WO2020093836A1 - 伊立替康脂质体制剂及其制备与应用 - Google Patents

伊立替康脂质体制剂及其制备与应用 Download PDF

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WO2020093836A1
WO2020093836A1 PCT/CN2019/110542 CN2019110542W WO2020093836A1 WO 2020093836 A1 WO2020093836 A1 WO 2020093836A1 CN 2019110542 W CN2019110542 W CN 2019110542W WO 2020093836 A1 WO2020093836 A1 WO 2020093836A1
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irinotecan
liposome
sulfonate
solution
aqueous phase
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French (fr)
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魏晓慧
姜炳奇
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Highfield Biopharmaceutical Corp
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Highfield Biopharmaceutical Corp
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Priority to JP2021522421A priority Critical patent/JP7395579B2/ja
Priority to US17/290,263 priority patent/US20220031618A1/en
Priority to EP19881055.8A priority patent/EP3878435B1/en
Publication of WO2020093836A1 publication Critical patent/WO2020093836A1/zh
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    • 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—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/20—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing sulfur, e.g. dimethyl sulfoxide [DMSO], docusate, sodium lauryl sulfate or aminosulfonic acids
    • 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
    • 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
    • A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
    • 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—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1277—Preparation processes; Proliposomes
    • 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 invention belongs to the technical field of nano drug delivery, and particularly relates to an irinotecan liposome preparation and its preparation and application.
  • Irinotecan (irinotecan, CPT-11), a semi-synthetic derivative of camptothecin, is an effective drug for the treatment of metastatic colon cancer. Camptothecin can specifically bind to topoisomerase I, which induces reversible single-strand breaks, thereby unwinding the DNA double-stranded structure; irinotecan and its active metabolite SN-38 can bind to topoisomerase I-DNA complex binding, thereby preventing reconnection of broken single strands.
  • irinotecan hydrochloride injection is the hydrochloride salt of irinotecan and has good water solubility.
  • irinotecan hydrochloride powder injection has greater toxic and side effects, mainly manifested as neutropenia and delayed diarrhea.
  • Onivyde uses the triethylamine salt of sucrose octasulfate ( Figure 1a) as the internal aqueous phase, and uses the gradient of triethylamine between the internal and external aqueous phase of the liposome to encapsulate irinotecan in the aqueous phase of the liposome to form irinote.
  • Kang-sucrose octasulfate salt to achieve the effect of sustained release in vivo .).
  • sucrose octasulfate triethylamine is not only expensive but also complicated to prepare: First, the sodium salt of sucrose octasulfate must be converted to sucrose octasulfate by ion exchange resin, and then reused Triethylamine is titrated to prepare sucrose octasulfate triethylamine salt. In addition, there are reports that sucrose octasulfate may have activity similar to growth factors (Qiu Yongfeng et al., Application Review of Sucrose octasulfate, "Medical and Health (Citation Version)" 2017 Vol. 5, page 286).
  • the drug loading gradient used is concentrated on the ammonium sulfate gradient (such as CN103120645B patent “Irinotecan or irinotecan hydrochloride liposomes and their preparation Method ”) and proton concentration gradient method (such as CN1960729B" Irinotecan Preparation ").
  • Irinotecan or irinotecan hydrochloride liposomes prepared with ammonium sulfate gradient and proton concentration gradient cannot effectively achieve sustained release.
  • the object of the present invention is to provide an irinotecan liposome preparation and its preparation and application, which are used to solve the problems in the prior art.
  • the first aspect of the present invention provides an irinotecan liposome preparation containing irinotecan liposomes
  • the irinotecan liposomes include: irinotecan and lipids Body carrier and the inner water phase located inside the liposome membrane and the outer water phase located outside the liposome membrane, the irinotecan is encapsulated in the inner water phase; inside the liposome membrane There is a sulfonate gradient between the water phase and the outer water phase outside the membrane.
  • the liposome is a unicompartmental liposome.
  • the particle size of the liposome ranges from 30 nm to 200 nm.
  • the particle size of the liposome ranges from 50 nm to 120 nm.
  • the particle size of the liposome ranges from 90 nm to 110 nm.
  • the D95 of the liposome is 200 nm or less; wherein, D95 is the corresponding particle size value when the cumulative distribution percentage of the liposome from small to large reaches 95%, that is, the lipid In the plastids, the number of liposome particles smaller than D95 accounts for 95% of the total number of liposome particles.
  • the D95 of the liposome is less than or equal to 120 nm; wherein, D95 is the corresponding particle size value when the cumulative distribution percentage of the liposome from small to large reaches 95%, that is, the lipid In the body, the number of liposome particles smaller than D95 accounts for 95% of the total number of liposome particles.
  • the D95 of the liposome is less than or equal to 110 nm; wherein, D95 is the corresponding particle size value when the cumulative distribution percentage of the liposome from small to large reaches 95%, ie In the body, the number of liposome particles smaller than D95 accounts for 95% of the total number of liposome particles.
  • the inner aqueous phase includes an aqueous sulfonate solution; the outer aqueous phase is a physiological isotonic solution.
  • the cation of irinotecan and the sulfonate anion in the internal water phase form an insoluble salt.
  • the sulfonate is selected from monovalent sulfonate and / or disulfonate.
  • the monovalent sulfonate can be selected from one of ammonium methanesulfonate, ammonium 4-hydroxybenzenesulfonate, triethylamine methanesulfonate, and triethylamine 4-hydroxybenzenesulfonate. One or more.
  • the disulfonate may be selected from one or more of ammonium ethanedisulfonate, ammonium malonate, triethylamine ethanesulfonate, and triethylamine malonate.
  • the concentration of monovalent sulfonate ions in the aqueous phase in the liposome is 100 mM to 800 mM.
  • the concentration of monovalent sulfonate ions in the aqueous phase in the liposome is 200 mM to 700 mM.
  • the concentration of disulfonate ions in the aqueous phase in the liposome is 50 mM to 500 mM.
  • the concentration of disulfonate ions in the aqueous phase in the liposome is 100 mM to 400 mM.
  • the physiological isotonic solution is selected from a 5% (w / v) aqueous glucose solution, a 10% (w / v) aqueous sucrose solution, or a 0.9% (w / v) aqueous sodium chloride solution.
  • the pH value of the aqueous phase in the liposome is 4.0-9.0.
  • the pH value of the aqueous phase in the liposome is 4.5-8.0.
  • the concentration of the irinotecan is greater than or equal to 0.86 mg / ml.
  • the drug lipid molar ratio between the irinotecan and the liposome is greater than or equal to 0.1.
  • the components of the liposome include phospholipids, cholesterol, and pegylated phospholipids.
  • the molecular weight of the polyethylene glycol in the PEGylated phospholipid is 50-10000.
  • the molecular weight of the polyethylene glycol in the PEGylated phospholipid is 2000.
  • the molar ratio between the phospholipid, the cholesterol, and the pegylated phospholipid is (30-80): (0.1-40): (0.1-30).
  • the molar ratio between the phospholipid, the cholesterol, and the pegylated phospholipid is 55: 40: 5.
  • the aqueous phase of the lipid in vitro is a sucrose solution with a mass concentration of 10%.
  • a second aspect of the present invention provides a method for preparing an irinotecan liposome preparation.
  • the method is an active drug loading method.
  • the active drug loading method includes the following steps:
  • step (3) Mix the blank liposome obtained in step (2) with the irinotecan soluble saline solution and incubate to remove free irinotecan soluble salt to obtain irinotecan liposomes.
  • the sulfonate is selected from monovalent sulfonate and / or disulfonate.
  • the monovalent sulfonate can be selected from one of ammonium methanesulfonate, ammonium 4-hydroxybenzenesulfonate, triethylamine methanesulfonate, and triethylamine 4-hydroxybenzenesulfonate. One or more.
  • the disulfonate can be selected from one or more of ammonium ethanedisulfonate, ammonium malonate, triethylamine ethanesulfonate, and triethylamine malonate.
  • the sulfonate concentration in the aqueous sulfonate solution is 50 mM to 800 mM.
  • step (1) and step (2) the concentration of cations in the aqueous sulfonate solution is 50-800 mM.
  • step (1) and step (2) the pH value of the sulfonate aqueous solution is 4.0-9.0.
  • step (1) and step (2) the pH value of the sulfonate aqueous solution is 4.5-8.0.
  • the concentration of sulfonate ion in the aqueous solution of sulfonate is 100 mM to 800 mM.
  • step (1) and step (2) when a monovalent sulfonate is selected, the concentration of the sulfonate ion in the aqueous sulfonate solution is 200 mM to 700 mM.
  • the concentration of cations in the aqueous solution of sulfonate is 50 mM to 500 mM.
  • the concentration of cations in the aqueous solution of sulfonate is 100 mM to 400 mM.
  • the physiological isotonic solution may be selected from: 5% (w / v) aqueous glucose solution, 10% (w / v) sucrose aqueous solution or 0.9% (w / v) Sodium chloride aqueous solution.
  • step (2) the pH value of the aqueous phase in the liposome is 4.0 to 9.0.
  • step (2) the pH value of the aqueous phase in the liposome is 4.5-8.0.
  • step (3) the molar ratio of drug-lipids is greater than or equal to 0.1.
  • the irinotecan soluble salt is selected from irinotecan hydrochloride.
  • the present invention provides the use of the irinotecan liposomes described above in the preparation of tumor therapeutic drugs for colorectal cancer, lung cancer, breast cancer or pancreatic cancer.
  • the irinotecan liposome preparation in the use, can allow more irinotecan to exist in the blood circulation in the form of active lactone ring, and reach the tumor site and be converted into SN- 38, thereby further improving the anti-tumor effect.
  • the irinotecan liposome preparation proposed by the present invention improves the availability of raw materials and reduces the cost of the preparation, and more easily realizes the stable loading of irinotecan, and has the advantages of high encapsulation rate and good storage stability At the same time, the preparation has obvious sustained release, which is conducive to ensuring the stability and effectiveness of irinotecan hydrochloride in vivo and improving the efficacy of drugs.
  • the irinotecan liposome of the present invention has a long half-life in vivo and high bioavailability.
  • Figure 1A Chemical structure of monovalent sulfonic acid (left: methanesulfonic acid; right: 4-hydroxybenzenesulfonic acid).
  • Figure 1B Chemical structure of disulfonic acid (left: ethanedisulfonic acid; right: propanedisulfonic acid)
  • Figure 3 Encapsulation efficiency of irinotecan liposomes prepared with different gradients at different drug-lipid ratios (0.1, 0.3 and 0.5, respectively) in three groups;
  • the inventors of the present invention have found through extensive exploration experiments that the anion of the sulfonate in the aqueous phase of the liposome can form an insoluble salt with the irinotecan cation, and then encapsulate irinotecan in the internal aqueous phase of the liposome stably. On this basis, the present invention has been completed.
  • the irinotecan liposome preparation of the present invention contains irinotecan liposomes.
  • the irinotecan liposomes include: irinotecan and liposome carriers.
  • the liposome has a liposome membrane with a bilayer structure.
  • the liposome membrane is similar to a biological membrane.
  • the carrier material of the liposome contains phospholipids and cholesterol.
  • the carrier material and content of liposomes are not particularly limited, as long as they can form a stable, leak-free liposome membrane with a bilayer structure. These are within the scope of knowledge known to those skilled in the art.
  • the phospholipid is selected from hydrogenated soybean lecithin (HSPC) and distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG).
  • HSPC hydrogenated soybean lecithin
  • DSPE-PEG distearoylphosphatidylethanolamine-polyethylene glycol
  • the molecular weight of PEG in the DSPE-PEG may range from 50 to 10,000.
  • the molecular weight of PEG in the DSPE-PEG is 2000.
  • the molar ratio between HSPC, CHOL (cholesterol), and DSPE-PEG can range from (30 to 80): (0.1 to 40): (0.1 to 30).
  • the molar ratio between HSPC, CHOL, and DSPE-PEG can range from 55: 40: 5.
  • the irinotecan lipid preparation further includes: an internal aqueous phase located inside the liposome membrane and an external aqueous phase located outside the liposome membrane.
  • the irinotecan is encapsulated in the inner aqueous phase.
  • the ammonium salt gradient means that there is a difference between the ammonium salt concentration gradient and the pH.
  • the gradient of triethylamine salt means that there is a difference between the concentration gradient of triethylamine salt and pH.
  • the inner water phase is an aqueous solution of sulfonate, and the cation of the irinotecan forms an insoluble salt with the sulfonate group, so that the irinotecan is encapsulated in the inner water phase.
  • the external water phase is a physiological isotonic solution.
  • the physiological isotonic solution is selected from: 5% (w / v) aqueous glucose solution, 10% (w / v) aqueous sucrose solution or 0.9% (w / v) aqueous sodium chloride solution.
  • % (W / w) refers to the mass percentage concentration, that is, the mass of solute contained in every 100 g of the solution.
  • % (W / v) refers to the mass volume percentage concentration, that is, the mass of solute contained in every 100 ml of solution.
  • the irinotecan liposome preparation may be a preparation for injection administration.
  • the preparation for injection administration may be selected from a subcutaneous injection form, an intravenous injection form, an intramuscular injection form, or a pelvic injection form.
  • the cation of the sulfonate can be selected from ammonium ion and triethylamine ion.
  • the sulfonate can be selected from monovalent sulfonates and / or disulfonates.
  • the monovalent sulfonate can be selected from one or more of ammonium methanesulfonate, ammonium 4-hydroxybenzenesulfonate, triethylamine methanesulfonate, and triethylamine 4-hydroxybenzenesulfonate.
  • the disulfonate may be selected from one or more of ammonium ethanedisulfonate, ammonium malonate, triethylamine ethanesulfonate, and triethylamine malonate.
  • the pH of the inner water phase may be 4.0-9.0. It may further be 4.5 to 8.0.
  • the concentration of sulfonate ions in the internal water phase may be 100-800 mM. Furthermore, it may be 200 mM to 700 mM.
  • the concentration of sulfonate ions in the internal water phase may be 50 mM to 500 mM. Furthermore, it may be 100 mM to 400 mM.
  • the concentration of irinotecan in the internal aqueous phase may be 0.1 mg / ml or more.
  • the preparation method of the irinotecan liposome preparation of the present invention includes the following steps:
  • step (3) Mix the blank liposome obtained in step (2) with the irinotecan soluble saline solution and incubate to remove free irinotecan soluble salt to obtain irinotecan liposomes.
  • the sulfonate is selected from monovalent sulfonate and / or disulfonate.
  • the monovalent sulfonate may be selected from one or more of ammonium methanesulfonate, ammonium 4-hydroxybenzenesulfonate, triethylamine methanesulfonate, and triethylamine 4-hydroxybenzenesulfonate.
  • the disulfonate may be selected from one or more of ammonium ethanedisulfonate, ammonium malonate, triethylamine ethanesulfonate, and triethylamine malonate.
  • step (1) and step (2) when a monovalent sulfonate is selected, the concentration of the sulfonate ion in the aqueous sulfonate solution is 100 mM to 800 mM.
  • step (1) and step (2) when a monovalent sulfonate is selected, the concentration of the sulfonate ion in the aqueous sulfonate solution is 200 mM to 700 mM.
  • the concentration of cations in the aqueous solution of sulfonate is 50 mM to 500 mM.
  • the concentration of cations in the aqueous solution of sulfonate is 100 mM to 400 mM.
  • the pH value of the sulfonate aqueous solution may be 4.0-9.0. It can also be 4.5 to 8.0.
  • the physiological isotonic solution may be selected from: 5% (w / v) aqueous glucose solution, 10% (w / v) sucrose water
  • the pH of the external water phase is 4.0-9.0. It can also be 5.0 to 8.0.
  • the drug-lipid ratio (that is, the molar ratio between irinotecan and liposome) may be 0.1 or more.
  • the drug-lipid ratio can also be 0.15 or more.
  • the drug-lipid ratio can also be 0.20 or more.
  • the drug-lipid ratio can also be 0.25 or more.
  • the drug-lipid ratio can also be 0.3 or more.
  • the drug-lipid ratio can also be 0.5 or more.
  • Can be 0.1-0.15. Can be 0.1-0.2. It can be 0.1-0.25.
  • the irinotecan soluble salt is selected from irinotecan hydrochloride.
  • the concentration of irinotecan may be greater than or equal to 10 mg / ml.
  • the concentration of irinotecan can be greater than or equal to 12mg / ml.
  • the concentration of irinotecan can be greater than or equal to 15 mg / ml.
  • it may be 10 mg / ml, 12 mg / ml, 15 mg / ml.
  • It can be 10 mg / ml-12 mg / ml.
  • It can be 12mg / ml-15mg / ml.
  • It can be 10mg / ml-15mg / ml.
  • the present invention utilizes the principle of active drug loading: there is a sulfonate ion gradient between the aqueous phase in the liposome and the external; the irinotecan hydrochloride in the external aqueous phase is partially ionized, wherein the non-dissociative form of irinotecan diffuses passively Into the aqueous phase of the liposome and combine with the hydrogen ions generated by the ionization of ammonium ions or triethylamine ions in the internal aqueous phase to form irinotecan ions, which in turn form crystals or amorphous insoluble salts with the sulfonate, so that Irinotecan is encapsulated in the liposome aqueous phase.
  • the ammonia gas generated by the ionization of ammonium ions in the aqueous phase of the liposome, or the triethylamine generated by the ionization of triethylamine ions continuously escapes from the aqueous phase of the liposome, maintaining the The concentration of hydrogen ions causes the irinotecan molecules in the aqueous phase of the liposome to continuously combine with the hydrogen ions to form irinotecan cations and form a precipitate with the sulfonate until almost all of the irinotecan in the external water phase is Encapsulated into the aqueous phase in the liposome.
  • the liposome has high drug loading, high stability, and has an excellent sustained-release effect.
  • Irinotecan is a semi-synthetic water-soluble camptothecin derivative, which can be used for tumor treatment including colorectal cancer, lung cancer, breast cancer and pancreatic cancer.
  • the irinotecan liposome preparation of the present invention can make more irinotecan exist in the blood circulation in the form of active lactone ring, and reach the tumor site and be converted into SN-38, thereby further improving the anti-tumor effect.
  • the irinotecan liposome of the present invention has a long half-life in vivo and high bioavailability.
  • UV detection and analysis method Ultraviolet (UV) detection and analysis method is adopted: the detection instrument is TECAN 200 PRO; detection wavelength is 369nm; detection temperature is 24 °C; detection orifice plate is 96 well plates, UV-transparent; detection volume is 200 ⁇ l.
  • Example 2 Preparation and characterization of irinotecan liposomes with ammonium methanesulfonate as internal aqueous phase
  • the phospholipids used in this example were purchased from the German Lipoid company. Specifically, hydrogenated soybean lecithin (HSPC, molecular weight 783.8); pegylated phospholipid is distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000); cholesterol (CHOL, molecular weight 386.7); Ritican was purchased from Aladdin; the methanesulfonic acid used was purchased from Sigma-Aldrich; the ammonia used was purchased from Aladdin.
  • HSPC hydrogenated soybean lecithin
  • pegylated phospholipid is distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000); cholesterol (CHOL, molecular weight 386.7);
  • Ritican was purchased from Aladdin; the methanesulfonic acid used was purchased from Sigma-Aldrich; the ammonia used was purchased from Aladdin.
  • the phospholipids used in the subsequent examples are all hydrogenated soybean phospholipids from Lipoid; the pegylated phospholipids used are all distearoylphosphatidylethanolamine-polyethylene glycol from Lipoid; The cholesterol used was Lipoid cholesterol; the irinotecan hydrochloride used was purchased from Aladdin.
  • Step (1) Weigh accurately 479.9mg hydrogenated soybean lecithin, 160.9mg cholesterol and 160.8mg distearoylphosphatidylethanolamine-polyethylene glycol 2000, add an appropriate amount of ethanol to fully dissolve and mix to obtain a lipid ethanol mixed solution;
  • Step (2) accurately measure 2.131 ml of methanesulfonic acid solution, titrate it to pH 4.0 ⁇ 6.0 with ammonia water, and dilute to 50ml with double distilled water (ddH 2 O) to obtain 650mM ammonium methanesulfonate with a pH of 4.0 ⁇ 6.0 Buffer solution
  • step (3) add the ethanol mixed solution of the lipid obtained in step (1) to 10 ml of 650 mM ammonium methanesulfonate buffer with a pH of 4.0 to 6.0, and place it in a water bath at 70 degrees Celsius for 30 minutes to make the liposomes full Hydrate to obtain a more uniform liposome suspension;
  • step (4) using a liposome extruder, the liposome suspension obtained in step (3) was sequentially extruded through polycarbonate membranes with different pore diameters for 10 times, and finally the particle size was 100 nm. Blank liposome with even distribution;
  • step (5) the liposome prepared in step (4) was placed in a dialysis bag with a molecular weight cut-off of 10,000, and 10% sucrose aqueous solution was used as the dialysis medium, and dialyzed at 4 degrees Celsius overnight.
  • the volume ratio of the sample to the dialysis medium is 1: 1000, and the dialysate is changed three times during dialysis to completely remove the ammonium methanesulfonate in the external aqueous phase of the lipid, to obtain an external aqueous phase composed of 10% sucrose and methylsulfonic acid
  • the ammonium solution is an internal aqueous phase and a blank liposome composed of a phospholipid bimolecular layer.
  • the aqueous phase of the liposome and the external has a certain pH and ammonium methanesulfonate concentration gradient.
  • the aqueous phase of the blank liposome is a 650 mM ammonium methanesulfonate aqueous solution (pH 4.0-6.0), and the aqueous phase of the blank liposome is a 10% sucrose aqueous solution.
  • the internal aqueous phase obtained in step (5) is a blank liposome suspension of ammonium methanesulfonate solution, and a 10 mg / ml aqueous solution of irinotecan hydrochloride at a volume ratio of 1 : 1 Mix and incubate at 50-60 degrees Celsius for 10-30 minutes to obtain liposomes containing irinotecan in the internal water phase.
  • the irinotecan liposome prepared in this example was diluted 50 times with ddH 2 O, and the particle size was analyzed using Zetasizer ZS90 (Malvern, UK). The specific results are shown in Table 3.
  • Table 3 shows that the particle size of liposomes after drug loading is around 100 nm, and the particle size distribution (PDI) is less than 0.1.
  • the particle size of the blank liposomes before drug loading is also around 100 nm, and the particle size distribution (PDI) is less than 0.1.
  • EE encapsulation efficiency
  • M inter is the content of irinotecan in the liposome preparation after the resin adsorbs the free drug, that is, the content of irinotecan encapsulated by the liposome;
  • M total is the liposome preparation of irinotecan before resin adsorption
  • the content of irinotecan in middle is the dosage of irinotecan.
  • Table 2 shows that irinotecan liposomes prepared according to this method have a good encapsulation rate and can achieve high drug-to-lipid drug loading.
  • the methanesulfonic acid used in this example was purchased from Sigma-Aldrich; the triethylamine used was purchased from Sinopharm Group.
  • Step (3) 10 ml of 650 mM methanesulfonic acid triethylamine buffer solution with a pH of 4.0 to 6.0 is added to the lipid ethanol solution obtained in step (1) without adding methanesulfonic acid. Ammonium buffer.
  • Example 2 The characterization method of irinotecan liposomes is referred to Example 2. The results are shown in Table 3. Table 3 shows that the encapsulation efficiency of the irinotecan liposome prepared in Example 3 is about 70%. Compared with the results of Example 2, the encapsulation rate of triethylamine methanesulfonate as the aqueous phase in the liposome has decreased, indicating that the contribution of the ammonium salt ion concentration gradient in active drug loading is better than that of triethylamine ion Concentration gradient.
  • the 4-hydroxybenzenesulfonic acid used in this example was purchased from Macklin Company; the ammonia used was purchased from Aladdin Company.
  • step (3) to the lipid ethanol solution obtained in step (1) is added 10ml of 650mM 4-hydroxybenzenesulfonate ammonium buffer (pH 4.0 ⁇ 6.0), without adding methylsulfonate Ammonium acid buffer.
  • Example 2 The characterization method of irinotecan liposomes is referred to Example 2. The results are shown in Table 4. Table 4 shows that the encapsulation efficiency of the irinotecan liposome prepared in Example 4 is good.
  • Example 5 Preparation and characterization of irinotecan liposomes with triethylamine 4-hydroxybenzenesulfonate solution as internal aqueous phase
  • the triethylamine 4-hydroxybenzenesulfonate used in this example was purchased from Macklin Company; the triethylamine used was purchased from Sinopharm Group.
  • step (2) Preparation of irinotecan liposomes, the specific preparation process refers to Example 2. The difference is that in step (2), add 10 ml of 650 mM 4-hydroxybenzenesulfonic acid triethylamine buffer solution with a pH of 4.0 to 6.0 to the lipid ethanol solution obtained in step (1) without adding methylsulfonate Ammonium acid buffer.
  • Example 2 The method for characterizing irinotecan liposomes is referred to Example 2. The results are shown in Table 5. Table 5 shows that the encapsulation efficiency of irinotecan liposome prepared in Example 5 is good.
  • Example 6 Preparation and characterization of irinotecan liposomes with ammonium ethanedisulfonate solution as internal aqueous phase
  • the ethanedisulfonic acid used in this example was purchased from Alfa Aesar; the ammonia used was purchased from Aladdin.
  • Step (2) Preparation of irinotecan liposomes, the specific preparation process refers to Example 2. The difference is that, in step (2), 10 ml of 325 mM ammonium ethanedisulfonate buffer with a pH of 4.0 to 6.0 is added to the lipid ethanol solution obtained in step (1) without adding ammonium methanesulfonate buffer. .
  • Example 6 For the method of characterizing irinotecan liposomes, refer to Example 2. The results are shown in Table 6. Table 6 shows that the encapsulation efficiency of irinotecan liposome prepared in Example 6 is good.
  • Example 7 Preparation and characterization of irinotecan liposomes with triethylamine ethanedisulfonate solution as internal aqueous phase
  • the ethanedisulfonic acid used in this example was purchased from Alfa Aesar; the triethylamine used was purchased from Sinopharm Group.
  • step (2) Preparation of irinotecan liposomes, the specific preparation process refers to Example 2. The difference is that in step (2) to the lipid ethanol solution obtained in step (1), 10 ml of 325 mM triethylamine buffer of ethanedisulfonic acid with a pH of 4.0 to 6.0 is added without adding ammonium methanesulfonate Buffer.
  • Example 7 The method for characterizing irinotecan liposomes is referred to Example 2. The results are shown in Table 7. Table 7 shows that the encapsulation efficiency of the irinotecan liposome prepared in Example 7 is good.
  • Example 8 Preparation and characterization of irinotecan liposomes with ammonium malonate solution as internal aqueous phase
  • the malonic disulfonic acid used in this example was purchased from Alfa Aesar; the ammonia used was purchased from Aladdin.
  • Step (2) Preparation of irinotecan liposomes, the specific preparation process refers to Example 2. The difference is that in step (2), 10 ml of 325 mM ammonium malonate disulfonate buffer with a pH of 4.0 to 6.0 is added to the lipid ethanol solution obtained in step (1) without adding ammonium methanesulfonate buffer .
  • Example 2 The method for characterizing irinotecan liposomes is referred to Example 2. The results are shown in Table 8. Table 8 shows that the encapsulation efficiency of irinotecan liposome prepared in Example 8 is good.
  • Example 9 Preparation and characterization of irinotecan liposomes with triethylamine malonate solution as internal aqueous phase
  • the malonic acid used in this example was purchased from Alfa Aesar; the triethylamine used was purchased from Sinopharm Group.
  • Step (2) Preparation of irinotecan liposomes, the specific preparation process refers to Example 2. The difference is that in step (2), 10 ml of 325 mM triethylamine malonate buffer with a pH of 4.0 to 6.0 is added to the lipid ethanol solution obtained in step (1) without adding ammonium methanesulfonate Buffer.
  • Example 9 The method for characterizing irinotecan liposomes is described in Example 2. The results are shown in Table 9. Table 9 shows that the encapsulation efficiency of the irinotecan liposome prepared in Example 9 is good.
  • Example 10 In vitro release study of irinotecan liposomes with different sulfonates as internal aqueous phase
  • the concentration of the drug in the aqueous phase of the liposome is different, so the insoluble salt formed by the drug and the sulfonate ion is likely to have a different structure, and thus may have a different drug release rate.
  • the in vitro release rate of irinotecan liposomes prepared with different sulfonates (including ammonium salt and triethylamine salt) as the internal aqueous phase in this example, the irinotecan grease for in vitro release studies The plastids all had the same drug-lipid ratio (0.3), the drug loading concentration was 2.58 mg / ml, and the lipid concentration was 8.91 mg / ml.
  • Bovine serum albumin (BSA) used in in vitro release studies was purchased from Biotechnology (Shanghai) Co., Ltd .; Dowex resin was purchased from Sigma-Aldrich.
  • Step (1) Weigh 4.0152g of bovine serum albumin (BSA) powder accurately, dissolve it in 100ml of physiological saline, and prepare a 40mg / ml aqueous solution of albumin physiological saline (the protein concentration is equivalent to the protein concentration in 50% plasma);
  • BSA bovine serum albumin
  • Step (2) taking the irinotecan liposome prepared in Example 2-9 by using different monovalent sulfonate solutions or disulfonate (pH 4.0-6.0) solutions as the internal aqueous phase; wherein, the lipid The concentration was 8.91 mg / ml, and the drug-lipid ratio (molar ratio of drug to lipid) was 0.3.
  • Step (3) the liposomes were diluted 80 times with 40mg / ml albumin physiological saline solution, and enough Dowex resin was added to adsorb free drug, forming a leak tank condition, shaking at 100 rpm (THZ-C constant temperature oscillator) , China). Samples were taken at different time points (0 hours, 1 hour, 3 hours, 6 hours, 9 hours and 24 hours), the supernatant was taken after standing, and the content of irinotecan wrapped in liposomes was determined by UV method and calculated Cumulative release rate of irinotecan at different time points.
  • the present invention also made irinotecan liposomes with sucrose octathioglycolipid triethylamine as the internal aqueous phase according to the instructions of the commercially available Onivyde.
  • the lipid concentration is 8.91 mg / ml and the drug-lipid ratio is 0.3; and irinotecan liposomes with 325 mM ammonium sulfate at pH 4.0-6.0 as the internal aqueous phase, wherein the lipid concentration is 8.91 mg / ml, drug-lipid ratio is 0.3.
  • the in vitro release rate of irinotecan liposomes with sucrose octasulfate triethylamine and ammonium sulfate as the internal aqueous phase in albumin saline was measured in parallel, and was compared with the irinotecan lipid proposed by the present invention
  • the release rates of plastid preparations were compared.
  • FIG. 4 shows that in albumin physiological saline solution, ammonium methanesulfonate, ammonium 4-hydroxybenzenesulfonate, triethylamine 4-hydroxybenzenesulfonate, ammonium ethanedisulfonate, triethylamine ethanedisulfonate.
  • the liposome irinotecan with ammonium malonate and triethylamine malonate solution as the internal aqueous phase has good sustained-release effect in vitro.
  • the ammonium methanesulfonate group reached 10.03%
  • the ammonium 4-hydroxybenzenesulfonate group reached 11.35%
  • the triethylamine 4-hydroxybenzenesulfonate group reached 10.32%
  • the ethanedisulfonate It reached 1.03% in the ammonium acid group, 3.49% in the triethylamine ethanesulfonate group, 2.31% in the ammonium malonate group, and 1.96% in the triethylamine malonate group.
  • the methanesulfonic acid and hydroxybenzenesulfonic acid used in this example were purchased from Sigma-Aldrich.
  • the melting point can reflect the regularity of the internal structure of the compound.
  • irinotecan was formed by directly mixing irinotecan hydrochloride and sulfonate solution Sulfonate, and the melting point of each salt was measured.
  • Table 10 shows that the melting temperature of each irinotecan sulfonate is between 86 ° C and 99 ° C.
  • thermodynamic behavior of irinotecan liposomes prepared in 2-9 was compared with that of self-made irinotecan liposomes with sucrose octasulfate triethylamine and ammonium sulfate as the internal aqueous phase.
  • All irinotecan liposome samples characterized by DSC had a drug-to-lipid ratio of 0.3, a lipid concentration of 8.9 mg / ml, an average particle size of 100 nm, and a PDI of less than 0.1.
  • the differential scanning calorimeter (DSC) used in this example is a capillary DSC (Capillary DSC) of GE Corporation.
  • the scanning temperature is 10-120 degrees Celsius, and the scanning speed is 1 degree / minute.
  • thermodynamic parameters of irinotecan hydrochloride liposomes in each group are shown in Table 11.
  • the present invention also prepared and characterized other types of irinotecan liposomes with reference to Examples 2 and 6.
  • Type 1 is different from the preparation method of irinotecan liposome in Example 2 in that step (2) is to take ammonium methanesulfonate solution to adjust the pH to prepare 800 mM ammonium methanesulfonate with a pH of 9.0 Buffer; in step (4), a blank liposome with a particle size of 200 nm is obtained. In step (5), it is mixed with an aqueous solution of irinotecan hydrochloride at a concentration of 15 mg / ml, and the rest are the same.
  • Type 2 is different from the preparation method of irinotecan liposome in Example 2 in that step (2) is to take ammonium methanesulfonate solution to adjust the pH to prepare 100mM ammonium methanesulfonate with a pH of 4.5 Buffer; in step (4), a blank liposome with a particle size of 30 nm is obtained. In step (5), it is mixed with an aqueous solution of irinotecan hydrochloride at a concentration of 10 mg / ml, and the rest are the same.
  • Type 3 is different from the preparation method of irinotecan liposome in Example 2 in that step (2) is to take ammonium methanesulfonate solution to adjust the pH to prepare 200mM ammonium methanesulfonate with a pH of 5.0 Buffer; in step (4), a blank liposome with a particle size of 50 nm is obtained. In step (5), it is mixed with an aqueous solution of irinotecan hydrochloride at a concentration of 13 mg / ml, and the rest are the same.
  • Type 4 is different from the preparation method of irinotecan liposome in Example 2 in that step (2) is to take ammonium methanesulfonate solution to adjust the pH to prepare 700 mM ammonium methanesulfonate with a pH of 8.0 Buffer; in step (4), a blank liposome with a particle size of 120 nm is obtained. In step (5), it is mixed with an aqueous solution of irinotecan hydrochloride at a concentration of 14 mg / ml, and the rest are the same.
  • Type 5 is different from the preparation method of irinotecan liposome in Example 6 in that step (2) is to take a solution of ethanedisulfonic acid hydrate to adjust the pH to prepare 500mM ethanedisulfonic acid with a pH of 6.0 Ammonium buffer; in step (4), a blank liposome with a particle size of 90 nm is obtained. In step (5), it is mixed with an aqueous solution of irinotecan hydrochloride at a concentration of 14 mg / ml, and the rest are the same.
  • Type 6 is different from the preparation method of irinotecan liposome in Example 6 in that step (2) is to adjust the pH by taking a solution of ethanedisulfonic acid hydrate to prepare a 50 mM ethanedisulfonic acid with a pH of 4.0 Ammonium buffer; in step (4), a blank liposome with a particle size of 110 nm is obtained. In step (5), it is mixed with an aqueous solution of irinotecan hydrochloride at a concentration of 12 mg / ml, and the rest are the same.
  • Type 7 is different from the preparation method of irinotecan liposome in Example 6 in that step (2) is to take a solution of ethanedisulfonic acid hydrate to adjust the pH to prepare 100 mM ethanedisulfonic acid with a pH of 4.5 Ammonium buffer; in step (4), a blank liposome with a particle size of 80 nm is obtained. In step (5), it is mixed with an aqueous solution of irinotecan hydrochloride at a concentration of 13 mg / ml, and the rest are the same.
  • Type 8 is different from the preparation method of irinotecan liposome in Example 6 in that step (2) is to take a solution of ethanedisulfonic acid hydrate to adjust the pH to prepare 400 mM ethanedisulfonic acid with a pH of 7.5. Ammonium buffer; in step (4), a blank liposome with a particle size of 150 nm is obtained. In step (5), it is mixed with an aqueous solution of irinotecan hydrochloride at a concentration of 13 mg / ml, and the rest are the same.
  • Type 1 the particle size and encapsulation efficiency of irinotecan liposome with ammonium methanesulfonate solution as the internal water phase
  • Type 3 The particle size and encapsulation efficiency of irinotecan liposome with ammonium methanesulfonate solution as the internal water phase
  • Type 4 Particle size and encapsulation efficiency of irinotecan liposomes with ammonium methanesulfonate solution as the internal water phase
  • Type 7 Particle size and encapsulation efficiency of irinotecan liposomes with ammonium ethanedisulfonate solution as the internal aqueous phase
  • Type 8 The particle size and encapsulation efficiency of irinotecan liposome with ammonium ethanedisulfonate solution as the internal water phase

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Abstract

一种伊立替康脂质体制剂、制备方法和用途，所述伊立替康脂质体包括：伊立替康和脂质体载体以及位于脂质体膜内的内水相和位于脂质体膜外的外水相，所述伊立替康被包封于所述内水相中；所述脂质体膜内的内水相和膜外的外水相之间存在磺酸盐梯度。该脂质体以单价磺酸盐和二磺酸盐为内水相，将伊立替康以不溶性磺酸盐和二磺酸盐的形式，包封在脂质体内水相中，具有缓释效果。

Description

伊立替康脂质体制剂及其制备与应用 技术领域
本发明属于纳米药物输送技术领域，具体涉及一种伊立替康脂质体制剂及其制备与应用。
背景技术
伊立替康(irinotecan，CPT-11)，为喜树碱的半合成衍生物，它是一种治疗转移性结肠癌的有效药物。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，从而使DNA双链断裂。
目前已上市的伊立替康产品有盐酸伊立替康注射液、冻干粉针制剂和脂质体注射剂。其中，盐酸伊立替康是伊立替康的盐酸盐，具有较好的水溶性。盐酸伊立替康注射液经静脉给药后，在偏碱性的生理环境下，游离伊立替康的内酯环易开环形成羧酸盐形式，从而失去活性，降低了药物的疗效。同时，盐酸伊立替康粉针剂的毒副作用较大，主要表现为中性粒细胞减少和迟发性腹泻。
2015年10月，FDA批准了伊立替康脂质体药物Onivyde，用于晚期胰腺癌治疗。Onivyde以蔗糖八硫酸酯(图1a)的三乙胺盐作为内水相，利用脂质体内外水相之间的三乙胺梯度将伊立替康包裹在脂质体内水相中，形成伊立替康-蔗糖八硫酸酯盐，达到体内缓释的效果(Drummond,D.C.等，Development of a highly active nanoliposomal irinotecan using a novel intraliposomal stabilization strategy[J].Cancer Research,2006,66(6),3271-3277.)。Onivyde脂质体载药所必需的关键辅料，蔗糖八硫酸酯三乙胺不仅原料昂贵，而且制备复杂：首先需用通过离子交换树脂将蔗糖八硫酸酯钠盐转化为蔗糖八硫酸酯，再用三乙胺滴定，制备为蔗糖八硫酸酯三乙胺盐。此外，还有报道，蔗糖八硫酸酯可能具有类似生长因子的活性(邱永峰等，蔗糖八硫酸酯的应用综述，《医药卫生(引文版)》2017年第5卷，286页)。
在目前国内已公开的盐酸伊立替康或伊立替康脂质体专利中，所采用的载药梯度集中在硫酸铵梯度(如CN103120645B专利“伊立替康或盐酸伊立替康脂质体及其制备方法”)及质子浓度梯度法(如CN1960729B“伊立替康制剂”)。采用硫酸铵梯度和质子浓度梯度制备的伊立替康或盐酸伊立替康脂质体并不能有效实现缓释，如采用硫酸铵梯度法制备的伊立替康脂质体(CN105796495A)在大鼠体内的药物半衰期是8.90小时。
因此有必要发展基于更为易得的其他类型的铵盐或三乙胺盐的主动载药梯度，用于伊立 替康脂质体的制备，并达到较好的体内缓释效果。
发明内容
为了克服现有技术中所存在的问题，本发明的目的在于提供一种伊立替康脂质体制剂及其制备与应用，用于解决现有技术中的问题。
为实现上述目的及其他相关目的，本发明第一方面提供了一种伊立替康脂质体制剂，含有伊立替康脂质体，所述伊立替康脂质体包括：伊立替康和脂质体载体以及位于脂质体膜内的内水相和位于脂质体膜外的外水相，所述伊立替康被包封于所述内水相中；所述脂质体膜内的内水相和膜外的外水相之间存在磺酸盐梯度。
于本发明的一个实施例中，所述脂质体为单室脂质体。
于本发明的一个实施例中，所述脂质体的粒径范围是30nm～200nm。
于本发明的一个实施例中，所述脂质体的粒径范围是50nm～120nm。
于本发明的一个实施例中，所述脂质体的粒径范围是90nm～110nm。
于本发明的一个实施例中，所述脂质体的D95为小于等于200nm；其中，D95为所述脂质体从小到大累积分布百分数达到95％时对应的粒径值，即所述脂质体中，粒径小于D95的脂质体颗粒数占所述脂质体总颗粒数的95％。
于本发明的一个实施例中，所述脂质体的D95小于等于120nm；其中，D95为所述脂质体从小到大累积分布百分数达到95％时对应的粒径值，即所述脂质体中，粒径小于D95的脂质体颗粒数占所述脂质体总颗粒数的95％.。
于本发明的一个实施例中，所述脂质体的D95小于等于110nm；其中，D95为所述脂质体从小到大累积分布百分数达到95％时对应的粒径值，即所述脂质体中，粒径小于D95的脂质体颗粒数占所述脂质体总颗粒数的95％。
于本发明的一个实施例中，所述内水相包括磺酸盐水溶液；所述外水相为生理等渗溶液。
于本发明的一个实施例中，所述内水相中的伊立替康的阳离子与磺酸盐阴离子形成不溶性盐。
于本发明的一个实施例中，所述磺酸盐选自单价磺酸盐和/或二磺酸盐。
于本发明的一个实施例中，单价磺酸盐可选自甲基磺酸铵、4-羟基苯磺酸铵、甲基磺酸三乙胺、4-羟基苯磺酸三乙胺中的一种或多种。
于本发明的一个实施例中，二磺酸盐可选自乙二磺酸铵、丙二磺酸铵、乙二磺酸三乙胺、丙二磺酸三乙胺中的一种或多种。
于本发明的一个实施例中，所述脂质体内水相中单价磺酸根离子浓度为100mM～800mM。
于本发明的一个实施例中，所述脂质体内水相中单价磺酸根离子浓度为200mM～700mM。
于本发明的一个实施例中，所述脂质体内水相中二磺酸根离子浓度为50mM～500mM。
于本发明的一个实施例中，所述脂质体内水相中二磺酸根离子浓度为100mM～400mM。
于本发明的一个实施例中，所述生理等渗溶液选自5％(w/v)葡萄糖水溶液、10％(w/v)蔗糖水溶液或0.9％(w/v)氯化钠水溶液。
于本发明的一个实施例中，所述脂质体内水相的pH值为4.0～9.0。
于本发明的一个实施例中，所述脂质体内水相的pH值为4.5～8.0。
于本发明的一个实施例中，在所述伊立替康脂质体制剂中，所述伊立替康的浓度大于等于0.86mg/ml。
于本发明的一个实施例中，所述伊立替康和所述脂质体之间的药脂摩尔比大于等于0.1。
于本发明的一个实施例中，所述脂质体的组分包括磷脂、胆固醇和聚乙二醇化磷脂。
于本发明的一个实施例中，所述聚乙二醇化磷脂中的聚乙二醇的分子量为50～10000。
于本发明的一个实施例中，所述聚乙二醇化磷脂中的聚乙二醇的分子量为2000。
于本发明的一个实施例中，所述磷脂、所述胆固醇和所述聚乙二醇化磷脂之间的摩尔比为(30～80)：(0.1～40)：(0.1～30)。
于本发明的一个实施例中，所述磷脂、所述胆固醇和所述聚乙二醇化磷脂之间的摩尔比为55:40:5。
于本发明的一个实施例中，所述脂质体外水相为质量浓度10％的蔗糖溶液。
本发明第二方面提供了一种伊立替康脂质体制剂的制备方法，所述方法为主动载药法，所述主动载药法包括以下步骤：
(1)制备内水相和外水相均含磺酸盐水溶液的空白脂质体；
(2)制备内水相含有磺酸盐水溶液、外水相含有生理等渗溶液的空白脂质体，以形成空白脂质体内外水相磺酸盐浓度梯度；
(3)将步骤(2)所得空白脂质体与伊立替康可溶性盐水溶液混合，孵育，去除游离伊立替康可溶性盐，获得伊立替康脂质体。
于本发明的一个实施例中，所述磺酸盐选自单价磺酸盐和/或二磺酸盐。
于本发明的一个实施例中，单价磺酸盐可选自甲基磺酸铵、4-羟基苯磺酸铵、甲基磺酸三乙胺、4-羟基苯磺酸三乙胺中的一种或多种。
于本发明的一个实施例中，二磺酸盐可选自乙二磺酸铵、丙二磺酸铵、乙二磺酸三乙胺、 丙二磺酸三乙胺中的一种或多种。
于本发明的一个实施例中，步骤(1)和步骤(2)中，所述磺酸盐水溶液中的磺酸盐浓度为50mM～800mM。
步骤(1)和步骤(2)中，所述磺酸盐水溶液中阳离子的浓度是50～800mM。
于本发明的一个实施例中，步骤(1)和步骤(2)中，所述磺酸盐水溶液的pH值为4.0～9.0。
于本发明的一个实施例中，步骤(1)和步骤(2)中，所述磺酸盐水溶液的pH值为4.5～8.0。
于本发明的一个实施例中，步骤(1)和步骤(2)中，选用单价磺酸盐时，所述磺酸盐水溶液中磺酸根离子的浓度是100mM～800mM。
进一步的，步骤(1)和步骤(2)中，选用单价磺酸盐时，所述磺酸盐水溶液中磺酸根离子的浓度是200mM～700mM。
于本发明的一个实施例中，选用二磺酸盐时，步骤(1)和步骤(2)中，所述磺酸盐水溶液中阳离子的浓度是50mM～500mM。
进一步的，选用二价磺酸盐时，步骤(1)和步骤(2)中，所述磺酸盐水溶液中阳离子的浓度是100mM～400mM。
于本发明的一个实施例中，步骤(2)中，所述生理等渗溶液可选自：5％(w/v)葡萄糖水溶液、10％(w/v)蔗糖水溶液或0.9％(w/v)氯化钠水溶液。
于本发明的一个实施例中，步骤(2)中，所述脂质体内水相的pH值为4.0～9.0。
进一步的，步骤(2)中，所述脂质体内水相的pH值为4.5～8.0。
于本发明的一个实施例中，步骤(3)中，药脂摩尔比大于等于0.1。
于本发明的一个实施例中，步骤(3)中，所述伊立替康可溶性盐选自盐酸伊立替康。
本发明提供了上文所述的伊立替康脂质体在制备结直肠癌、肺癌、乳腺癌或胰腺癌的肿瘤治疗药物中的用途。
于本发明的一个实施例中，所述用途中，伊立替康脂质体制剂可使更多的伊立替康以活性内酯环形式存在于血液循环中，并到达肿瘤部位，转化为SN-38，从而进一步改善抗肿瘤效果。
本发明的有益效果为：
本发明提出的伊立替康脂质体制剂，在改善原料的易得性、降低制剂成本的同时，更为简便地实现伊立替康的稳定装载，具有高包封率和储存稳定性好的优点；同时，该制剂具有明显的缓释性，有利于保证盐酸伊立替康的体内稳定性和有效性，提升药物疗效。本发明所述的伊立替康脂质体体内半衰期长，生物利用度高。
附图说明
图1A：单价磺酸(左：甲基磺酸；右：4-羟基苯磺酸)的化学结构。
图1B：二磺酸(左：乙二磺酸；右：丙二磺酸)的化学结构
图2盐酸伊立替康水溶液UV标准曲线图；
图3三组不同投药药脂比时(分别为0.1，0.3和0.5)，采用不同梯度制备的伊立替康脂质体的包封率；
图4以不同磺酸盐溶液为内水相的伊立替康脂质体在模拟血浆中的累积释放速率图(TEA：三乙胺；MS：甲基磺酸；HBS：4-羟基苯磺酸；EDS：乙二磺酸；PDS：丙二磺酸)；
图5采用不同磺酸盐梯度、三乙胺硫糖脂梯度以及硫酸铵梯度制备的伊立替康脂质体的差示热扫描图。
具体实施方式
本发明发明人经过大量探索实验发现，脂质体内水相中的磺酸盐的阴离子可以与伊立替康阳离子形成不溶盐，进而将伊立替康稳定包封在脂质体的内水相中，在此基础上完成了本发明。
本发明的伊立替康脂质体制剂，含有伊立替康脂质体，所述伊立替康脂质体包括：伊立替康和脂质体载体。
所述脂质体具有呈双分子层结构的脂质体膜。所述脂质体膜类似于生物膜。
所述脂质体的载体材料含有磷脂和胆固醇。本发明对于脂质体的载体材料及含量并无特别的限制，只要能够形成稳定的、无泄漏的呈双分子层结构的脂质体膜即可。这些均在本领域技术人员所能知晓的知识范围内。
本发明实施例中列举了：所述磷脂选用氢化豆磷脂(HSPC)和二硬脂酰磷脂酰乙醇胺-聚乙二醇(DSPE-PEG)。但是，并不仅限于此。
所述DSPE-PEG中PEG的分子量范围可以是50～10000。例如，所述DSPE-PEG中PEG的分子量是2000。
HSPC、CHOL(胆固醇)、DSPE-PEG之间的摩尔比例范围可以是(30～80)：(0.1～40)：(0.1～30)。例如，HSPC、CHOL、DSPE-PEG之间的摩尔比例范围可以是55:40:5。
所述伊立替康脂质制剂还包括：位于脂质体膜内的内水相和位于脂质体膜外的外水相。所述伊立替康被包封于所述内水相中。
所述脂质体膜内的内水相和膜外的外水相之间存在磺酸盐梯度。
所述铵盐梯度是指存在铵盐浓度梯度与pH的差异。
所述三乙胺盐梯度是指存在三乙胺盐浓度梯度与pH的差异。
所述内水相为磺酸盐水溶液，所述伊立替康的阳离子与所述磺酸根形成不溶性盐，从而使伊立替康被包封于所述内水相中。所述外水相为生理等渗溶液。
所述生理等渗溶液选自：5％(w/v)葡萄糖水溶液、10％(w/v)蔗糖水溶液或0.9％(w/v)氯化钠水溶液。
％(w/w)是指质量百分浓度，亦即每100g的溶液中含有的溶质的质量。
％(w/v)是指质量体积百分浓度，亦即每100ml的溶液中含有的溶质的质量。
所述伊立替康脂质体制剂可以为注射给药制剂。
所述注射给药制剂可选自皮下注射剂型、静脉注射剂型、肌肉注射剂型或盆腔注射剂型。
所述磺酸盐的阳离子可选自铵根离子、三乙胺离子。
所述磺酸盐可选自单价磺酸盐和/或二磺酸盐。其中单价磺酸盐可选自甲基磺酸铵、4-羟基苯磺酸铵、甲基磺酸三乙胺、4-羟基苯磺酸三乙胺中的一种或多种。二磺酸盐可选自乙二磺酸铵、丙二磺酸铵、乙二磺酸三乙胺、丙二磺酸三乙胺中的一种或多种。
所述内水相的pH可以是4.0～9.0。进一步可以是4.5～8.0。
选用单价磺酸盐时，所述内水相中磺酸根离子的浓度可以是100～800mM。进一步还可以是200mM～700mM。
选用二磺酸盐时，所述内水相中磺酸根离子的浓度可以是50mM～500mM。进一步还可以是100mM～400mM。
所述内水相中伊立替康的浓度可以是0.1mg/ml以上。
本发明所述的伊立替康脂质体制剂的制备方法，包括如下步骤：
(1)制备内水相和外水相均含磺酸盐水溶液的空白脂质体；
(2)制备内水相含有磺酸盐水溶液、外水相含有生理等渗溶液的空白脂质体，以形成空白脂质体内外水相磺酸盐浓度梯度；
(3)将步骤(2)所得空白脂质体与伊立替康可溶性盐水溶液混合，孵育，去除游离伊立替康可溶性盐，获得伊立替康脂质体。
所述磺酸盐选自单价磺酸盐和/或二磺酸盐。
单价磺酸盐可选自甲基磺酸铵、4-羟基苯磺酸铵、甲基磺酸三乙胺、4-羟基苯磺酸三乙胺中的一种或多种。
二磺酸盐可选自乙二磺酸铵、丙二磺酸铵、乙二磺酸三乙胺、丙二磺酸三乙胺中的一种或多种。
步骤(1)和步骤(2)中，选用单价磺酸盐时，所述磺酸盐水溶液中磺酸根离子的浓度是100mM～800mM。
进一步的，步骤(1)和步骤(2)中，选用单价磺酸盐时，所述磺酸盐水溶液中磺酸根离子的浓度是200mM～700mM。
选用二磺酸盐时，步骤(1)和步骤(2)中，所述磺酸盐水溶液中阳离子的浓度是50mM～500mM。
进一步的，选用二价磺酸盐时，步骤(1)和步骤(2)中，所述磺酸盐水溶液中阳离子的浓度是100mM～400mM。
步骤(1)和步骤(2)中，所述磺酸盐水溶液的pH值可以为4.0～9.0。还可以为4.5～8.0。
步骤(2)中，所述生理等渗溶液可选自：5％(w/v)葡萄糖水溶液、10％(w/v)蔗糖水
溶液或0.9％(w/v)氯化钠水溶液。
于本发明的一个实施例中，步骤(2)中，所述外水相的pH是4.0～9.0。还可以为5.0～8.0。
于本发明的一个实施例中，步骤(3)中，药脂比(亦即伊立替康与脂质体之间的摩尔比)可以是0.1及以上。药脂比还可以是0.15及以上。药脂比还可以是0.20及以上。药脂比还可以是0.25及以上。药脂比还可以是0.3及以上。药脂比还可以是0.5及以上。可以是0.1-0.15。可以是0.1-0.2。可以是0.1-0.25。可以是0.1-0.3。可以是0.15-0.3。可以是0.3-0.5。可以是0.2-0.5。
于本发明的一个实施例中，步骤(3)中，所述伊立替康可溶性盐选自盐酸伊立替康。
所述伊立替康可溶性盐水溶液中，伊立替康的浓度可以大于等于10mg/ml。伊立替康的浓度可以大于等于12mg/ml。伊立替康的浓度可以大于等于15mg/ml。例如可以是10mg/ml、12mg/ml、15mg/ml。可以是10mg/ml-12mg/ml。可以是12mg/ml-15mg/ml。可以是10mg/ml-15mg/ml。
本发明利用主动载药原理：在脂质体内外水相之间存在磺酸盐离子梯度；外水相中的盐酸伊立替康发生部分电离，其中，非解离形式的伊立替康以被动扩散方式进入脂质体内水相中，与内水相中铵根离子或三乙胺离子电离所产生的氢离子结合，形成伊立替康离子，进而与磺酸根形成晶体或无定型不溶性盐，从而使伊立替康包封在脂质体内水相中。同时，脂质体内水相中的铵根离子电离所产生的氨气，或三乙胺离子电离所产生的三乙胺不断从脂质体内水相逸出，维持了脂质体内水相中的氢离子的浓度，使脂质体内水相中的伊立替康分子持续不断地与氢离子结合，形成伊立替康阳离子，并与磺酸根形成沉淀，直到外水相中几乎所有的伊立替康被包封到脂质体内水相中。该脂质体具有高载药量，高稳定性，并且具备优异 的缓释效果。
伊立替康脂质体制剂的用途
伊立替康是一种半合成水溶性喜树碱类衍生物，可用于包括结直肠癌、肺癌、乳腺癌及胰腺癌的肿瘤治疗。
本发明所述的伊立替康脂质体制剂可使更多的伊立替康以活性内酯环形式存在于血液循环中，并到达肿瘤部位，转化为SN-38，从而进一步改善抗肿瘤效果。本发明所述的伊立替康脂质体体内半衰期长，生物利用度高。
在进一步描述本发明具体实施方式之前，应理解，本发明的保护范围不局限于下述特定的具体实施方案；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围；在本发明说明书和权利要求书中，除非文中另外明确指出，单数形式“一个”、“一”和“这个”包括复数形式。
当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
除非另外说明，本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的药剂学，药物分析学，药物化学，分析化学，分子生物学、生物化学及相关领域的常规技术。这些技术在现有文献中已有完善说明。
实施例1、盐酸伊立替康的检测方法
本实施例中建立盐酸伊立替康标准曲线，具体如下。
采用紫外(ultraviolet，UV)检测分析方法：检测仪器为TECAN
Figure PCTCN2019110542-appb-000001
200 PRO；检测波长为369nm；检测温度为24℃；检测孔板为
Figure PCTCN2019110542-appb-000002
96 well plates,UV-transparent；检测体积为200μl。
精密称量2.5001mg盐酸伊立替康溶解于25ml容量瓶中得到浓度为0.1000mg/ml的盐酸伊立替康水溶液；将盐酸伊立替康水溶液与超纯水混合，梯度稀释，得到浓度为5μg/ml、6.25μg/ml、7.5μg/ml、10μg/ml、12.5μg/ml、15μg/ml、20μg/ml和25μg/ml的盐酸伊立替康标准溶液。采用UV法检测上述浓度的盐酸伊立替康标准溶液，检测结果如表1所示。
表1不同浓度的盐酸伊立替康水溶液UV检测值
Figure PCTCN2019110542-appb-000003
Figure PCTCN2019110542-appb-000004
根据表1所示的结果建立标准曲线并考察回收率。工作曲线如图2所示。盐酸伊立替康水溶液的UV标准曲线为为Y＝0.0262X+0.0105(n＝8)，R 2＝0.9991，回收率均在95％～105％，标曲拟合良好。
实施例2、以甲基磺酸铵为内水相的伊立替康脂质体的制备和表征
本实施例中使用的磷脂均购自德国Lipoid公司。具体为氢化豆磷脂(HSPC，分子量783.8)；聚乙二醇化磷脂具体为二硬脂酰磷脂酰乙醇胺-聚乙二醇2000(DSPE-PEG2000)；胆固醇(CHOL，分子量386.7)；使用的盐酸伊立替康购自Aladdin公司；使用的甲基磺酸购自西格玛奥德里奇(Sigma-Aldrich)公司；使用的氨水购自Aladdin公司。
需要说明的是，在后续的实施例中所使用的磷脂均为Lipoid公司的氢化豆磷脂；所使用的聚乙二醇化磷脂均为Lipoid公司的二硬脂酰磷脂酰乙醇胺-聚乙二醇；所使用的胆固醇均为Lipoid的胆固醇；所使用的盐酸伊立替康均购自Aladdin公司。
2.1伊立替康脂质体的制备
具体制备过程如下：
步骤(1)，精密称量479.9mg氢化豆磷脂，160.9mg胆固醇和160.8mg二硬脂酰磷脂酰乙醇胺-聚乙二醇2000，加入适量乙醇充分溶解混合，得到脂质的乙醇混合溶液；
步骤(2)，精密量取2.131ml甲磺酸溶液，用氨水滴定至pH 4.0～6.0，双蒸水(ddH 2O)定容至50ml，得到pH为4.0～6.0的650mM甲基磺酸铵缓冲液；
步骤(3)，将步骤(1)所得脂质的乙醇混合溶液加入10ml pH为4.0～6.0的650mM甲基磺酸铵缓冲液，并置于70摄氏度中搅拌水浴30分钟，使脂质体充分水合，得到较均匀的脂质体混悬液；
步骤(4)，利用脂质体挤出仪，将步骤(3)所得脂质体混悬液依次挤压通过不同孔径 的聚碳酯膜各10次，最终得到粒径大小为100nm，粒径分布均匀的空白脂质体；
步骤(5)，将步骤(4)所制得的脂质体置于截留分子量为10000的透析袋中，以10％蔗糖水溶液作为透析介质，4摄氏度透析过夜。样品与透析介质体积比为1:1000，透析期间换三次透析液，完全除去脂质体外水相中的甲基磺酸铵，得到由10％的蔗糖组成的外水相、由甲基磺酸铵溶液为内水相，以及由磷脂双分子层构成空白脂质体，脂质体内外水相具有一定的pH和甲基磺酸铵浓度梯度。具体地，空白脂质体内水相为650mM甲基磺酸铵水溶液(pH4.0～6.0)、空白脂质体外水相为质量分数10％蔗糖水溶液。
步骤(6)，将步骤(5)中所得的内水相为甲基磺酸铵溶液的空白脂质体混悬液，与浓度为10mg/ml的盐酸伊立替康水溶液，按照体积比为1:1混合，并于50-60摄氏度中孵育10-30分钟，得到内水相含伊立替康的脂质体。
2.2、伊立替康脂质体的表征
2.2.1、伊立替康脂质体的粒径测定
将本实施例制备的伊立替康脂质体，用ddH 2O稀释50倍，利用Zetasizer ZS90(马尔文公司，英国)对粒径进行分析，具体结果如表3所示。表3表明，载药后的脂质体粒径均在100nm左右，粒度分布(PDI)均小于0.1。此外，载药前空白脂质体的粒径也在100nm左右，粒度分布(PDI)均小于0.1。
2.2.2、伊立替康脂质体包封率的测定
在制备得到的伊立替康脂质体中加入适量Dowex树脂(西格玛奥德里奇(Sigma-Aldrich)公司)，充分振摇，吸附未包裹的伊立替康。静置后取上清200ul，采用UV检测分析方法(实施例1)，测定加入树脂前后脂质体中的伊立替康的含量。
伊立替康脂质体的包封率(EE)按以下公式计算：
Figure PCTCN2019110542-appb-000005
其中，M inter是树脂吸附游离药物后的脂质体制剂中伊立替康的含量，即被脂质体包封的伊立替康的含量；M total是树脂吸附前的伊立替康脂质体制剂中伊立替康的含量，即伊立替康的投药量。结果如表2所示。表2表明，按照本方法制备的伊立替康脂质体的包封率良好，可实现高药脂比载药。
表2以甲基磺酸铵溶液为内水相的伊立替康脂质体粒径和包封率
Figure PCTCN2019110542-appb-000006
Figure PCTCN2019110542-appb-000007
实施例3、以甲基磺酸三乙胺溶液为内水相的伊立替康脂质体的制备和表征
本实施例中使用的甲磺酸购自西格玛奥德里奇(Sigma-Aldrich)公司；使用的三乙胺购自国药集团。
精密量取2.131ml甲磺酸溶液，用三乙胺滴定至pH 4.0～6.0，双蒸水(ddH 2O)定容至50ml，得到pH为4.0～6.0的650mM甲基磺酸三乙胺缓冲液；
制备伊立替康脂质体，具体制备过程参照实施例2。不同之处在于，在步骤(3)往步骤(1)所得到的脂质的乙醇溶液加入10ml、pH为4.0～6.0的650mM甲基磺酸三乙胺缓冲液，而不加入甲基磺酸铵缓冲液。
伊立替康脂质体的表征方法参照实施例2，结果如表3所示。表3表明，实施例3制备的伊立替康脂质体的包封率约在70％左右。与实施例2结果相比，以甲基磺酸三乙胺作为脂质体内水相其包封率有所下降，说明铵盐离子浓度梯度在主动载药中的贡献要优于三乙胺离子浓度梯度。
表3以甲基磺酸三乙胺溶液为内水相的伊立替康脂质体粒径和包封率
Figure PCTCN2019110542-appb-000008
实施例4、以4-羟基苯磺酸铵溶液为内水相的伊立替康脂质体的制备和表征
本实施例中使用的4-羟基苯磺酸购自麦克林(Macklin)公司；使用的氨水购自Aladdin公司。
精密量取6.513ml 4-羟基苯磺酸溶液，用氨水滴定至pH 4.0～6.0，双蒸水(ddH 2O)定容至50ml，得到pH为4.0～6.0的650mM 4-羟基苯磺酸铵缓冲液；
制备伊立替康脂质体，具体制备过程参照实施例2。不同之处在于，在步骤(3)往步骤(1)所得到的脂质乙醇溶液加入10ml浓度为650mM 4-羟基苯磺酸铵缓冲液(pH为4.0～6.0)，而不加入甲基磺酸铵缓冲液。
伊立替康脂质体的表征方法参照实施例2，结果如表4所示。表4表明，实施例4制备的伊立替康脂质体的包封率良好。
表4以4-羟基苯磺酸铵溶液为内水相的伊立替康脂质体粒径和包封率
Figure PCTCN2019110542-appb-000009
实施例5、以4-羟基苯磺酸三乙胺溶液为内水相的伊立替康脂质体的制备和表征
本实施例中使用的4-羟基苯磺酸三乙胺购自麦克林(Macklin)公司；使用的三乙胺购自国药集团。
精密量取6.513ml 4-羟基苯磺酸溶液，用三乙胺滴定至pH 4.0～6.0，双蒸水(ddH 2O)定容至50ml，得到pH为4.0～6.0的650mM 4-羟基苯磺酸三乙胺缓冲液；
制备伊立替康脂质体，具体制备过程参照实施例2。不同之处在于，在步骤(2)往步骤(1)所得到的脂质乙醇溶液加入10ml、pH为4.0～6.0的650mM 4-羟基苯磺酸三乙胺缓冲液，而不加入甲基磺酸铵缓冲液。
表征伊立替康脂质体的方法参照实施例2，结果如表5所示。表5表明，实施例5制备得伊立替康脂质体的包封率良好。
表5以4-羟基苯磺酸三乙胺溶液为内水相的伊立替康脂质体粒径和包封率
Figure PCTCN2019110542-appb-000010
实施案例2、3、4、5的四种内水相成分对伊立替康脂质体载药能力的影响于图3所示。
实施例6、以乙二磺酸铵溶液为内水相的伊立替康脂质体的制备和表征
本实施例中使用的乙二磺酸购自阿法埃莎(Alfa Aesar)公司；使用的氨水购自Aladdin公司。
精密称量3.1538mg乙二磺酸水合物溶于双蒸水(ddH 2O)，用氨水滴定至pH 4.0～6.0，双蒸水定容至50ml，得到pH为4.0～6.0的325mM乙二磺酸铵缓冲液；
制备伊立替康脂质体，具体制备过程参照实施例2。不同之处在于，在步骤(2)往步骤(1)所得到的脂质乙醇溶液加入10ml、pH为4.0～6.0的325mM乙二磺酸铵缓冲液，而不加入甲基磺酸铵缓冲液。
表征伊立替康脂质体的方法参照实施例2，结果如表6所示。表6表明，实施例6制备得伊立替康脂质体的包封率良好。
表6以乙二磺酸铵溶液为内水相的伊立替康脂质体粒径和包封率
Figure PCTCN2019110542-appb-000011
实施例7、以乙二磺酸三乙胺溶液为内水相的伊立替康脂质体的制备和表征
本实施例中使用的乙二磺酸购自阿法埃莎(Alfa Aesar)公司；使用的三乙胺购自国药集团。
精密称量3.1535mg乙二磺酸水合物溶于双蒸水(ddH 2O)，用三乙胺滴定至pH 4.0～6.0，双蒸水定容至50ml，得到pH为4.0～6.0的325mM乙二磺酸三乙胺缓冲液；
制备伊立替康脂质体，具体制备过程参照实施例2。不同之处在于，在步骤(2)往步骤(1)所得到的脂质乙醇溶液加入10ml、pH为4.0～6.0的325mM乙二磺酸三乙胺缓冲液，而不加入甲基磺酸铵缓冲液。
表征伊立替康脂质体的方法参照实施例2，结果如表7所示。表7表明，实施例7制备得伊立替康脂质体的包封率良好。
表7以乙二磺酸三乙胺溶液为内水相的伊立替康脂质体粒径和包封率
Figure PCTCN2019110542-appb-000012
实施例8、以丙二磺酸铵溶液为内水相的伊立替康脂质体的制备和表征
本实施例中使用的丙二磺酸购自阿法埃莎(Alfa Aesar)公司；使用的氨水购自Aladdin公司。
精密量取4.741ml丙二磺酸溶液，用氨水滴定至pH 4.0～6.0，双蒸水(ddH 2O)定容至50ml，得到pH为4.0～6.0的325mM丙二磺酸铵缓冲液；
制备伊立替康脂质体，具体制备过程参照实施例2。不同之处在于，在步骤(2)往步骤(1)所得到的脂质乙醇溶液加入10ml、pH为4.0～6.0的325mM丙二磺酸铵缓冲液，而不加入甲基磺酸铵缓冲液。
表征伊立替康脂质体的方法参照实施例2，结果如表8所示。表8表明，实施例8制备得伊立替康脂质体的包封率良好。
表8以丙二磺酸铵溶液为内水相的伊立替康脂质体粒径和包封率
Figure PCTCN2019110542-appb-000013
实施例9、以丙二磺酸三乙胺溶液为内水相的伊立替康脂质体的制备和表征
本实施例中使用的丙二磺酸购自阿法埃莎(Alfa Aesar)公司；使用的三乙胺购自国药集团。
精密量取4.735ml 4-羟基苯磺酸溶液，用三乙胺滴定至pH 4.0～6.0，双蒸水(ddH 2O)定容至50ml，得到pH为4.0～6.0的325mM丙二磺酸三乙胺缓冲液；
制备伊立替康脂质体，具体制备过程参照实施例2。不同之处在于，在步骤(2)往步骤(1)所得到的脂质乙醇溶液加入10ml、pH为4.0～6.0的325mM丙二磺酸三乙胺缓冲液，而不加入甲基磺酸铵缓冲液。
表征伊立替康脂质体的方法参照实施例2，结果如表9所示。表9表明，实施例9制备得伊立替康脂质体的包封率良好。
表9以丙二磺酸三乙胺溶液为内水相的伊立替康脂质体粒径和包封率
Figure PCTCN2019110542-appb-000014
实施例2～9的八种磺酸盐梯度对伊立替康脂质体的包封率的影响如图3所示。
实施例10、以不同磺酸盐为内水相的伊立替康脂质体的体外释放研究
不同药脂比时，脂质体内水相中药物的浓度不同，因此药物与磺酸根离子所形成的不溶性盐很可能具有不同的结构，因而可能具有不同的药物释放速率。为了比较以不同的磺酸盐(包括铵盐和三乙胺盐)为内水相所制备的伊立替康脂质体的体外释放速率，本实施例中，进行体外释放研究的伊立替康脂质体均具有相同的药脂比(0.3)，载药浓度均为2.58mg/ml、脂质浓度均为8.91mg/ml。
用于体外释放研究的牛血清白蛋白(BSA)购自于生工生物工程(上海)股份有限公司；Dowex树脂购自西格玛奥德里奇(Sigma-Aldrich)公司。
伊立替康脂质体的体外释放实验具体过程如下：
步骤(1)，精密称量4.0152g牛血清白蛋白(BSA)粉末，溶解于100ml生理盐水，配制40mg/ml白蛋白生理盐水溶液(蛋白浓度与50％血浆中蛋白浓度相当)；
步骤(2)，取实施例2-9中以不同单价磺酸盐溶液或二磺酸盐(pH为4.0～6.0)溶液为内水相所制备的伊立替康脂质体；其中，脂质浓度为8.91mg/ml、药脂比(药物和脂质的摩尔比)均为0.3。
步骤(3)，用40mg/ml白蛋白生理盐水溶液将脂质体稀释80倍，加入足量的Dowex树脂吸附游离药物，形成漏槽条件，于37℃100rpm摇床(THZ-C恒温振荡器，中国)中震荡。在不同的时间点(0小时、1小时、3小时、6小时、9小时和24小时)取样，静置后取上清，UV法测定包裹在脂质体中的伊立替康的含量，计算不同时间点伊立替康的累积释放速率。
作为比较，本发明还按照市售Onivyde的说明书自制了以蔗糖八硫糖脂三乙胺为内水相的伊立替康脂质体。其中，脂质浓度为8.91mg/ml、药脂比为0.3；以及以pH4.0-6.0的325mM的硫酸铵为内水相的伊立替康脂质体，其中，脂质浓度为8.91mg/ml、药脂比为0.3。按照上述方法，平行测定了以蔗糖八硫酸脂三乙胺和硫酸铵为内水相的伊立替康脂质体在白蛋白生理盐水中的体外释放速率，并与本发明提出的伊立替康脂质体制剂的释放速率进行了比较。
各组伊立替康脂质体的体外累积释放曲线如图4所示。图4表明，在白蛋白生理盐水溶液中，以甲基磺酸铵、4-羟基苯磺酸铵、4-羟基苯磺酸三乙胺、乙二磺酸铵、乙二磺酸三乙胺、丙二磺酸铵和丙二磺酸三乙胺溶液为内水相的脂质体伊立替康均具备较好的体外缓释效果。其中，从24小时累积释放百分率来看，甲基磺酸铵组达到10.03％，4-羟基苯磺酸铵组达到11.35％，4-羟基苯磺酸三乙胺组达到10.32％，乙二磺酸铵组达到1.03％，乙二磺酸三乙胺组达到3.49％，丙二磺酸铵组达到2.31％，丙二磺酸三乙胺组达到1.96％。同时平行测定蔗糖八硫酸脂三乙胺组24小时累积释放百分率达到8.91％，硫酸铵组达到了15.34％。该体 外释放研究结果表明，以单价磺酸盐和二磺酸盐为内水相的伊立替康脂质体的释放速率明显慢于以硫酸铵为内水相的脂质体的释放速率。
实施例11、伊立替康磺酸盐的熔点测定
本实施例使用的甲磺酸和羟基苯磺酸购自西格玛奥德里奇(Sigma-Aldrich)公司。
熔点可以反映化合物内部结构的规整性。盐的熔点越高，其分子排列很可能越规整。例如晶体的熔点往往高于无定型。为了考察脂质体内水相中，不同的磺酸盐与伊立替康所形成的盐的可能结构，本实施例中通过将盐酸伊立替康与磺酸盐溶液直接混合的方式，形成伊立替康磺酸盐，并测定了各个盐的熔点。
具体过程如下：
(1)精密量取2.131ml甲磺酸溶液，加入2.431ml氨水，双蒸水(ddH 2O)定容至50ml，得到浓度为650mM的甲基磺酸铵水溶液；精密称量50.6mg盐酸伊立替康加入1ml 650mM的甲基磺酸铵水溶液中，摇匀；
(2)精密量取2.131ml甲磺酸溶液，加入4.572ml三乙胺，双蒸水(ddH 2O)定容至50ml，得到浓度为650mM的甲基磺酸三乙胺水溶液；精密称量50.6mg盐酸伊立替康加入1ml650mM的甲基磺酸三乙胺水溶液中，摇匀；
(3)精密量取6.513ml 4-羟基苯磺酸溶液，加入2.432ml氨水，双蒸水(ddH 2O)定容至50ml，得到浓度为650mM的羟基苯磺酸铵水溶液；精密称量50.6mg盐酸伊立替康加入1ml 650mM的羟基苯磺酸铵水溶液中，摇匀；
(4)精密量取6.513ml 4-羟基苯磺酸溶液，加入5.866ml三乙胺，双蒸水(ddH 2O)定容至50ml，得到浓度为650mM的羟基苯磺酸三乙胺水溶液；精密称量50.6mg盐酸伊立替康加入1ml 650mM的羟基苯磺酸三乙胺水溶液中，摇匀；
将上述盐酸伊立替康与磺酸盐溶液的混悬液，油浴加热，测定熔点，结果如表10所示。
表10不同伊立替康磺酸盐的熔点测定结果
Figure PCTCN2019110542-appb-000015
表10表明，各伊立替康磺酸盐的熔融温度在86℃～99℃之间。将熔融实验结果结合后续 DSC实验结果分析，可以得出以这些磺酸盐溶液作为脂质体内水相时，与脂质体内水相中的伊立替康离子形成了晶型或无定型结构的不溶性盐，从而达到对伊立替康的稳定包封，并增加伊立替康脂质体的储存稳定性。
实施例12伊立替康脂质体的高分辨DSC表征
研究表明，脂质体内水相中的纳米晶体的形成，有利于提高脂质体制剂的储存稳定性，并减缓药物释放速率(Wei,X.等,Cardinal role of intraliposome doxorubicin-sulfate nanorod crystal in doxil properties and performance[J].ACS Omega 2018,3(3),2508-2517)。
为了考察单价磺酸盐和二磺酸盐在脂质体内水相中，与伊立替康所形成的盐的微观结构，在实施例11熔点测定的基础上，我们采用高分辨DSC研究了实施例2～9中所制备的伊立替康脂质体的热力学行为，并与自制的以蔗糖八硫酸酯三乙胺和硫酸铵为内水相的伊立替康脂质体进行了对比。所有进行DSC表征的伊立替康脂质体样品的投药药脂比均为0.3，脂质浓度为8.9mg/ml，平均粒径为100nm且PDI小于0.1。
本实施例使用的差示扫描量热仪(DSC)为美国GE公司的毛细管DSC(Capillary DSC)。扫描温度为10-120摄氏度，扫描速度为1度/分钟。
具体过程如下：
取实施例2-9中以不同磺酸盐溶液为内水相制备的伊立替康脂质体，0.22um滤膜过滤后，手动加入毛细管DSC的样品池中(约200ul)，用10％蔗糖溶液作为对照，加热扫描并记录热力学图谱。按照文献方法(Wei,X.等,Insights into composition/structure/function relationships of
Figure PCTCN2019110542-appb-000016
gained from"high-sensitivity"differential scanning calorimetry[J].Eur J Pharm Biopharm 2016,104,260-270)处理所得的热力学图谱并依据各脂质体中所包裹的伊利替康的摩尔浓度计算热力学参数。结果如图5所示。各组盐酸伊立替康脂质体的主要热力学参数如表11所示。
表11以不同磺酸盐溶液为内水相制备的伊立替康脂质体的主要药动学参数
Figure PCTCN2019110542-appb-000017
Figure PCTCN2019110542-appb-000018
DSC实验结果表明，伊立替康脂质体内水相中的磺酸盐与药物形成了较为规整的结构，具有较高的熔点。这种形式有利于药物的稳定包载和制剂的缓释。结合实施例10的体外释放结果，以甲基磺酸铵、4-羟基苯磺酸铵、4-羟基苯磺酸三乙胺溶液以及四组二磺酸盐溶液为内水相的伊立替康脂质体具备较好的体外缓释效果。
实施例12
本发明还参照实施例2和实施例6制备了其他种类的伊立替康脂质体，并进行表征。
类型1、与实施例2中伊立替康脂质体制备方法的不同之处在于，步骤(2)为，取甲基磺酸铵溶液调pH，制得pH为9.0的800mM甲基磺酸铵缓冲液；步骤(4)中，得到粒径大小为200nm的空白脂质体，步骤(5)中，与浓度为15mg/ml的盐酸伊立替康水溶液混合，其余皆相同。
类型2、与实施例2中伊立替康脂质体制备方法的不同之处在于，步骤(2)为，取甲基磺酸铵溶液调pH，制得pH为4.5的100mM甲基磺酸铵缓冲液；步骤(4)中，得到粒径大小为30nm的空白脂质体，步骤(5)中，与浓度为10mg/ml的盐酸伊立替康水溶液混合，其余皆相同。
类型3、与实施例2中伊立替康脂质体制备方法的不同之处在于，步骤(2)为，取甲基磺酸铵溶液调pH，制得pH为5.0的200mM甲基磺酸铵缓冲液；步骤(4)中，得到粒径大小为50nm的空白脂质体，步骤(5)中，与浓度为13mg/ml的盐酸伊立替康水溶液混合，其余皆相同。
类型4、与实施例2中伊立替康脂质体制备方法的不同之处在于，步骤(2)为，取甲基磺酸铵溶液调pH，制得pH为8.0的700mM甲基磺酸铵缓冲液；步骤(4)中，得到粒径大小为120nm的空白脂质体，步骤(5)中，与浓度为14mg/ml的盐酸伊立替康水溶液混合， 其余皆相同。
类型5、与实施例6中伊立替康脂质体制备方法的不同之处在于，步骤(2)为，取乙二磺酸水合物溶液调pH，制得pH为6.0的500mM乙二磺酸铵缓冲液；步骤(4)中，得到粒径大小为90nm的空白脂质体，步骤(5)中，与浓度为14mg/ml的盐酸伊立替康水溶液混合，其余皆相同。
类型6、与实施例6中伊立替康脂质体制备方法的不同之处在于，步骤(2)为，取乙二磺酸水合物溶液调pH，制得pH为4.0的50mM乙二磺酸铵缓冲液；步骤(4)中，得到粒径大小为110nm的空白脂质体，步骤(5)中，与浓度为12mg/ml的盐酸伊立替康水溶液混合，其余皆相同。
类型7、与实施例6中伊立替康脂质体制备方法的不同之处在于，步骤(2)为，取乙二磺酸水合物溶液调pH，制得pH为4.5的100mM乙二磺酸铵缓冲液；步骤(4)中，得到粒径大小为80nm的空白脂质体，步骤(5)中，与浓度为13mg/ml的盐酸伊立替康水溶液混合，其余皆相同。
类型8、与实施例6中伊立替康脂质体制备方法的不同之处在于，步骤(2)为，取乙二磺酸水合物溶液调pH，制得pH为7.5的400mM乙二磺酸铵缓冲液；步骤(4)中，得到粒径大小为150nm的空白脂质体，步骤(5)中，与浓度为13mg/ml的盐酸伊立替康水溶液混合，其余皆相同。
类型1、以甲基磺酸铵溶液为内水相的伊立替康脂质体粒径和包封率
Figure PCTCN2019110542-appb-000019
类型2、以甲基磺酸铵溶液为内水相的伊立替康脂质体粒径和包封率
Figure PCTCN2019110542-appb-000020
类型3、以甲基磺酸铵溶液为内水相的伊立替康脂质体粒径和包封率
Figure PCTCN2019110542-appb-000021
类型4、以甲基磺酸铵溶液为内水相的伊立替康脂质体粒径和包封率
Figure PCTCN2019110542-appb-000022
类型5、以乙二磺酸铵溶液为内水相的伊立替康脂质体粒径和包封率
Figure PCTCN2019110542-appb-000023
类型6、以乙二磺酸铵溶液为内水相的伊立替康脂质体粒径和包封率
Figure PCTCN2019110542-appb-000024
类型7、以乙二磺酸铵溶液为内水相的伊立替康脂质体粒径和包封率
Figure PCTCN2019110542-appb-000025
类型8、以乙二磺酸铵溶液为内水相的伊立替康脂质体粒径和包封率
Figure PCTCN2019110542-appb-000026
上述实施例仅例示性说明本发明的原理及其功效，而非用于限制本发明。任何熟悉此技术的人士皆可在不违背本发明的精神及范畴下，对上述实施例进行修饰或改变。因此，举凡所属技术领域中具有通常知识者在未脱离本发明所揭示的精神与技术思想下所完成的一切等效修饰或改变，仍应由本发明的权利要求所涵盖。

Claims (10)

  1. 一种伊立替康脂质体制剂，含有伊立替康脂质体，所述伊立替康脂质体包括：伊立替康和脂质体载体以及位于脂质体膜内的内水相和位于脂质体膜外的外水相，所述伊立替康被包封于所述内水相中；所述脂质体膜内的内水相和膜外的外水相之间存在磺酸盐梯度。
  2. 根据权利要求1所述的伊立替康脂质体制剂，其特征在于，所述内水相包括磺酸盐水溶液；所述外水相为生理等渗溶液。
  3. 根据权利要求2所述的伊立替康脂质体制剂，其特征在于，所述内水相中的伊立替康的阳离子与磺酸盐阴离子形成不溶性盐。
  4. 根据权利要求2所述的伊立替康脂质体制剂，其特征在于，所述磺酸盐包括单价磺酸盐和/或二磺酸盐。
  5. 根据权利要求4所述的伊立替康脂质体制剂，其特征在于，还包括以下特征中的一项或多项：a.所述单价磺酸盐选自甲基磺酸铵、4-羟基苯磺酸铵、甲基磺酸三乙胺和4-羟基苯磺酸三乙胺中的一种或多种；b.所述二磺酸盐选自乙基二磺酸铵、丙基二磺酸铵、乙基二磺酸三乙胺和丙基二磺酸三乙胺中的一种或多种。
  6. 根据权利要求1所述的伊立替康脂质体制剂，其特征在于，还包括以下特征中的一项或多项：a.所述脂质体内水相的pH值为4.0～9.0；b.所述伊立替康脂质体制剂中，伊立替康的浓度大于等于0.1mg/ml；c.所述伊立替康脂质体制剂中，伊立替康的包封率大于等于85％。
  7. 一种如权利要求1-6任一项所述的伊立替康脂质体制剂的制备方法，其特征在于，所述方法为主动载药法，所述主动载药法包括以下步骤：
    (1)制备内水相和外水相均含磺酸盐水溶液的空白脂质体；
    (2)制备内水相含有磺酸盐水溶液、外水相含有生理等渗溶液的空白脂质体，以形成空白脂质体内外水相磺酸盐浓度梯度；
    (3)将步骤(2)所得空白脂质体与伊立替康可溶性盐水溶液混合，孵育，去除游离伊立替康可溶性盐，获得伊立替康脂质体。
  8. 如权利要求7所述的伊立替康脂质体制剂的制备方法，其特征在于，所述方法还包括以下特征中的一项或多项：
    a.步骤(1)和步骤(2)中，所述磺酸盐水溶液中的磺酸盐浓度为100mM-800mM；
    b.步骤(1)和步骤(2)中，所述磺酸盐水溶液的pH值为4.0～9.0；
    c.步骤(1)和步骤(2)中，所述磺酸盐水溶液中磺酸根离子的浓度是50mM～800mM；
    d.步骤(1)和步骤(2)中，所述磺酸盐水溶液中阳离子的浓度是50～800mM；
    e.步骤(2)中，所述生理等渗溶液可选自：5％(w/v)葡萄糖水溶液、10％(w/v)蔗糖水溶液或0.9％(w/v)氯化钠水溶液；
    f.步骤(2)中，所述外水相的pH是4.0～9.0；
    g.步骤(3)中，药脂摩尔比大于等于0.1；
    h.步骤(3)中，所述伊立替康可溶性盐选自盐酸伊立替康。
  9. 一种由如权利要求7-8任一项所述方法制备获得的伊立替康脂质体制剂。
  10. 如权利要求1-6任一项所述伊立替康脂质体制剂在制备结直肠癌、肺癌、乳腺癌或胰腺癌的肿瘤治疗药物中的用途。
PCT/CN2019/110542 2018-11-05 2019-10-11 伊立替康脂质体制剂及其制备与应用 Ceased WO2020093836A1 (zh)

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