  • the neutral phospholipid used in the present invention may be selected from materials such as hydrogenated soybean lecithin (HSPC), egg yolk phospholipid (EPC), and soybean phospholipid (SPC), and particularly when the neutral phospholipid is hydrogenated soybean lecithin, the effect is the best.
  • HSPC hydrogenated soybean lecithin
  • EPC egg yolk phospholipid
  • SPC soybean phospholipid
  • the neutral phospholipid is 2 to 5 parts, preferably 2.5 to 4 parts.
  • the liposome of the present invention can be prepared according to a liposome preparation method conventional in the art, and for the liposome of the present invention, it is preferably prepared by an ion gradient method. Using an ion ladder In the method, the ion phase between the aqueous phase and the outer aqueous phase of the liposome has an ion gradient formed by the buffer, and preferably the water in the liposome has an ion gradient having a higher ion concentration than the outer aqueous phase, which can improve the storage period.
  • the particle size of the liposome is stable, and the drug effect is better maintained, which can control the small and uniform average particle size of the liposome, and can minimize the change of the liposome particle size during storage.
  • the invention can reduce the change of the liposome particle size during storage by adding a lipid derivative of a hydrophilic polymer to the formulation, and the addition of the polyethylene glycol derivative can prolong the liposome Cycle time in the body.
  • the polyethylene glycol derivative is selected from the group consisting of polyethylene glycol 2000-distearoyl phosphatidylethanolamine (DSPE-PEG 2QQQ ) polyethylene glycol 5000 -distearoylphosphatidylethanolamine, polyethylene glycol 2000-dipalmitoyl Phosphatidylethanolamine, polyethylene glycol 5000-dipalmitoylphosphatidylethanolamine.
  • the present application preferably adds a lipid derivative of a hydrophilic polymer to the liposome, and based on the prescription ratio, the use of DSPE-PEG 2000 is most effective.
  • the weight ratio of the lipid derivative to irinotecan or irinotecan hydrochloride is from 0.2 to 0.4.
  • the liposome can further contain a charged phospholipid, and the charged phospholipid is selected from the group consisting of dilauroylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, dimyristoylphosphatidylglycerol, dioleate phospholipid
  • acylserine dioleoylphosphatidylglycerol, dilauroylphosphatidic acid, dimyristoylphosphatidic acid or distearoylphosphatidic acid
  • the weight ratio of charged phospholipid to neutral phospholipid is 1 :5 ⁇ 1: 100.
  • Preferred liposomes of the invention comprise the following components in a weight ratio:
  • the cholesterol is 0.8-0.95 parts, and the ratio of cholesterol to hydrogenated soybean lecithin is 1:4.
  • the present application also provides a process for preparing irinotecan or irinotecan hydrochloride liposomes, which can be prepared by a conventional liposome preparation method.
  • Those skilled in the art can prepare according to the prescription of the liposome provided by the present invention by various methods.
  • the preferred method of preparation is the ion gradient method.
  • the preparation method comprises the following steps:
  • a neutral phospholipid, cholesterol is dissolved in a mixed solvent of absolute ethanol or absolute ethanol-tert-butanol, mixed with a buffer, and then a blank liposome is prepared by a high pressure homogenizer or/and an extrusion device.
  • Required particle size
  • a buffer medium was added to the irinotecan hydrochloride liposome, and the unencapsulated drug was removed by a tangential flow device while the sample was concentrated to an appropriate volume.
  • the present application also provides liposome injections containing the above liposomes.
  • a stabilizer used in the present invention may also be selected from commonly used stabilizers such as vitamin E, ethylenediaminetetraacetic acid, etc., and these stabilizers all contribute to the stability of the formulation.
  • ethylenediaminetetraacetic acid or its salt has the best effect relative to other stabilizers, and is most beneficial for improving the stability of liposomes. Therefore, ethylenediaminetetraacetic acid can be used. And one or more of ethylenediaminetetraacetic acid disodium salt and ethylenediaminetetraacetic acid dicalcium salt, and the stabilizer is added in a ratio of 0% to 0.5 w/v% and the lower limit is not 0%.
  • the composition of the present invention contains an antioxidant, and the antioxidant may be selected from water-soluble anti-drugs.
  • An oxygen-soluble or oil-soluble antioxidant wherein the oil-soluble antioxidant is selected from the group consisting of ⁇ -tocopherol, ⁇ -tocopheryl succinate, ⁇ -tocopherol tocopherol or a mixture thereof, and the water-soluble antioxidant is selected from the group consisting of ascorbic acid , sodium hydrogen sulfite, sodium sulfite, sodium metabisulfite, L-cysteine or a mixture thereof, the antioxidant is added in a proportion of 0% to 0.5 w/v % and the lower limit is not 0%.
  • the injection can be in the form of an injection or a lyophilized powder.
  • the preparation may contain an osmotic pressure adjusting agent selected from the group consisting of glucose, sucrose, sorbitol, mannitol, sodium chloride, glycerin, histidine and its hydrochloride, glycine and its hydrochloride, lysine. Or one or more of serine, glutamic acid, arginine or valine, and the osmotic pressure adjusting agent is added in a ratio of 0% to 5 w/v% and the lower limit is not 0%.
  • the injection is further filled with a lyophilized protective agent, and lyophilized to obtain a liposome lyophilized powder.
  • the lyoprotectant is selected from one or more of the group consisting of glucose, sucrose, trehalose, mannitol, dextran or lactose.
  • Preferred injectable liposomes of the invention comprise the following components by weight:
  • the preparation method of the above injection comprises the following steps:
  • a neutral phospholipid, cholesterol is dissolved in a mixed solvent of absolute ethanol or absolute ethanol-tert-butanol, mixed with a buffer, and then a blank liposome is prepared by a high pressure homogenizer or/and an extrusion device.
  • Required particle size
  • step 3) "Preparation of drug-containing liposome"
  • steps may be further included: 4) removal of free drug and concentration of sample: buffer medium is added to irinotecan hydrochloride liposome, The tangential flow device removes the unencapsulated drug while concentrating the sample to the appropriate volume.
  • the liposome drug concentration can be adjusted, the volume is adjusted, the filter is sterilized, and the capsule is filled in a vial to obtain a liposome injection; or a lyoprotectant is added to the liposome drug sample. Adjust the drug concentration, make up the volume, filter and sterilize, enclose in the bottle, freeze-dry, and obtain the freeze-dried powder.
  • the irinotecan hydrochloride preparation is prepared into a liposome preparation to overcome the deficiencies of the existing products and technologies, and the drug is encapsulated in the aqueous phase of the liposome to improve the stability of the drug, so that the drug exists in the form of a lactone ring in the body, and can be long. Time maintains the concentration of active metabolite SN-38 in plasma to increase the efficacy of the formulation and reduce the toxic side effects of the drug.
  • the irinotecan or irinotecan hydrochloride liposome preparation of the invention can solve the problem of low drug loading of liposome by controlling the ratio of specific drugs, phospholipids and cholesterol, and the ratio of drug to lipid (w/w) can be Up to 0.25 or more, at the same time, the encapsulation efficiency of the drug can reach more than 90%, and the preferred prescription can reach more than 95%; the invention selects the relationship between the amount of cholesterol and phospholipid by further selecting the relationship between the prepared liposome and the lipid. Stability of the plastid.
  • the invention also improves the stability of the liposome by screening a stabilizer, preferably by adding a certain proportion of ethylenediaminetetraacetate; the particle size of the liposome is between 10 nm and 220 nm, and the distribution is uniform; Stable, irinotecan or irinotecan hydrochloride liposome injection factors
  • Stable, irinotecan or irinotecan hydrochloride liposome injection factors The experimental results showed that there was no significant change in particle size and encapsulation rate after 10 days at 40 °C, and all indicators met the requirements; irinotecan Or irinotecan hydrochloride liposome injection compared to a commercial preparation, The tumor inhibition rate was significantly improved and the toxicity was significantly reduced.
  • Fig. 1 shows the particle size distribution of the irinotecan or irinotecan hydrochloride liposome injection of the present invention.
  • Fig. 2 is a view showing the morphology of the irinotecan or irinotecan hydrochloride liposome injection of the present invention.
  • Fig. 3 shows the results of an in vivo antitumor efficacy test of the irinotecan or irinotecan hydrochloride liposome injection of the present invention.
  • HSPC hydrogenated soybean lecithin
  • CHOL cholesterol
  • a lipid solution A prescribed amount of hydrogenated soybean lecithin (HSPC) and cholesterol (CHOL) were dissolved in an appropriate amount of absolute ethanol to obtain a lipid solution, and mixed with 100 ml of an ammonium sulfate solution, and ethanol was removed under reduced pressure to obtain a crude liposome. Thereafter, the high-pressure homogenizer was used to homogenize for 5 cycles, and then the liposome was extruded through an extrusion apparatus to control the particle size (2 ⁇ . ⁇ extruded film, extruded 5 times). The prepared DSPE-PEG O aqueous solution was then added and incubated for 20 minutes with stirring. The blank liposome was dialyzed by a tangential flow ultrafiltration device, and the injection water was continuously supplied in the middle to obtain a blank liposome.
  • HSPC hydrogenated soybean lecithin
  • CHOL cholesterol
  • aqueous solution of irinotecan hydrochloride was prepared by using water for injection, and was added to the above-mentioned blank liposome dispersion having an ion gradient according to a weight ratio of irinotecan hydrochloride to HSPC of 1:3.5.
  • the mixture was heated and stirred at 60 ° C, and the drug-loaded liposome was obtained by incubating for 20 minutes.
  • the unencapsulated drug was removed using a tangential flow ultrafiltration device while the sample was concentrated to about 50 ml and 0.45 g of sodium chloride was added to adjust the osmotic pressure. Adjust the concentration of the drug, adjust the volume, 0.22 ⁇ filter to filter and sterilize, and fill with nitrogen in a vial to obtain irinotecan hydrochloride liposome injection.
  • a prescribed amount of hydrogenated soybean lecithin and cholesterol were dissolved in an appropriate amount of absolute ethanol to obtain a lipid solution, and mixed with 100 ml of an ammonium sulfate solution, and ethanol was removed under reduced pressure to obtain a crude liposome. Thereafter, a high-pressure homogenizer 1000 bar was homogenized for 5 cycles, and then the liposome was extruded through an extrusion apparatus to control the particle size (extruder was spread 2 ⁇ . ⁇ extruded film, extruded 5 times). Then, the prepared aqueous solution of DSPE-PEG 2QQQ was added and incubated for 20 minutes with stirring. The blank liposome was dialyzed by a tangential flow ultrafiltration device, and the injection water was continuously supplied in the middle to obtain a blank liposome.
  • aqueous solution of irinotecan hydrochloride is prepared by using water for injection, and is added to the above blank liposome dispersion having an ion gradient according to the weight ratio of irinotecan hydrochloride to HSPC of 1:3.5, heated and stirred at 60 ° C, and incubated for 20 minutes. Drug-loaded liposomes. The tangential flow ultrafiltration device was used to remove the unencapsulated drug while the sample was concentrated to about 50 ml, adjusted by adding 0.45 g of sodium chloride.
  • the blank liposome formulation and preparation method are the same as in the case of Example 2, except that the weight ratios of irinotecan hydrochloride and HSPC are 1:1.5, 1:2, 1:3.5, 1:4 and 1:5, respectively.
  • the liposome was prepared, and the encapsulation efficiency and particle size of irinotecan hydrochloride liposome samples are shown in the following table.
  • the blank liposome and the drug-loaded liposome were prepared according to the dosage of the prescription component in Example 2, and the preparation method was the same as that described in Example 2, except that high-purity egg yolk phospholipid (EPC) and high-purity soybean were respectively used.
  • Phospholipids (SPC) are prepared by replacing HSPC in the prescription.
  • the prepared liposome samples were examined for stability at 25 °C. See the table below for the results. The results showed that the liposome samples prepared by HSPC had the best stability, and there was no significant change in the main indexes at 25 °C for 2 months.
  • Time PC composition Encapsulation efficiency (%) Drug loading (mg/ml)
  • Particle size nm
  • Ethanol injection method a prescribed amount of hydrogenated soybean lecithin, DSPE-PEG 2QQQ , cholesterol is dissolved in an appropriate amount of absolute ethanol to obtain a lipid solution, which is injected into a physiological saline solution of irinotecan hydrochloride, and the ethanol is removed under reduced pressure to obtain a lipid. Rough body. Thereafter, a high-pressure homogenizer of 1000 bar was used for homogenization for 5 cycles, and then the liposome was extruded through an extrusion apparatus to control the particle size (extruder 2 ⁇ . ⁇ extruded film, extruded 5 times). Adjust the concentration of the drug, adjust the volume, 0.22 ⁇ filter to filter and sterilize, and fill with nitrogen in a vial to obtain irinotecan hydrochloride liposome injection.
  • Membrane dispersion method a prescribed amount of hydrogenated soybean lecithin, DSPE-PEG 2QQQ , cholesterol is dissolved in a proper amount of chloroform to obtain a lipid solution, and the lipid solution is rotary evaporated into a film, and chloroform is removed, and then a physiological saline solution of irinotecan hydrochloride is added. The hydration was incubated for about 1 h. Then, a high-pressure homogenizer was used to homogenize for 5 cycles, and the liposome was extruded through an extrusion device to control the particle size (2 ⁇ . ⁇ extrusion film was extruded and extruded 5 times). Adjust the concentration of the drug, adjust the volume, 0.22 ⁇ filter to filter and sterilize, and fill with nitrogen in a vial to obtain irinotecan hydrochloride liposome injection.
  • Example 2 99.4 85.9 Preparation method ⁇ 1> 15.3 87.9
  • Preparation method ⁇ 2> 17.8 90.2 The results show that the target product can be prepared by using passive drug loading method such as ethanol injection method and thin film evaporation method to prepare irinotecan hydrochloride liposome.
  • passive drug loading method such as ethanol injection method and thin film evaporation method to prepare irinotecan hydrochloride liposome.
  • the encapsulation efficiency of the sample is low, and most of the drugs do not enter the liposome; the sample prepared by the active drug loading method (Example 2) has high encapsulation efficiency, high drug loading, and small and uniform particle size. Therefore, the active drug loading method is adopted.
  • the preparation of irinotecan hydrochloride liposomes by the ion gradient method has a very good effect.
  • PEG ⁇ -DSPE o. i g 0.1 g 0.1 g 0.1 g Vitamin E 1 0.02 g 1 0.02 g
  • EDTA-2Na 1 1 0.02 g 0.02 g ammonium sulfate solution (300 mM) 100 ml 100 ml 100 ml 100 ml irinotecan hydrochloride 0.3 g 0.3 g 0.3 g 0.3 g Preparation method:
  • Blank liposomes Inject lipid ethanol. Homogenization 1000bar, 6 times; 200nm extrusion 3 times, 100nm extrusion 5 times; force into PEG 2000 -DSPE, 60 °C for 30 min. Dialysis was performed 3 times in tangential flow, 50 ml each time. Wherein VE is added to the phospholipid organic solvent, and EDTA is added to the ammonium sulfate solution.
  • Drug-loaded liposomes A 10 mg/ml aqueous solution of irinotecan hydrochloride was added to the blank liposome and given at 60 ° C for 15 min. The sample was concentrated with a tangential flow to about 50 ml, which is a 5 mg/ml sample.
  • a prescribed amount of hydrogenated soybean lecithin and cholesterol were dissolved in an appropriate amount of absolute ethanol to obtain a lipid solution, and mixed with 100 ml of a manganese sulfate solution, and ethanol was removed under reduced pressure to obtain a crude liposome. Thereafter, the liposome was extruded through an extrusion apparatus to control the particle size (2 ⁇ . ⁇ extruded film, extruded 5 times).
  • the blank liposome was dialyzed by a tangential flow ultrafiltration device, and the injection water was continuously supplied in the middle to obtain a blank liposome.
  • aqueous solution of irinotecan hydrochloride was prepared by using water for injection, added to a blank liposome dispersion having an ion gradient, heated and stirred at 50 ° C, and incubated for 20 minutes to obtain a drug-loaded liposome.
  • the unencapsulated drug was removed using a tangential flow ultrafiltration device while the sample was concentrated to about 50 ml and 2.5 g of mannitol was added to adjust the osmotic pressure. Adjust the concentration of the drug, adjust the volume, 0.22 ⁇ filter to filter and sterilize, and fill with nitrogen in a vial to obtain irinotecan hydrochloride liposome injection.
  • the particle size of the liposome was 89.3 nm and the encapsulation efficiency was 97.5%.
  • a prescribed amount of hydrogenated egg yolk lecithin, cholesterol was dissolved in an appropriate amount of absolute ethanol to obtain a lipid solution, and mixed with 100 ml of a magnesium sulfate solution. Thereafter, the liposome was extruded through an extrusion apparatus to control the particle size (2 extruders, ⁇ extruded film, and extruded 5 times).
  • the blank liposome was dialyzed by a tangential flow ultrafiltration device, and the injection water was continuously supplied in the middle to obtain a blank liposome.
  • aqueous solution of irinotecan hydrochloride was prepared by using water for injection, added to a blank liposome dispersion having an ion gradient, heated and stirred at 50 ° C, and incubated for 20 minutes to obtain a drug-loaded liposome.
  • the unencapsulated drug was removed using a tangential flow ultrafiltration device while the sample was concentrated to about 50 ml and 2.5 g of histidine was added to adjust the osmotic pressure. Adjust the concentration of the drug, adjust the volume, 0.22 ⁇ filter to filter and sterilize, and fill with nitrogen in a vial to obtain irinotecan hydrochloride liposome injection.
  • the particle size of the liposome was 87.6 nm and the encapsulation efficiency was 98.1%.
  • a prescribed amount of hydrogenated soybean lecithin and cholesterol were dissolved in an appropriate amount of absolute ethanol to obtain a lipid solution, and mixed with 100 ml of an ammonium sulfate solution, and ethanol was removed under reduced pressure to obtain a crude liposome. After that, the high-pressure homogenizer was homogenized for 5 cycles, and then the prepared DSPE-PEG 2QQQ aqueous solution was added and stirred for 20 minutes. Dialysis air using tangential flow ultrafiltration White liposomes, with uninterrupted replenishment of water for injection, give blank liposomes.
  • aqueous solution of irinotecan hydrochloride was prepared by using water for injection, added to a blank liposome dispersion having an ion gradient, heated and stirred at 60 ° C, and incubated for 20 minutes to obtain a drug-loaded liposome.
  • the unencapsulated drug was removed using a tangential flow ultrafiltration device while the sample was concentrated to about 50 ml and 0.45 g of sodium chloride was added to adjust the osmotic pressure. Adjust the concentration of the drug, adjust the volume, 0.22 ⁇ filter to filter and sterilize, and fill with nitrogen in a vial to obtain irinotecan hydrochloride liposome injection.
  • the particle size of the liposome was 87.3 nm and the entrapment efficiency was 99.2%.
  • a prescribed amount of hydrogenated soybean lecithin, cardiolipin, DSPE-PEG 5QQQ , cholesterol, ⁇ -tocopherol is dissolved in an appropriate amount of absolute ethanol to obtain a lipid solution, mixed with 100 ml of citric acid solution, and ethanol is removed under reduced pressure to obtain a blank lipid. Rough body. After that, a high-pressure homogenizer of 1000 bar was used, and the homogenization was carried out 5 times. The blank liposome was dialyzed by a tangential flow ultrafiltration device, and 400 ml of a 0.9% sodium chloride solution was continuously supplied in the middle to obtain a blank liposome.
  • the irinotecan hydrochloride solution was prepared by using water for injection, added to a blank liposome dispersion having an ion gradient, heated and stirred at 60 ° C, and incubated for 20 minutes to obtain a drug-loaded liposome.
  • the unencapsulated drug was removed using a tangential flow ultrafiltration device while the sample was concentrated to approximately 50 ml. Adjust the concentration of the drug, adjust the volume, 0.22 ⁇ filter to filter and sterilize, and fill with nitrogen in a vial to obtain irinotecan hydrochloride liposome injection.
  • the particle size of the liposome was 85.8 nm and the entrapment efficiency was 98.6%.
  • Example 9 Irinotecan hydrochloride 0.8g
  • a prescribed amount of DPPC, DPPG, cholesterol was dissolved in an appropriate amount of absolute ethanol to obtain a lipid solution, and mixed with 100 ml of an ammonium sulfate solution (containing disodium edetate), and ethanol was removed under reduced pressure to obtain a crude liposome. Then use a high pressure homogenizer 1000bar, homogenized 5 times.
  • the blank liposome was dialyzed by a tangential flow ultrafiltration device, and 400 ml of a 0.9% sodium chloride solution was continuously supplied in the middle to obtain a blank liposome.
  • the irinotecan hydrochloride solution was prepared by using water for injection, added to a blank liposome dispersion having an ion gradient, heated and stirred at 60 ° C, and incubated for 20 minutes to obtain a drug-loaded liposome.
  • the unencapsulated drug was removed using a tangential flow ultrafiltration device while the sample was concentrated to about 50 ml and ascorbic acid was added. Adjust the concentration of the drug, adjust the volume, 0.22 ⁇ filter to filter and sterilize, and fill with nitrogen in a vial to obtain irinotecan hydrochloride liposome injection.
  • the particle size of the liposome was 89.4 nm and the entrapment efficiency was 97.2%.
  • a prescribed amount of hydrogenated soybean lecithin, cholesterol, and ⁇ -tocopherol were dissolved in an appropriate amount of absolute ethanol to obtain a lipid solution, and mixed with 100 ml of an ammonium sulfate solution, and ethanol was removed under reduced pressure to obtain a crude liposome. Then, using a high-pressure homogenizer 1000bar, homogenizing 5 times, and then extruding the liposome through an extrusion device (extruding 5 sheets of 100 nm extruded film, extruding 5 times), and then adding the prepared DSPE-PEG 5QQQ The aqueous solution was incubated for 20 minutes with stirring.
  • the blank liposome was dialyzed by a tangential flow ultrafiltration device, and 400 ml of a 0.9% sodium chloride solution was continuously supplied in the middle to obtain a blank liposome.
  • the irinotecan hydrochloride solution was prepared by using water for injection, added to a blank liposome dispersion having an ion gradient, heated and stirred at 60 ° C, and incubated for 20 minutes to obtain a drug-loaded liposome.
  • the unencapsulated drug was removed using a tangential flow ultrafiltration device while the sample was concentrated to about 50 ml, and sucrose and mannitol were added to make the mixture uniform.
  • Adjust the concentration of the drug adjust the volume, 0.22 ⁇ filter membrane filter sterilization, canned in a vial, freeze-dried, that is, irinotecan hydrochloride liposome freeze-dried powder needle.
  • the liposome lyophilized powder was hydrated, and the liposome particle size was determined to be 90.8 nm, and the encapsulation efficiency was 97.5%.
  • Test example 1
  • Encapsulation rate determination method Pipette 1mL sample solution into 10mL volumetric flask, dilute to the mark with water, shake well, set 8010 ultrafilter (MILLIPORE company) ultrafiltration, discard the primary filtrate, take the filtrate as the test sample Solution.
  • Encapsulation rate w ° W x lOO%
  • the irinotecan hydrochloride liposome (CPT-11 liposome) prepared according to Example 2 was provided by Shanghai Hengrui Pharmaceutical Co., Ltd., irinotecan hydrochloride injection (CPT-11, Jiangsu Hengrui Pharmaceutical Co., Ltd. Provided by the company)
  • Preparation method All the physiological saline is used to prepare the desired concentration.
  • mice BALB/cA-nude nude mice, 6-7 weeks, $, purchased from Shanghai Slack Laboratory Animals LLC. Certificate No.: SCXK (Shanghai) 2007-0005. Breeding environment: SPF level.
  • Test method Nude mice were subcutaneously inoculated with human colon cancer Ls-174t cells. After the tumors were grown to 150-300 mm 3 , the animals were randomly divided into groups (10:). The dosages and dosing schedules are shown in the table below. 2 - 3 tumor volume, weighing the mouse, record the data. The tumor volume (V) is calculated as:
  • V l/2x a xb 2
  • a and b represent length and width, respectively.
  • CPT-11 liposome and CPT-11 significantly inhibited the growth of human colon cancer Ls-174t nude mice, and CPT-1 was dose-dependent. At high dose (3 mg/kg), there were 4/ 14 tumors partially resolved, low dose (lmg 10mg / kg equivalent, suggesting that CPT-11 liposome may be at least 10 times more effective than its injection, with

Description

伊立替康或盐酸伊立替康脂质体及其制备方法 技术领域
本发明涉及一种伊立替康或盐酸伊立替康脂质体及其制备方法， 以及含有该脂质体的注射剂及其制备方法。 背景技术
伊立替康是喜树碱的半合成衍生物。 喜树碱可特异性地与拓扑异 构酶 I结合， 后者诱导可逆性单链断裂， 从而使 DNA双链结构解旋； 伊立替康及其活性代谢物 SN-38可与拓扑异构酶 I-DNA复合物结合， 从而阻止断裂单链的再连接。 现有研究提示， 伊立替康的细胞毒作用 归因于 DNA合成过程中， 复制酶与拓扑异构酶 I-DNA-伊立替康 （或 SN-38) 三联复合物相互作用， 从而引起 DNA双链断裂。
盐酸伊立替康药理作用明显， 临床疗效确切， 在恶性肿瘤治疗中 应用广泛， 但是， 它和其他喜树碱类药物一样， 同样存在的一个问题 就是， 其结构中的饱和内酯环具有 pH依赖性， 在生理条件下会可逆的 转变为其羧酸盐形式， 而使抗肿瘤活性减弱。 盐酸伊立替康的现有市 售制剂为盐酸伊立替康注射液及其冻干粉针制剂， 临床上经静脉给药 后， 游离药物直接处在偏碱性的生理环境下， 其结构中的内酯环易发 生水解反应转变为羧酸盐形式， 从而失去活性， 间接的降低了药物的 疗效。 而且， 制剂的毒副作用较大， 主要表现为中性粒细胞减少和迟 发性腹泻。
脂质体作为近年来研究较为广泛的一种药物载体， 其主要特点是 可以保护被包封药物， 增加药物稳定性， 改变药物在体内的分布行为， 携载药物被动或主动靶向到病变部位。 脂质体作为抗肿瘤药物的良好 载体可有效的提高药物疗效， 降低毒副作用。
国际申请 WO2005/117878 公开了伊立替康脂质体的处方和制备 方法， 该制剂中含有伊立替康或盐酸伊立替康、 选自氢化大豆磷脂酰 胆碱、 磷脂酰乙醇胺、 卵磷脂、 心磷脂等磷脂、 胆固醇。 同样,中国专 利申请 CN1994279A也公开了伊立替康脂质体的处方和制备方法， 其 中实施例 5的使用磷脂酰胆碱作为磷脂制备了脂质体。
虽然上述专利文献中记载的处方已经具有较好效果， 但是如果制 备成适合人体使用的制剂， 该脂质体在稳定性、 粒径等方面仍不能令 人满意。 发明内容
本发明的目的是提供一种载药量较高， 且能同时满足包封率高， 稳定性好、 适合制备成制剂的伊立替康或盐酸伊立替康脂质体。
虽然 目前一些文献 （例如国际申请 WO2005/117878 和 CN1994279A)中记载了伊立替康的脂质体组成和制备方法， 其中个别 方案的部分指标较好。 但针对稳定性、 粒径等方面的控制并未提供任 何信息。 申请人经对脂质体进一歩研究， 令人惊奇的发现当选择处方 的辅料和用量关系符合某些条件时， 特别胆固醇的用量对脂质体粒径 和稳定性有一定的影响， 在选择中性磷和胆固醇的基础上控制二者之 间的比例， 能够使脂质体的粒径变得小而均匀， 并且提高了脂质体的 稳定性。 与其他处方相比， 本申请脂质体的稳定性等指标明显提高。 另外本发明与国际申请 WO2005/117878和 CN1994279A等技术相比， 产品中不含碱性官能团化合物和阳离子脂质， 处方组成简单， 载药量 高， 稳定性好， 本发明的脂质体有很好的抗肿瘤效果。
本发明的脂质体含有伊立替康或盐酸伊立替康、 中性磷脂以及胆 固醇， 其中所述胆固醇与中性磷脂的重量比例为 1 : 3〜5， 优选比例为 1： 3.5-4.5 , 最优选为 1 : 4。
本发明所使用的中性磷脂可选择氢化大豆卵磷脂 （HSPC)、 蛋黄 磷脂 （EPC )、 大豆磷脂 （SPC ) 等材料， 特别是当中性磷脂使用氢化 大豆卵磷脂时， 其效果最好。 当进一歩控制药物和磷脂之间的重量配 比关系为下述关系时， 脂质体的载药量极大地提高：
伊立替康或盐酸伊立替康 1份
中性磷脂 2〜5份， 优选 2.5-4份。 本发明的脂质体可根据本领域常规的脂质体制备方法制备得到， 对于本发明的脂质体来说， 优选使用离子梯度法制备。 在使用离子梯 度法时， 所述脂质体内水相与外水相之间具有缓冲剂形成的离子梯度， 优选所述脂质体内水相比外水相具有离子浓度高的离子梯度， 这可提 高贮存期间脂质体的粒径稳定， 更好的维持药效， 这能够控制脂质体 平均粒径小而均匀， 可使脂质体粒径在贮存期的变化减小到最低程度。
本发明通过在配方中加入亲水性高分子的脂质衍生物可使脂质体 粒径在贮存期的变化减小到最低程度， 同时聚乙二醇衍生物的加入可 以延长脂质体在体内的循环时间。聚乙二醇衍生物选自聚乙二醇 2000- 二硬酯酰磷脂酰乙醇胺（DSPE-PEG2QQQ)聚乙二醇 5000-二硬脂酰磷脂 酰乙醇胺、 聚乙二醇 2000-二棕榈酰磷脂酰乙醇胺、 聚乙二醇 5000-二 棕榈酰磷脂酰乙醇胺。 因此为了提高药物的长效性， 本申请优选在脂 质体中添加亲水性高分子的脂质衍生物， 在该处方比例基础上， 使用 DSPE-PEG2000是效果最明显的。 优选脂质衍生物与伊立替康或盐酸伊 立替康的重量比为 0.2〜0.4。
脂质体可进一歩含有带电荷磷脂， 带电荷磷脂选自二月桂酰磷脂 酰甘油、 二棕榈酰磷脂酰甘油、 二硬脂酰磷脂酰甘油、 二肉豆蔻酰磷 脂酰甘油、 二油酸磷脂酰丝氨酸、 二油酰磷脂酰甘油、 二月桂酰磷脂 酸、 二肉豆蔻酰磷脂酸或二硬脂酰磷脂酸中的一种或多种， 且带电荷 磷脂与中性磷脂的重量比例为 1 :5〜1 : 100。
本发明优选的脂质体含有以下重量配比的成分：
盐酸伊立替康 1份
氢化大豆卵磷脂 3.4-3.8份
聚乙二醇 2000-二硬酯酰磷脂酰乙醇胺 0.34-0.38份
胆固醇 0.8-0.95份， 且胆固醇与氢化大豆卵磷脂的比例为 1 : 4。 本申请还提供了伊立替康或盐酸伊立替康脂质体的制备方法， 本 发明的脂质体可选择通常的脂质体制备方法制备得到。 本领域技术人 员可根据本发明提供的脂质体的处方， 选择各种方法制备得到。 对于 本发明的脂质体所选择的处方而言， 优选的制备方法是离子梯度法。 该制备方法包含下述歩骤：
1)通过下述 A至 D中任何一种方法制备空白脂质体： A、 根据配方选用中性磷脂、 胆固醇溶于无水乙醇或无水乙醇-叔 丁醇混合溶剂中， 与缓冲剂混合， 减压除掉乙醇后得到空白脂质 体粗品， 之后采用高压均质机或 /和挤压设备制备空白脂质体至所 需的粒度；
B、 根据配方选用中性磷脂、 胆固醇溶于氯仿或氯仿 -甲醇混合溶 剂， 旋转蒸发形成脂质膜， 加入缓冲剂水化得到空白脂质体粗品， 之后采用高压均质机或 /和挤压设备制备空白脂质体至所需的粒
C、 根据配方选用中性磷脂、 胆固醇与缓冲剂混合， 之后采用高压 均质机或 /和挤压设备制备空白脂质体至所需的粒度；
D、 根据配方选用中性磷脂、 胆固醇溶于无水乙醇或无水乙醇-叔 丁醇混合溶剂中， 与缓冲剂混合， 之后采用高压均质机或 /和挤压 设备制备空白脂质体至所需的粒度；
2)脂质体膜内外水相离子梯度的产生：置换空白脂质体外水相，使 脂质体内水相与外水相产生离子梯度；
3)含药脂质体制备：配制盐酸伊立替康水溶液，加入到具有离子梯 度的空白脂质体分散液中， 加热搅拌， 孵育一定时间即得。
在歩骤 3 ) "含药脂质体制备"歩骤后， 还可包含以下歩骤：
4)游离药物的去除及样品的浓縮:在盐酸伊立替康脂质体中加入缓 冲介质， 采用切向流装置除去未包封的药物， 同时将样品浓縮至适当 的体积。 本申请还提供了含有上述脂质体的脂质体注射剂。 在将脂质体制 备成适合使用的注射剂时， 添加稳定剂是有益的。 本发明所使用的稳 定剂也可选择通常使用的稳定剂， 例如维生素 E、 乙二胺四乙酸等， 这 些稳定剂都对制剂的稳定性有所帮助。 对于上述处方而言， 通过对稳 定剂的研究发现乙二胺四乙酸或其盐相对于其他稳定剂效果最好， 对 于提高脂质体的稳定性最有好处， 因此可选用乙二胺四乙酸、 乙二胺 四乙酸二钠盐、 乙二胺四乙酸二钙盐中的一种或多种， 且稳定剂的加 入比例为 0%〜0.5 w/v %且下限不为 0%。
本发明的组合物中优选含有抗氧剂， 所述抗氧剂可选自水溶性抗 氧剂或油溶性抗氧剂， 所述油溶性抗氧剂选自 α-生育酚、 α-生育酸琥 珀酸酯、 α-醋酸生育酚或其混合物， 所述水溶性抗氧剂选自抗坏血酸、 亚硫酸氢钠、 亚硫酸钠、 焦亚硫酸钠、 L-半胱氨酸或其混合物， 抗氧 剂的加入比例为 0%〜0.5 w/v %且下限不为 0 %。
注射剂可以是注射液或冻干粉针形式。 制剂中可含有渗透压调节 剂， 所述渗透压调节剂选自葡萄糖、 蔗糖、 山梨醇、 甘露醇、 氯化钠、 甘油、 组氨酸及其盐酸化物、 甘氨酸及其盐酸化物、 赖氨酸、 丝氨酸、 谷氨酸、 精氨酸或缬氨酸中的一种或多种， 且渗透压调节剂的加入比 例为 0%〜5 w/v %且下限不为 0%。
对于冻干粉形式的制剂来说， 注射剂进一歩含有冻干保护剂， 进 行冷冻干燥后制得脂质体冻干粉针。 冻干保护剂选自葡萄糖、 蔗糖、 海藻糖、 甘露醇、 右旋糖酐或乳糖中的一种或多种。
本发明优选的注射剂脂质体含有以下重量配比的成分：
盐酸伊立替康 1份
氢化大豆卵磷脂 3.4-3.8份
聚乙二醇 2000-二硬酯酰磷脂酰乙醇胺 0.34-0.38份
胆固醇 0.8-0.95份
乙二胺四乙酸二钠 0.05-0.09份，
上述注射剂的制备方法包含下述歩骤：
1)通过下述 A至 D中任何一种方法制备空白脂质体：
A、 根据配方选用中性磷脂、 胆固醇溶于无水乙醇或无水乙醇-叔 丁醇混合溶剂中， 与缓冲剂混合， 减压除掉乙醇后得到空白脂质 体粗品， 之后采用高压均质机或 /和挤压设备制备空白脂质体至所 需的粒度；
B、 根据配方选用中性磷脂、 胆固醇溶于氯仿或氯仿 -甲醇混合溶 剂， 旋转蒸发形成脂质膜， 加入缓冲剂水化得到空白脂质体粗品， 之后采用高压均质机或 /和挤压设备制备空白脂质体至所需的粒
C、 根据配方选用中性磷脂、 胆固醇与缓冲剂混合， 之后采用高压 均质机或 /和挤压设备制备空白脂质体至所需的粒度；
D、 根据配方选用中性磷脂、 胆固醇溶于无水乙醇或无水乙醇-叔 丁醇混合溶剂中， 与缓冲剂混合， 之后采用高压均质机或 /和挤压 设备制备空白脂质体至所需的粒度；
2)脂质体膜内外水相离子梯度的产生：置换空白脂质体外水相，使 脂质体内水相与外水相产生离子梯度；
3)含药脂质体制备：配制盐酸伊立替康水溶液，加入到具有离子梯 度的空白脂质体分散液中， 加热搅拌， 孵育一定时间即得。
在歩骤 3 ) "含药脂质体制备"歩骤后， 还可包含以下歩骤： 4)游离药物的去除及样品的浓縮:在盐酸伊立替康脂质体中加入缓 冲介质， 采用切向流装置除去未包封的药物， 同时将样品浓縮至适当 的体积。
在得到脂质体后， 还可通过调整脂质体药物浓度， 定容， 过滤除 菌， 灌封于小瓶中， 得脂质体注射液； 或者向脂质体药物样品中加入 冻干保护剂， 调整药物浓度， 定容， 过滤除菌， 灌封于瓶中， 冷冻干 燥， 得冻干粉针。
本发明的有益效果：
将盐酸伊立替康制成脂质体制剂， 克服现有产品和技术的不足， 将 药物包裹于脂质体内水相中， 提高药物稳定性， 使药物在体内以内酯 环的状态存在， 能够长时间维持活性代谢物 SN-38在血浆中的浓度， 以达到增加制剂疗效， 降低药物的毒副作用。
本发明的伊立替康或盐酸伊立替康脂质体制剂通过控制特定的药 物、 磷脂和胆固醇的比例关系， 可达到解决脂质体载药量低的问题， 药脂比 （w/w) 可达 0.25 以上， 同时药物包封率可达 90%以上， 优选 的处方可达 95%以上； 本发明通过进一歩选择胆固醇和磷脂的用量关 系， 制备的脂质体粒径较小， 提高了脂质体的稳定性。 本发明还通过 对稳定剂的筛选， 优选出加入一定比例的乙二胺四乙酸盐明显提高了 脂质体的稳定性； 脂质体的粒径在 10nm-220nm之间， 分布均匀； 性 质稳定， 伊立替康或盐酸伊立替康脂质体注射剂影响因素实验结果表 明， 40°C度条件下放置 10天， 粒径和包封率均无显著变化， 各项指标 符合要求； 伊立替康或盐酸伊立替康脂质体注射剂与市售制剂相比， 抑瘤率显著提高， 毒性显著降低。 附图说明
图 1 显示本发明伊立替康或盐酸伊立替康脂质体注射剂的粒径分 布。
图 2显示本发明伊立替康或盐酸伊立替康脂质体注射剂的形态图。 图 3 显示本发明伊立替康或盐酸伊立替康脂质体注射剂的体内抗 肿瘤药效试验的结果。 具体实施方式
下面实施例用于进一歩说明本发明， 但本发明并不仅限于此实施 例。
实施例 1
处方
Figure imgf000008_0001
制备方法：
将处方量的氢化大豆卵磷脂（HSPC)、 胆固醇（CHOL )溶于适量 无水乙醇得脂质溶液， 与 100ml硫酸铵溶液混合， 减压除掉乙醇得到 空白脂质体粗品。之后采用高压均质机 1000bar均质 5次循环， 之后通 过挤出设备挤出脂质体控制其粒径 （挤出器铺 2张 Ο. ΐμιη挤出膜， 挤 出 5次）。之后加入配制好的 DSPE-PEG O水溶液，搅拌孵育 20分钟。 采用切向流超滤装置透析空白脂质体， 中间不间断的补充注射用水， 即得空白脂质体。
用注射用水配制盐酸伊立替康水溶液，按照盐酸伊立替康与 HSPC 的重量比例为 1 :3.5加入到上述具有离子梯度的空白脂质体分散液中， 60°C加热搅拌， 孵育 20分钟即得载药脂质体。 采用切向流超滤装置除 去未包封的药物， 同时将样品浓縮至约 50ml， 加入 0.45g氯化钠调节 渗透压。 调整药物浓度， 定容， 0.22 μιη滤膜过滤除菌， 充氮气灌封于 小瓶中， 即得盐酸伊立替康脂质体注射剂。
HSPC: CHOL 制备方法 平均粒径
均质后 138.7
6： 1
Ο. ΐμηι挤出 5次 92.26
均质后 136.2
5： 1
Ο. ΐμηι挤出 5次 89.5
均质后 123.4
4： 1
Ο. ΐμηι挤出 5次 87.26
均质后 145,1
3： 1
Ο. ΐμηι挤出 5次 Q '¾ 4
均质后 142
2.5： ： 1
Ο. ΐμηι挤出 5次 98.56 制备的各处方粒径变化如下表所示， 结果表明， 磷脂与胆固醇重 量比为 4: 1时样品粒径最小。
将不同磷脂与胆固醇重量比制备的样品在 25°C条件下放置， 考察 稳定性。 结果如下表， 25°C条件下放置 60天， 磷脂与胆固醇重量比为 4:1样品粒径及包封率无明显变化， 其它比例的样品粒径增大， 包封率 也有所下降， 可见， 磷脂与胆固醇重量比为 4:1的样品稳定性较好。 粒径
HSPC/ 放置时间 外观 电位 冷兽
口里 心水 溶血
EN% (z-v)
CHOL (25 °C，天） 性状 (mv) (mg/ml) 质0 /0 磷脂 nm
类白色
0 98.86 92.3 -30.5 5.05 0.58 0.39 混悬液
类白色
6： 1 30 98.56 94.3 -26.8 5.04 0.75 0.56 混悬液
类白色
60 98.20 95.9 -24.9 5.06 0.85 0.66 混悬液
类白色
0 99.37 87.3 -32.1 5.10 0.55 0.40 混悬液
4： 1 类白色
30 99.25 87.5 -30.9 5.11 0.64 0.50 混悬液
60 类白色 99.18 87.8 -28.6 5.09 0.70 0.62 混悬液
类白色
0 99.27 98.5 -35.8 5.12 0.60 0.38 混悬液
类白色
.5 1 30 98.75 100.2 -28.6 5.09 0.73 0.51 混悬液
类白色
60 98.19 101.7 -25.3 5.07 0.84 0.67 混悬液 结论：综合各项指标，结果证明胆固醇与磷脂之间的比例控制在 1 : 3〜5可达到比较好的使用效果， 最佳配比为 1 : 4。 实施例 2
处方
盐酸伊立替康 0.28g
氢化大豆卵磷脂 （HSPC) lg
醇 2000-二硬酯酰磷脂酰乙醇胺
O.lg
(DSPE-PEG
胆固醇 0.25g
硫酸铵 5g
乙二胺四乙酸二钠 0.02g
氯化钠 0.45g
注射用水 加至所需体积 制备方法：
将处方量的氢化大豆卵磷脂、 胆固醇溶于适量无水乙醇得脂质溶 液， 与 100ml硫酸铵溶液混合， 减压除掉乙醇得到空白脂质体粗品。 之后采用高压均质机 lOOObar均质 5次循环，之后通过挤出设备挤出脂 质体控制其粒径（挤出器铺 2张 Ο.ΐμιη挤出膜， 挤出 5次）。 之后加入 配制好的 DSPE-PEG2QQQ水溶液，搅拌孵育 20分钟。采用切向流超滤装 置透析空白脂质体， 中间不间断的补充注射用水， 即得空白脂质体。
用注射用水配制盐酸伊立替康水溶液，按照盐酸伊立替康与 HSPC 的重量比例为 1:3.5加入到上述具有离子梯度的空白脂质体分散液中， 60°C加热搅拌， 孵育 20分钟即得载药脂质体。 采用切向流超滤装置除 去未包封的药物， 同时将样品浓縮至约 50ml， 加入 0.45g氯化钠调节 实施例 3
空白脂质体处方及制备方法与实施例 2相同， 不同的是按照盐酸 伊立替康与 HSPC的重量比例分别为为 1:1.5、 1:2、 1:3.5、 1:4及 1:5 进行制备脂质体， 盐酸伊立替康脂质体样品包封率及粒径见下表。
CPT11/HSPC 包封率 （％) 载药量 （mg/ml) 粒径 (nm)
1:1.5 83.2 5.11 87.1
1:2 90.8 5.15 86.5
1:3.5 99.4 5.08 85.9
1:4 99.1 4.81 85.4
1:5 99.4 4.25 86.7 结果说明， 当盐酸伊立替康与 HSPC的比例为 1:1.5时包封率显著 降低， 而当其比例为 1:5时载药量下降明显，不适合制备成临床应用的 制剂， 当其比例在 1:2与 1:4之间时包封率及载药量均较高。 实施例 4
按照实施例 2中处方组份的用量来制备空白脂质体及载药脂质体， 制备方法同实施例 2 所述方法， 所不同的是分别采用高纯蛋黄磷脂 (EPC)、 高纯大豆磷脂 （SPC) 替换处方中的 HSPC来制备。 将制备 的脂质体样品置于 25°C条件下考察稳定性。 结果见下表。 试验结果表 明， 采用 HSPC制备的脂质体样品稳定性最好， 25°C条件下放置 2个 月主要指标无明显变化。 时间 PC组成 包封率 （％) 载药量 （mg/ml) 粒径 (nm)
HSPC 99.4 5.08 85.9
0M EPC 99.5 5.10 87.5
SPC 99.2 5.01 86.9
HSPC 99.5 5.10 85.5
1M EPC 92.4 5.07 88.2
SPC 93.9 5.05 87.3 HSPC 98.7 5.07 86.5
2M EPC 85.8 5.06 93.2
SPC 89.6 5.02 91.5
、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、 实施例 5
处方
盐酸伊立替康 0.28g
氢化大豆卵磷脂 （HSPC) lg
聚乙二醇 2000-二硬酯酰磷脂酰乙醇胺
O.lg
(DSPE-PEG誦）
胆固醇 0.25g
生理盐水溶液 50ml
注射用水 加至所需体积 制备方法 <1> ：
乙醇注入法： 将处方量的氢化大豆卵磷脂、 DSPE-PEG2QQQ、 胆固 醇溶于适量无水乙醇得脂质溶液， 注入到盐酸伊立替康的生理盐水溶 液中， 减压除掉乙醇得到脂质体粗品。 之后采用高压均质机 lOOObar 均质 5次循环， 之后通过挤出设备挤出脂质体控制其粒径（挤出器铺 2 张 Ο.ΐμιη挤出膜， 挤出 5次）。 调整药物浓度， 定容， 0.22 μιη滤膜过 滤除菌， 充氮气灌封于小瓶中， 即得盐酸伊立替康脂质体注射剂。
制备方法 <2>：
薄膜分散法： 将处方量的氢化大豆卵磷脂、 DSPE-PEG2QQQ、 胆固 醇溶于适量氯仿得脂质溶液， 将脂质溶液旋转蒸发成膜， 除尽氯仿然 后加入盐酸伊立替康的生理盐水溶液水化孵育约 lh。 之后采用高压均 质机 1 OOObar均质 5次循环，再通过挤出设备挤出脂质体控制其粒径 (挤 出器铺 2张 Ο.ΐμιη挤出膜， 挤出 5次）。调整药物浓度， 定容， 0.22 μιη 滤膜过滤除菌， 充氮气灌封于小瓶中， 即得盐酸伊立替康脂质体注射 剂。
测定制备方法 <1>、 <2>以及实施例 2制备的盐酸伊立替康脂质体 包封率及粒径。 实施例 2 99.4 85.9 制备方法 <1> 15.3 87.9
制备方法 <2> 17.8 90.2 结果表明， 采用被动载药法如乙醇注入法及薄膜蒸发法制备盐酸 伊立替康脂质体时， 可以制备出目的产品。 但其样品包封率较低， 大 部分药物都没有进入到脂质体中； 而采用主动载药法制备的样品 （实 施例 2) 包封率高， 载药量高， 粒径小而均匀， 所以采用主动载药法。 本发明中即采用离子梯度法制备盐酸伊立替康脂质体有非常好的效 果。
实施例 6 处方 处方 1 处方 2 处方 3 处方 4
HSPC i g i g i g i g 胆固醇 250 mg 250 mg 250 mg 250 mg
PEG誦 -DSPE o. i g 0.1 g 0.1 g 0.1 g 维生素 E 1 0.02 g 1 0.02 g
EDTA-2Na 1 1 0.02 g 0.02 g 硫酸铵溶液（300mM) 100 ml 100 ml 100 ml 100 ml 盐酸伊立替康 0.3 g 0.3 g 0.3 g 0.3 g 制备方法：
空白脂质体： 将脂质乙醇注入。 均质 1000bar， 6次； 200nm挤出 3次， 100 nm挤出 5次； 力口入 PEG2000-DSPE， 60°C赋予 30 min。 切向 流透析 3次， 每次加入 50 ml。 其中 VE加在磷脂有机溶剂中， EDTA 加在硫酸铵溶液中。
载药脂质体： 配制约 10mg/ml的盐酸伊立替康水溶液， 加入空白 脂质体中， 60°C赋予 15 min。用切向流浓縮样品至约 50ml，即为 5mg/ml 样品。
稳定性结果见下表， 可见， 单独加入乙二胺四乙酸二钠样品各项 指标无显著变化， 能显著提高了脂质体的稳定性， 而其它稳定剂对脂 质体的稳定性没有明显改善。
Figure imgf000014_0001
实施例 7
处方 ( 1 )
盐酸伊立替康 0.5g 氢化大豆卵磷脂 1.5g
胆固醇 0.4g
硫酸锰 10g
甘露醇 2-5g
注射用水 加至所需体积
制备方法：
将处方量的氢化大豆卵磷脂、 胆固醇溶于适量无水乙醇得脂质溶 液， 与 100ml硫酸锰溶液混合， 减压除掉乙醇得到空白脂质体粗品。 之后通过挤出设备挤出脂质体控制其粒径 （挤出器铺 2张 Ο. ΐμιη挤出 膜， 挤出 5次）。 采用切向流超滤装置透析空白脂质体， 中间不间断的 补充注射用水， 即得空白脂质体。 用注射用水配制盐酸伊立替康水溶 液， 加入到具有离子梯度的空白脂质体分散液中， 50°C加热搅拌， 孵 育 20分钟即得载药脂质体。 采用切向流超滤装置除去未包封的药物， 同时将样品浓縮至约 50ml， 加入 2.5g甘露醇调节渗透压。 调整药物浓 度， 定容， 0.22 μιη滤膜过滤除菌， 充氮气灌封于小瓶中， 即得盐酸伊 立替康脂质体注射剂。经过纳米粒度测定仪测得脂质体粒度为 89.3nm 包封率为 97.5% 处方 (2)
盐酸伊立替康 lg
氢化蛋黄卵磷脂 （HEPC) 3.45g
胆固醇 0.8g 硫酸镁 10g 组氨酸 2-5g 注射用水 加至所需体积
制备方法：
将处方量的氢化蛋黄卵磷脂、 胆固醇溶于适量无水乙醇得脂质溶 液， 与 100ml硫酸镁溶液混合。 之后通过挤出设备挤出脂质体控制其 粒径（挤出器铺 2张 Ο.ΐμιη挤出膜， 挤出 5次）。 采用切向流超滤装置 透析空白脂质体， 中间不间断的补充注射用水， 即得空白脂质体。 用 注射用水配制盐酸伊立替康水溶液， 加入到具有离子梯度的空白脂质 体分散液中， 50°C加热搅拌， 孵育 20分钟即得载药脂质体。 采用切向 流超滤装置除去未包封的药物， 同时将样品浓縮至约 50ml， 加入 2.5g 组氨酸调节渗透压。 调整药物浓度， 定容， 0.22 μιη滤膜过滤除菌， 充 氮气灌封于小瓶中， 即得盐酸伊立替康脂质体注射剂。 经过纳米粒度 测定仪测得脂质体粒度为 87.6nm， 包封率为 98.1%。 处方 (3 )
盐酸伊立替康 0.3g 氢化大豆卵磷脂 （HSPC) lg
聚乙二醇 2000-二硬酯酰磷脂酰乙
0.05g
醇胺 （DSPE-PEG2000)
胆固醇 0.25g
硫酸铵 5g 氯化钠 0.45g
注射用水 加至所需体积
制备方法：
将处方量的氢化大豆卵磷脂、 胆固醇溶于适量无水乙醇得脂质溶 液， 与 100ml硫酸铵溶液混合， 减压除掉乙醇得到空白脂质体粗品。 之后采用高压均质机 lOOObar均质 5 次循环后， 之后加入配制好的 DSPE-PEG2QQQ水溶液， 搅拌孵育 20分钟。采用切向流超滤装置透析空 白脂质体， 中间不间断的补充注射用水， 即得空白脂质体。 用注射用 水配制盐酸伊立替康水溶液， 加入到具有离子梯度的空白脂质体分散 液中， 60°C加热搅拌， 孵育 20分钟即得载药脂质体。 采用切向流超滤 装置除去未包封的药物， 同时将样品浓縮至约 50ml， 加入 0.45g氯化 钠调节渗透压。 调整药物浓度， 定容， 0.22 μιη滤膜过滤除菌， 充氮气 灌封于小瓶中， 即得盐酸伊立替康脂质体注射剂。 经过纳米粒度测定 仪测得脂质体粒度为 87.3nm， 包封率为 99.2%。 实施例 8
处方
盐酸伊立替康 0.5g 氢化大豆卵磷脂 （HSPC) ig
心肌磷脂 （CL) 0.5g
聚乙二醇 5000-二硬酯酰磷脂酰乙醇
0.5g
胺 （DSPE-PEG
α-生育酚 0.05g 胆固醇 0.35g
柠檬酸 5.76g
氯化钠 约 3.6g
注射用水 加至所需体积
制备方法：
将处方量的氢化大豆卵磷脂、 心肌磷脂、 DSPE-PEG5QQQ、 胆固醇、 α-生育酚溶于适量无水乙醇得脂质溶液， 与 100ml柠檬酸溶液混合， 减压除掉乙醇得到空白脂质体粗品。 之后采用高压均质机 lOOObar, 均 质 5次。采用切向流超滤装置透析空白脂质体，中间不间断的补充 0.9% 氯化钠溶液 400ml， 即得空白脂质体。用注射用水配制盐酸伊立替康溶 液， 加入到具有离子梯度的空白脂质体分散液中， 60°C加热搅拌， 孵 育 20分钟即得载药脂质体。 采用切向流超滤装置除去未包封的药物， 同时将样品浓縮至约 50ml。 调整药物浓度， 定容， 0.22 μιη滤膜过滤 除菌， 充氮气灌封于小瓶中， 即得盐酸伊立替康脂质体注射剂。 经过 纳米粒度测定仪测得脂质体粒度为 85.8nm， 包封率为 98.6%。 实施例 9 盐酸伊立替康 0.8g
棕榈酰磷脂酰胆碱 (DPPC) 2g
棕榈酰磷脂酰甘油 (DPPG) 0.2g
胆固醇 0.5g
抗坏血酸 0.05g
乙二胺四乙酸二钠 0.05g
硫酸铵 5g
氯化钠 约 3.6g
注射用水 加至所需体积
制备方法：
将处方量的 DPPC、 DPPG,胆固醇溶于适量无水乙醇得脂质溶液， 与 100ml硫酸铵溶液 （含乙二胺四乙酸二钠） 混合， 减压除掉乙醇得 到空白脂质体粗品。 之后采用高压均质机 1000bar， 均质 5次。 采用切 向流超滤装置透析空白脂质体， 中间不间断的补充 0.9%氯化钠溶液 400ml, 即得空白脂质体。 用注射用水配制盐酸伊立替康溶液， 加入到 具有离子梯度的空白脂质体分散液中， 60°C加热搅拌， 孵育 20分钟即 得载药脂质体。 采用切向流超滤装置除去未包封的药物， 同时将样品 浓縮至约 50ml， 加入抗坏血酸。 调整药物浓度， 定容， 0.22 μιη滤膜 过滤除菌， 充氮气灌封于小瓶中， 即得盐酸伊立替康脂质体注射剂。 经过纳米粒度测定仪测得脂质体粒度为 89.4nm， 包封率为 97.2%。 实施例 10
处方
盐酸伊立替康 0.5g
氢化大豆卵磷脂 （HSPC ) ig
聚乙二醇 5000-二硬酯酰磷脂酰乙
o.ig
醇胺 （DSPE-PEG2000)
α-生育酚 0.05g
胆固醇 0.3g
硫酸铵 5g
氯化钠 约 3.6g
蔗糖 2g 甘露醇 lg
注射用水 加至所需体积
制备方法：
将处方量的氢化大豆卵磷脂、胆固醇、 α-生育酚溶于适量无水乙醇 得脂质溶液， 与 100ml硫酸铵溶液混合， 减压除掉乙醇得到空白脂质 体粗品。 之后采用高压均质机 1000bar， 均质 5次， 再通过挤出设备挤 出脂质体（挤出器铺 5张 lOOnm挤出膜， 挤出 5次）， 之后加入配制好 的 DSPE-PEG5QQQ水溶液，搅拌孵育 20分钟。采用切向流超滤装置透析 空白脂质体， 中间不间断的补充 0.9%氯化钠溶液 400ml， 即得空白脂 质体。 用注射用水配制盐酸伊立替康溶液， 加入到具有离子梯度的空 白脂质体分散液中， 60°C加热搅拌， 孵育 20分钟即得载药脂质体。 采 用切向流超滤装置除去未包封的药物， 同时将样品浓縮至约 50ml， 加 入蔗糖和甘露醇使混合均匀。 调整药物浓度， 定容， 0.22 μηι滤膜过滤 除菌， 罐装于西林瓶中， 冷冻干燥， 即得盐酸伊立替康脂质体冻干粉 针。 将脂质体冻干粉针水化后测定脂质体粒度为 90.8nm， 包封率为 97.5%。 试验例 1
根据本发明制得的成品 （按实施例 2 ) 的理化特征：
【粒径分布】取本品适量， 以水稀释后用动态光散射法进行测定。 测定波长 λ=633ηιη， 测定角度 173°， 温度 25°C。 粒径大小以强度径 (Intensity) 数据表示。 粒径分布图见图 1， 平均粒径为 85.9nm。
【形态学考察】 吸取脂质体样品适量， 将铜网置于干净滤纸上， 向铜网上滴加样品， 用 2%磷钨酸染色， 待干燥后在 JEM2010透射电 镜 (日本电子株式会社）下观察本品。形态图见图 2。经透射电镜观察， 伊立替康脂质体形态均为典型的双分子层结构， 粒径大部分在 200 nm 以下， 与动态光散射法测定的结果相吻合。
【包封率测定】 药物含量测定方法： 色谱柱： Agilent ZORBAX Eclipse XDB-C18 (4.6χ 150ηιηι,5μηι) ; 流动相： 乙腈 - 0.05Μ磷酸二氢 钾缓冲液 （ρΗ 4， 含 1%三乙胺） = 20:80; 柱温： 40°C ·' 进样体积： 20 μ∑·, 流速： 1.0mL/min。 包封率测定方法： 吸取 lmL样品溶液至 10mL量瓶中， 加入水稀 释至刻度， 摇匀， 置 8010型超滤器 （MILLIPORE公司） 超滤， 弃去 初滤液， 取续滤液为供试品溶液。 分别吸取供试品溶液、 对照品溶液 20μΙ注入液相色谱仪，记录色谱图，以外标法计算制剂游离药物含量， 记为 W; 另按含量测定项下方法计算本品的药物总含量， 记为 WQ。 按 下式计算样品的包封率。
w - w
包封率 = w° W x lOO%
w0
测定结果： 本品包封率为 99.4。
【影响因素试验】 取本品置于不同条件下进行影响因素考察， 结 果如下表所示：
Figure imgf000020_0001
结果表明， 样品对光照敏感， 经过强光照射样品外观变黄， 含量 下降， 有关物质明显增大； 样品在高温 40°C时包封率和粒径无明显变 化， 但有关物质略增大； 低温和冷冻循环试验表明样品产生大粒子。 综合考虑磷脂在高温状态下的不稳定性， 结合影响因素试验结果， 本 品应低温避光贮存。
【体内抗肿瘤药效试验】
药物名称： 按实施例 2制备的盐酸伊立替康脂质体 （CPT-11脂质 体） 由上海恒瑞医药有限公司提供， 盐酸伊立替康注射液 （CPT-11 , 江苏恒瑞医药股份有限公司提供）
配制方法： 均用生理盐水配成所需浓度。
实验动物： BALB/cA-nude裸小鼠， 6-7周， $， 购自上海斯莱克 实验动物有限责任公司。 合格证号： SCXK (沪） 2007— 0005。 饲养环 境： SPF级。
试验方法： 裸小鼠皮下接种人结肠癌 Ls-174t细胞， 待肿瘤生长至 150-300mm3后， 将动物随机分组 ((10：)。 给药剂量和给药方案见下表。 每周测 2— 3次瘤体积， 称鼠重， 记录数据。 肿瘤体积 （V) 计算公式 为：
V= l/2xaxb2 其中 a、 b分别表示长、 宽。
体积簡 ϊ ) 体积(1議3) RTV
Veliicle DO, 5 IV 219.S ± 37.2 2013.7 303.1 ± 2.3
CPT-i l廳质偉 Ltoig kg DO, 3 W 212.2. ± 42.1 732..Z 162.6 ±： 0.7
CPT-U撤质体 3i mg/kg DO, 3 IV 205.0 ± 49.0 265..! 122.9 ± 0.4
CPT-I i I ffig¾ DO, 3 IV 204.6 ± 44.7 844, 197.5 ± 0.9 c!O: 第一'次给药 UT 1瘤体积; P value指与对照相比。 实验结果：
CPT-11脂质体及 CPT-11均明显抑制人结肠癌 Ls-174t裸小鼠移植瘤的生长， CPT-1 明显的剂量依赖性， 高剂量 （3mg/kg) 给药时， 有 4/14个肿瘤部分消退， 低剂量 （lmg 10mg/kg给药时相当， 提示 CPT-11脂质体的疗效有可能比其注射液至少提高 10倍， 具
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Claims

权利要求书-
1、 一种伊立替康或盐酸伊立替康脂质体， 其特征在于所述脂质体 含有伊立替康或盐酸伊立替康、 中性磷脂以及胆固醇， 其中所述胆固 醇与中性磷脂的重量比例为 1 : 3〜5。
2、 根据权利要求 1所述的脂质体， 其特征在于所述中性磷脂和伊 立替康或盐酸伊立替康符合以下重量配比：
伊立替康或盐酸伊立替康 1份
中性磷脂 2〜5份， 优选 2.5-4份。
3、 根据权利要求 1所述的脂质体， 其特征在于所述中性磷脂含有 氢化大豆卵磷脂。
4、 根据权利要求 3所述的脂质体， 其特征在于所述中性磷脂是氢 化大豆卵磷脂。
5、 根据前述任意一项权利要求所述的脂质体， 其特征在于所述胆 固醇与中性磷脂的比例为 1 : 3.5-4.5 , 最优选为 1 : 4。
6、 根据前述任意一项权利要求所述的脂质体， 其特征在于在所述 脂质体通过离子梯度法制备得到。
7、 根据权利要求 6所述的脂质体， 其特征在于在所述脂质体内水 相与外水相之间具有缓冲剂形成的离子梯度， 优选所述脂质体内水相 比外水相具有离子浓度高的离子梯度。
8、 根据前述任意一项权利要求所述的脂质体， 其特征在于所述脂 质体还含有亲水性高分子的脂质衍生物， 优选是 DSPE-PEG2QQQ。
9、 根据权利要求 8所述的脂质体， 其特征在于所述亲水性高分子 的脂质衍生物与伊立替康或盐酸伊立替康的重量比为 0.2〜0.4。
10、 根据前述任意一项权利要求所述的脂质体， 其特征在于所述 脂质体进一歩含有带电荷磷脂， 所述带电荷磷脂选自二月桂酰磷脂酰 甘油、 二棕榈酰磷脂酰甘油、 二硬脂酰磷脂酰甘油、 二肉豆蔻酰磷脂 酰甘油、 二油酸磷脂酰丝氨酸、 二油酰磷脂酰甘油、 二月桂酰磷脂酸、 二肉豆蔻酰磷脂酸或二硬脂酰磷脂酸中的一种或多种， 且带电荷磷脂 与中性磷脂的重量比例为 1 :5〜1 : 100。
11、 根据权利要求 1 所述的脂质体， 其特征在于所述脂质体含有 以下重量配比的成分：
盐酸伊立替康 1份
氢化大豆卵磷脂 3.4-3.8份
聚乙二醇 2000-二硬酯酰磷脂酰乙醇胺 0.34-0.38份
胆固醇 0.8-0.95份， 且胆固醇与氢化大豆卵磷脂的比例为 1 : 4。
12、 权利要求 1-11任意一项所述脂质体的制备方法， 其特征在于 该制备方法包含下述歩骤：
1)通过下述 A至 D中任何一种方法制备空白脂质体：
A.根据配方选用中性磷脂、 胆固醇溶于无水乙醇或无水乙醇 -叔丁 醇混合溶剂中， 与缓冲剂混合， 减压除掉乙醇后得到空白脂质体粗品， 之后采用高压均质机或 /和挤压设备制备空白脂质体至所需的粒度；
B.根据配方选用中性磷脂、胆固醇溶于氯仿或氯仿-甲醇混合溶剂， 旋转蒸发形成脂质膜， 加入缓冲剂水化得到空白脂质体粗品， 之后采 用高压均质机或 /和挤压设备制备空白脂质体至所需的粒度；
C.根据配方选用中性磷脂、 胆固醇与缓冲剂混合， 之后采用高压 均质机或 /和挤压设备制备空白脂质体至所需的粒度；
D.根据配方选用中性磷脂、 胆固醇溶于无水乙醇或无水乙醇 -叔丁 醇混合溶剂中， 与缓冲剂混合， 之后采用高压均质机或 /和挤压设备制 备空白脂质体至所需的粒度；
2)脂质体膜内外水相离子梯度的产生：置换空白脂质体外水相，使 脂质体内水相与外水相产生离子梯度； 3)含药脂质体制备：配制盐酸伊立替康水溶液，加入到具有离子梯 度的空白脂质体分散液中， 加热搅拌， 孵育， 即得。
13、 根据权利要求 12所述的制备方法， 其特征在于在 3 ) 含药脂 质体制备歩骤后， 还包含以下歩骤：
4)游离药物的去除及样品的浓縮:在盐酸伊立替康脂质体中加入缓 冲介质， 采用切向流装置除去未包封的药物， 同时将样品浓縮至适当 的体积。
14、根据权利要求 12-13任意一项所述的制备方法，其特征在于所 述缓冲剂选自含有 Na+、 K+、 Fe2+、 Ca2+、 Ba2+、 Mn2+、 Mg2+、 Li+、 NH4 + 或 H+离子盐的一种或多种。
15、 含有权利要求 1-11任意一项所述伊立替康或盐酸伊立替康脂 质体的脂质体注射剂。
16、 根据权利要求 15所述的脂质体注射剂， 其特征在于所述注射 剂含有稳定剂， 所述稳定剂选自乙二胺四乙酸、 乙二胺四乙酸二钠盐、 乙二胺四乙酸二钙盐中的一种或多种， 稳定剂的加入比例为 0%〜0.5 w/v %且下限不为 0%， 优选是乙二胺四乙酸二钠。
17、 根据权利要求 15所述的脂质体注射剂， 其特征在于所述注射 剂是注射液或冻干粉针。
18、 根据权利要求 15所述的脂质体注射剂， 其特征在于所述注射 剂含有渗透压调节剂， 所述渗透压调节剂选自葡萄糖、 蔗糖、 山梨醇、 甘露醇、 氯化钠、 甘油、 组氨酸及其盐酸化物、 甘氨酸及其盐酸化物、 赖氨酸、 丝氨酸、 谷氨酸、 精氨酸或缬氨酸中的一种或多种， 渗透压 调节剂的加入比例为 0%〜5 w/v %且下限不为 0%。
19、 根据权利要求 15所述的脂质体注射剂， 其特征在于所述注射 剂进一歩含有抗氧剂， 所述抗氧剂选自水溶性抗氧剂或油溶性抗氧剂， 所述油溶性抗氧剂选自 α-生育酚、 α-生育酸琥珀酸酯、 α-醋酸生育酚或 其混合物， 所述水溶性抗氧剂选自抗坏血酸、 亚硫酸氢钠、 亚硫酸钠、 焦亚硫酸钠、 L-半胱氨酸或其混合物， 抗氧剂的加入比例为 0%〜0.5 w/v %且下限不为 0%。
20、 根据权利要求 17所述的脂质体注射剂， 其特征在于所述注射 剂是冻干粉针， 其中含有冻干保护剂， 是通过冷冻干燥制得的脂质体 冻干粉针。
21、 根据权利要求 15所述的脂质体注射剂， 其特征在于所述注射 剂脂质体含有以下重量配比的成分：：
盐酸伊立替康 1份
氢化大豆卵磷脂 3.4-3.8份
聚乙二醇 2000-二硬酯酰磷脂酰乙醇胺 0.34-0.38份
胆固醇 0.8-0.95份
乙二胺四乙酸二钠 0.05-0.09份， 且胆固醇与氢化大豆卵磷脂的比例为 1 : 4。
22、权利要求 15-21任意一项所述脂质体注射剂的制备工艺，其特 征在于所述工艺包括权利要求 12-13任意一项的制备方法。
23、 根据权利要求 22的制备工艺， 其特征在于所述工艺还包括： 定容、 除菌、 分装： 调整脂质体药物浓度， 定容， 过滤除菌， 灌 封于小瓶中， 得脂质体注射液； 或者
向脂质体药物样品中加入冻干保护剂， 调整药物浓度， 定容， 过 滤除菌， 灌封于瓶中， 冷冻干燥， 得冻干粉针。
PCT/CN2009/075298 2009-12-03 2009-12-03 伊立替康或盐酸伊立替康脂质体及其制备方法 WO2011066684A1 (zh)

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ES09851784.0T ES2547698T3 (es) 2009-12-03 2009-12-03 Liposoma de irinotecán o su clorhidrato y método de preparación del mismo
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HUE09851784A HUE027467T2 (en) 2009-12-03 2009-12-03 Liposomes or hydrochlorides of irinotecan and their preparation method
RU2012123875/15A RU2526114C2 (ru) 2009-12-03 2009-12-03 Липосомы иринотекана или его солей, способ их получения
PT98517840T PT2508170E (pt) 2009-12-03 2009-12-03 Lipossoma de irinotecano ou o seu cloridrato e seu método de preparação
EP09851784.0A EP2508170B1 (en) 2009-12-03 2009-12-03 Liposome of irinotecan or its hydrochloride and preparation method thereof
DK09851784.0T DK2508170T3 (en) 2009-12-03 2009-12-03 LIPOSOM OF IRINOTECAN OR ITS HYDROCHLORIDE AND ITS PROCEDURE
CA2782911A CA2782911C (en) 2009-12-03 2009-12-03 Liposome of irinotecan or its hydrochloride and preparation method thereof
PL09851784T PL2508170T3 (pl) 2009-12-03 2009-12-03 Liposom zawierający irynotekan lub jego chlorowodorek i metoda jego otrzymywania
US13/512,048 US20120282325A1 (en) 2009-12-03 2009-12-03 Liposome of irinotecan or its hydrochloride and preparation method thereof
MX2012005775A MX2012005775A (es) 2009-12-03 2009-12-03 Liposoma de irinotecano o su clorhidrato y metodo para la preparacion de los mismos.
JP2012541296A JP5645954B2 (ja) 2009-12-03 2009-12-03 イリノテカン又はその塩酸塩のリポソーム及びその製造方法
BR112012012151A BR112012012151B8 (pt) 2009-12-03 2009-12-03 lipossoma de irinotecano ou cloridrato de irinotecano, método de preparação do mesmo, bem como injeção de lipossoma e processo de preparação da mesma
KR1020127015958A KR20120089754A (ko) 2009-12-03 2009-12-03 이리노테칸 또는 그의 하이드로클로라이드의 리포좀 및 그의 제조 방법
HK11113962.4A HK1159482A1 (en) 2009-12-03 2011-12-28 Liposome of irinotecan or its hydrochloride and preparation method thereof
ZA2012/03316A ZA201203316B (en) 2009-12-03 2012-05-08 Liposome of irinotecan or its hydrochloride and preparation method thereof
HRP20150911TT HRP20150911T1 (hr) 2009-12-03 2015-09-01 Liposom irinotekana ili njegovog hidroklorida te postupak njegove pripreme
SM201500245T SMT201500245B (it) 2009-12-03 2015-10-09 Liposoma di irinotecan o rispettivo cloridrato e procedimento di preparazione dello stesso
CY20151100943T CY1116811T1 (el) 2009-12-03 2015-10-21 Λιποσωμα ιρινοτεκανης ή του υδροχλωριδιου αυτης και μεθοδος παρασκευης αυτου
US15/464,922 US10022365B2 (en) 2009-12-03 2017-03-21 Liposome of irinotecan or irinotecan hydrochloride and preparation method thereof

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US10980795B2 (en) 2012-06-13 2021-04-20 Ipsen Biopharm Ltd. Methods for treating pancreatic cancer using combination therapies comprising liposomal irinotecan
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CN102935066B (zh) * 2011-08-16 2014-09-17 齐鲁制药有限公司 一种伊立替康脂质体制剂及其制备方法
CN102935066A (zh) * 2011-08-16 2013-02-20 齐鲁制药有限公司 一种伊立替康脂质体制剂及其制备方法
KR101842279B1 (ko) * 2012-03-29 2018-03-26 우석대학교 산학협력단 이리노테칸의 안정성증진을 위한 주사제용 조성물
JP2015520126A (ja) * 2012-04-10 2015-07-16 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア ビス−ポリマー脂質−ペプチド複合体及びそのナノ粒子
US9949927B2 (en) 2012-04-10 2018-04-24 The Regents Of The University Of California Bis-polymer lipid-peptide conjugates and nanoparticles thereof
JP2018150327A (ja) * 2012-04-10 2018-09-27 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア ビス−ポリマー脂質−ペプチド複合体及びそのナノ粒子
US10806702B2 (en) 2012-04-10 2020-10-20 The Regents Of The University Of California Bis-polymer lipid-peptide conjugates and nanoparticles thereof
US10980795B2 (en) 2012-06-13 2021-04-20 Ipsen Biopharm Ltd. Methods for treating pancreatic cancer using combination therapies comprising liposomal irinotecan
US11369597B2 (en) 2012-06-13 2022-06-28 Ipsen Biopharm Ltd. Methods for treating pancreatic cancer using combination therapies
CN103181898A (zh) * 2012-11-23 2013-07-03 杭州师范大学 一种奥沙利铂脂质体及其应用
CN104906586A (zh) * 2014-03-10 2015-09-16 中国科学院上海药物研究所 一种盐酸伊立替康复合磷脂组合物、制备方法及其应用
US11318131B2 (en) 2015-05-18 2022-05-03 Ipsen Biopharm Ltd. Nanoliposomal irinotecan for use in treating small cell lung cancer
US11844795B2 (en) 2015-08-20 2023-12-19 Ipsen Biopharm Ltd. Combination therapy for cancer treatment
US11344552B2 (en) 2015-08-21 2022-05-31 Ipsen Biopharm Ltd. Methods for treating metastatic pancreatic cancer using combination therapies comprising liposomal irinotecan and oxaliplatin
US10993914B2 (en) 2015-10-16 2021-05-04 Ipsen Biopharm Ltd. Stabilizing camptothecin pharmaceutical compositions
US11071726B2 (en) 2016-11-02 2021-07-27 Ipsen Biopharm Ltd. Treating gastric cancer using combination therapies comprising liposomal irinotecan, oxaliplatin, 5-fluorouracil (and leucovorin)
WO2018083470A1 (en) * 2016-11-02 2018-05-11 Ipsen Biopharm Ltd. Treating gastric cancer using combination therapies comprising liposomal irinotecan, oxaliplatin, 5-fluoruracil (and leucovorin)
AU2017354903B2 (en) * 2016-11-02 2023-04-13 Ipsen Biopharm Ltd. Treating gastric cancer using combination therapies comprising liposomal irinotecan, oxaliplatin, 5-fluoruracil (and leucovorin)
CN114177278A (zh) * 2021-10-18 2022-03-15 山东多美康生物医药有限公司 脂质体制备

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PL2508170T3 (pl) 2015-12-31
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ZA201203316B (en) 2013-11-27
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KR20120089754A (ko) 2012-08-13
US10022365B2 (en) 2018-07-17
BR112012012151B1 (pt) 2021-01-19
US20170189392A1 (en) 2017-07-06
CN102271659A (zh) 2011-12-07
AU2009356132B2 (en) 2015-01-22
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CN102271659B (zh) 2013-09-18
US20120282325A1 (en) 2012-11-08

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