HK40070304A - Stabilizing camptothecin pharmaceutical compositions - Google Patents

Description

Stable camptothecin pharmaceutical compositions

The application is a divisional application of an invention patent application with the filing date of 2016, 10 and 15, and the filing number of 201680073535.9 (International application No. PCT/US2016/057247) and the name of 'stable camptothecin pharmaceutical composition'.

Priority requirement

The present patent application claims the following benefits: U.S. provisional patent application serial nos. 62/242,835 (filed on 2015, 10-month 16-day), 62/242,873 (filed on 2015, 10-month 16-day), 62/244,061 (filed on 2015, 10-month 20-day), and 62/244,082 (filed on 2015, 10-month 20-day), each of which is incorporated by reference herein in its entirety.

Technical Field

The present application relates to stabilizing pharmaceutical compositions comprising camptothecin compounds, including liposomal camptothecin pharmaceutical formulations that are stabilized to reduce lysolipid formation during storage.

Background

Camptothecin compounds (e.g., irinotecan or topotecan) are useful for treating tumors and/or cancers in humans. For example, injectable liposomal pharmaceuticals for the treatment of certain forms of cancer can be prepared as liposomal dispersions encapsulating camptothecin compounds. Liposomal camptothecin compositions camptothecin compounds can be encapsulated with a polyanionic trapping agent in a liposome comprising cholesterol and one or more phospholipids ("PL"). However, hydrolysis of phospholipids and hydrolysis of active lactone structures in camptothecin can occur in camptothecin liposomes having one or more phospholipids. Hydrolytic breakdown of liposomal phospholipids, such as phosphatidylcholine ("PC" +) can alter the release of camptothecin compounds, such as irinotecan, from the liposomes. The first step of hydrolysis of PL (e.g. PC) may result in the formation of lyso-PL (e.g. lysophosphatidylcholine ("lyso-PC"), which is a glycerophosphocholine fatty acid monoester).

Liposomal camptothecin compositions are affected by pH in at least two ways. First, the hydrolytic breakdown of phospholipids of liposomal camptothecin (e.g., liposomal irinotecan) tends to be pH-dependent, with pH values of 6.0 or 6.5 being believed to minimize hydrolysis of phosphatidylcholine. Conditions with a pH greater than 6.5 tend to increase (1) the conversion of camptothecin compounds (e.g., irinotecan) to the less active carboxylate form and (2) the amount of lyso-PC in the liposomes. Second, camptothecin compounds undergo a pH-dependent transition between the less active carboxylic acid form (mainly at neutral and basic pH) and the predominantly active lactone form at acidic pH. For example, the conversion of the carboxylate form to the lactone form of irinotecan occurs primarily at pH 6.0 (about 85% of irinotecan is the active lactone form) to pH 7.5 (only about 15% of irinotecan is the active lactone form). At pH6.5, about 65% of irinotecan is in the active larger lactone form.

It was unexpectedly found that the stability of phospholipid-containing liposomal camptothecin prepared at pH6.5 was adversely affected by the formation of lyso-PC during storage under refrigerated conditions (2-8 ℃). For example, sample 12 (irinotecan sucrose octasulfate encapsulated in irinotecan liposomes comprising DSPC, cholesterol and MPEG-2000-DSPE prepared at pH6.5 in a molar ratio of 3:2: 0.015) subsequently produced greater than 30 mol% (relative to the total amount of phosphatidylcholine in the irinotecan liposome composition) of lyso-PC during the first 3 months of refrigerated storage (2-8 ℃) after preparation (and greater than 35 mol% lyso-PCR during the first 9 months).

Thus, there remains a need for stable pharmaceutical compositions of camptothecin. For example, there is a need for a more stable, improved liposomal formulation of irinotecan that produces less lyso-PC during refrigerated storage at 2-8 ℃ after manufacture. The present invention addresses this need.

Disclosure of Invention

The present invention provides novel camptothecin pharmaceutical compositions (e.g., liposomal irinotecan) having improved stability, including camptothecin liposomal compositions comprising an esterphospholipid, wherein the rate of formation of lysophospholipids ("lyso-PL") (e.g., lysophosphatidylcholine or "lyso-PC") is reduced. The present invention is based in part on the unexpected recognition that: liposomal compositions of camptothecin compounds (e.g., irinotecan) can be prepared to yield reduced amounts of lysophospholipids after prolonged storage at 2-8 ℃. The following unexpected findings make it possible to prepare such stable liposome compositions: controlling specific parameters during liposome preparation (the ratio of drug and phospholipid relative to the trapping dose, the pH of the liposome formulation, and the amount of trapping agent counter ions in the liposome formulation) synergistically reduces lysophospholipid formation during storage of the camptothecin liposome formulation. The present invention provides extremely valuable information for designing and identifying improved liposome compositions that are robust while reducing the costs associated with developing such compositions.

A stable camptothecin composition comprising one or more phospholipids, including PEG-containing phospholipids, preferably forms no more than 20 mol% of lyso-PL (relative to total liposomal phospholipids) during the first 6 months of storage at 4 ℃, and/or forms no more than 25 mol% of lyso-PL during the first 9 months of storage at 4 ℃. The stable liposome of irinotecan preferably forms lyso-PL at an average rate of less than about 2 mol% (e.g., 0.5-1.5 mol%) of lyso-PL per month during the first 9 months of storage at 4 ℃ after preparation of the camptothecin composition. Preferred stable camptothecin compositions include irinotecan or a salt thereof (e.g., irinotecan sucrose octasulfate) in a liposomal irinotecan composition comprising cholesterol and one or more phospholipids, including PEG-containing phospholipids, that forms no more than 20 mol% lyso-PC (relative to total liposomal phospholipids) during 6 months of storage at 4 ℃ and/or forms no more than 25 mol% lyso-PC during 9 months of storage at 4 ℃ (e.g., during the first 6 and/or 9 months of stability testing after preparation). Stable irinotecan liposomes can form lyso-PC at a rate of less than about 2 mol% (e.g., 0.5-1.5 mol%) of lyso-PC per month during storage at 4 ℃ (e.g., during the first 9 months of stability testing after preparation). The stable liposomal composition of irinotecan comprising phosphatidylcholine can produce less than 1mg of lyso-PC during the first 9 months of stability testing at 2-8 ℃ after preparation.

In a first embodiment, the stable liposomal camptothecin composition has a pH of greater than 6.5 (e.g., 7.0-7.5, including 7.25, 7.3, or 7.5) and comprises liposomes encapsulating irinotecan and a sulfate polyanionic trapping agent (e.g., irinotecan sulfatide or "SOS"), having an irinotecan/sulfate compound gram-equivalent ratio ("ER") of greater than 0.9 (e.g., 0.9-1.1). The ER of irinotecan SOS liposomal formulation can be calculated as follows: the formula is used: ER ═ I/(SN), the liposome composition was determined for the molar amount of liposomal co-encapsulated irinotecan (I) and sulfate compound (S) per unit (e.g., 1mL), where N is the valence of the sulfate compound anion (e.g., N is 8 for sulfatide, and SO for free sulfate)₄ ^2-And N is 2). Preferably, the sulfate compound (S) is sucrose octasulfate, which contains 8 sulfate moieties per mole SOS。

In a second embodiment, stable liposomal camptothecin compositions are obtained using a specific ratio of camptothecin, anion trapping agent, and liposome-forming phospholipid, the compositions preferably having a stability ratio ("SR") of greater than about 950 (e.g., 950-. This embodiment provides a preparation criterion for predicting liposome stability as reflected by a stability ratio, as explained more fully below. This embodiment of the invention is based in part on the discovery that: when phospholipid-based camptothecin-containing liposomes are prepared by reacting (1) a camptothecin compound (e.g., irinotecan, topotecan, etc.) with (2) liposomes encapsulating a polysulfated anionic capture agent (e.g., sucrose octasulfate), the stability of the resulting loaded liposomes depends on the initial concentration of sulfate groups in the capture agent-liposome and the ratio of encapsulated camptothecin to phospholipid in the liposome. The stability ratio is defined as follows: SR is A/B, wherein: a is the amount of the portion of irinotecan encapsulated in the trapping agent liposome during loading, which is equivalent in grams to irinotecan free anhydrous base per mole of phospholipid in the composition; and B is the concentration of sulfate groups in the solution of thiosugar ester (or other capture agent) used to prepare the capture agent liposomes, expressed in mol/L (based on the concentration of sulfate groups). The stability ratio surprisingly predicts a surprising reduction in lyso-PC formation in phospholipid-based camptothecin-containing liposomes, even at pH 6.5: after 9 months of storage at 4 ℃, the phosphatidylcholine-containing irinotecan liposomes having a prepared stability ratio of about 942 yielded about 36 mol% lyso-PC (i.e., the stability ratio increased by about 5%, resulting in a 34% reduction in lyso-PC production under these conditions) compared to irinotecan liposomes having a prepared stability ratio of about 990 (sample 2) yielded about 24 mol% lyso-PC. In contrast, the stability ratio of irinotecan liposomes increased by about 30% from 724 (sample 12) to 942 (sample 3), resulting in about 1% more lyso-PC after 9 months of storage at 4 ℃ (e.g., comparing 35.7 mol% lyso-PC in sample 3 to 35.4 mol% lyso-PC in sample 12).

In a third embodiment, a novel stable irinotecan-encapsulating liposome composition that produces a reduced amount of lysophosphatidylcholine (lyso-PC) during storage at 2-8 ℃ comprises an irinotecan composition of formula (I) below, wherein x is 8:

liposomal irinotecan can comprise a composition of formula (I) encapsulated in liposomes. Preferably, the composition of formula (I) is formed (e.g. precipitated) in liposomes comprising cholesterol and one or more phospholipids, including for example PEG-containing phospholipids. For example, a compound of formula (I) may be formed in liposomes as follows: in forming a stable liposomal irinotecan composition, (1) a camptothecin compound (e.g., irinotecan, topotecan, etc.) is reacted with (2) liposomes encapsulating a polysulfated anion trap (e.g., sucrose octasulfate). Preferably, the liposomal irinotecan composition has a pH greater than 6.5 (e.g., 7.0-7.5, including 7.25, 7.3, and 7.5).

Preferred stable camptothecin compositions include liposomal irinotecan compositions comprising irinotecan or a salt thereof (e.g., irinotecan sucrose octasulfate) encapsulated within irinotecan liposomes comprising cholesterol and the phospholipids 1, 2-distearoyl-sn-glycerol-3-phosphocholine (DSPC) and methoxy-capped polyethylene glycol-distearoyl phosphatidylethanolamine (e.g., MPEG-2000-DSPE) in an aqueous isotonic buffer, the liposomal irinotecan composition comprising (or forming) less than 10 mol% lysophosphatidylcholine (lyso-PC) after the first 3 months of storage at 2-8 ℃, less than 20 mol% lysophosphatidylcholine (lyso-PC) after the first 6 months (or 180 days) of storage at 2-8 ℃, and/or comprises (or forms) less than 25 mol% lysophosphatidylcholine (lyso-PC) after the first 9 months of storage at 2-8 ℃ (e.g., during the stability test of the first 9 months after preparation).

Irinotecan liposomes preferably contain cholesterol, 1, 2-distearoyl-sn-glycerol-3-phosphocholine (DSPC) and methoxy-capped polyethylene glycol-distearoyl phosphatidylethanolamine (e.g., MPEG-2000-DSPE) in a 3:2:0.015 molar ratio, encapsulating 500mg (+ -10%) irinotecan per mmol of total liposomal phospholipid. The stable liposomal irinotecan composition preferably comprises irinotecan liposomes providing a total of about 4.3mg of irinotecan moieties per mL of the liposomal irinotecan composition, wherein at least about 98% of the irinotecan is encapsulated in the irinotecan liposomes (e.g., as irinotecan sucrose octasulfate, e.g., a compound of formula (I) above). Certain preferred liposomal compositions are storage-stable liposomal irinotecan compositions having a pH of 7.00-7.50 (e.g., 7.0, 7.25, 7.3, 7.5) and comprising a dispersion of irinotecan liposomes encapsulating irinotecan sucrose octasulfate in unilamellar vesicles (unilamellar bilayer vesicles) consisting of cholesterol and the phospholipids 1, 2-distearoyl-sn-glycerol-3-phosphocholine (DSPC) and methoxy-capped polyethylene glycol (MW2000) -distearoyl phosphatidylethanolamine (MPEG-2000-DSPE), the liposomal irinotecan composition comprising irinotecan moiety at a concentration equivalent to 500mg (± 10%) irinotecan per mmol of total liposomal phospholipids and 4.3mg irinotecan per mL of the liposomal irinotecan composition in grams of anhydrous free base, the storage-stable liposomal irinotecan composition stabilizes to form less than 1mg/mL of lyso-PC during the first 6 months of storage at 4 ℃. For example, certain preferred pharmaceutical liposomal irinotecan compositions comprise irinotecan or a salt thereof encapsulated in irinotecan (e.g., irinotecan sucrose octasulfate) at 4.3mg/mL irinotecan moieties, 6.81mg/mL 1, 2-distearoyl-sn-glycerol-3-phosphocholine (DSPC), 2.22mg/mL cholesterol, and 0.12mg/mL methoxy-capped polyethylene glycol (MW2000) -distearoyl phosphatidylethanolamine (MPEG-2000-DSPE) in an aqueous isotonic buffer, the liposomal composition comprising less than 10 mol% lysophosphatidylcholine (lyso-PC) after 3 months of storage at 2-8 ℃, less than 20 mol% lysophosphatidylcholine (lyso-PC) after 6 months (or 180 days) of storage at 2-8 ℃, and/or comprises less than 25 mol% lysophosphatidylcholine (lyso-PC) after 9 months of storage at 2-8 ℃.

In some embodiments, the liposome composition is prepared by a method comprising: contacting a solution comprising irinotecan moieties with the trapping agent liposomes to form irinotecan SOS liposomes under conditions effective to load 500g (+ -10%) of irinotecan moieties per mol phospholipid into the trapping agent liposomes comprising PL and to allow release of TEA cations from the trapping agent liposomes, the trapping agent liposomes acting as TEA₈SOS encapsulates Triethylammonium (TEA) and Sucrose Octasulfate (SOS) trapping agent (preferably TEA) at a concentration of 0.4-0.5M based on sulfate group concentration₈SOS trapping agent solution), and (b) contacting irinotecan SOS liposomes with 2- [4- (2-hydroxyethyl) piperazin-1-yl]Ethanesulfonic acid (HEPES) to give a liposomal composition of irinotecan at a pH of 7.25-7.50, resulting in a liposomal composition of irinotecan stabilized to form less than 10 mol% lysophosphatidylcholine (lyso-PC) during storage at 4 ℃ for 3 months (relative to the total amount of phosphatidylcholine in the liposomal composition of irinotecan).

For example, the present invention provides a liposomal composition of irinotecan comprising stabilized irinotecan liposomes encapsulating irinotecan Sucrose Octasulfate (SOS) in unilamellar vesicles of unilamellar lipids, the vesicles having a diameter of about 110nm and consisting of 1, 2-distearoyl-sn-glycerol-3-phosphocholine (DSPC), cholesterol, and methoxy-capped polyethylene glycol (MW2000) -distearoyl phosphatidylethanolamine (MPEG-2000-DSPE), wherein the stabilized irinotecan liposomes are obtained by a process comprising the steps of: (a) contacting irinotecan with the capture liposomes under conditions effective to load 500g (+ -10%) of irinotecan moiety per mol phospholipid into the capture liposomes and allow release of TEA cations from the capture liposomes to form irinotecan SOS liposomes that act as TEA₈SOS encapsulates Triethylammonium (TEA) cations and Sucrose Octasulfate (SOS) trapping agent at a concentration of 0.4-0.5M (based on sulfate group concentration), and (b) contacting irinotecan SOS liposomes with 2- [4- (2-hydroxyethyl) piperazin-1-yl]Ethanesulfonic acid (HEPES) to give liposomal compositions of irinotecan with a pH of 7.25-7.50 to give storage at 4 deg.CThe liposome composition of irinotecan stabilized to form less than 10 mol% of lysophosphatidylcholine (lyso-PC) over a period of 3 months (relative to the total amount of phosphatidylcholine in the liposome composition of irinotecan).

Liposomal irinotecan compositions are useful for treating patients diagnosed with various forms of cancer. For example, liposomal irinotecan can be administered to treat Small Cell Lung Cancer (SCLC) without the need for other antineoplastic agents. In some embodiments, the liposomal irinotecan composition and the other antineoplastic agent are administered in combination. For example, a liposomal irinotecan composition, 5-fluorouracil, and leucovorin (without other antineoplastic agents) can be administered to treat patients diagnosed with metastatic pancreatic cancer as the disease progresses following gemcitabine-based therapy. Liposomal irinotecan compositions, 5-fluorouracil, leucovorin and oxaliplatin (without other antineoplastic agents) can be administered to treat patients diagnosed with previously untreated pancreatic cancer. Liposomal irinotecan compositions, 5-fluorouracil, leucovorin, and an EGFR inhibitor (e.g., an oligoclonal antibody EGFR inhibitor, e.g., MM-151) can be administered to treat patients diagnosed with colorectal cancer.

Unless stated otherwise in the specification, the liposome composition contains irinotecan (free base or salt form) in grams relative to the moles of phospholipid at a ratio equivalent to that provided by 471g or 500g (± 10%) irinotecan free base per mole of phospholipid.

As used herein (and unless otherwise specified) "irinotecan moiety" refers only to irinotecan lactone; i.e., irinotecan lactone as the anhydrous free base.

As used herein (and unless otherwise indicated), the term "camptothecin" includes camptothecin and camptothecin derivatives, including irinotecan, topotecan, lurtotecan, silatecan, irinotecan peg, TAS 103, 9-aminocamptothecin, 7-ethylcamptothecin, 10-hydroxycamptothecin, 9-nitrocamptothecin, 10, 11-methylenedioxycamptothecin, 9-amino-10, 11-methylenedioxycamptothecin, 9-chloro-10, 11-methylenedioxycamptothecin, (7- (4-methylpiperazinylmethylene) -10, 11-ethylenedioxy-20 (S) -camptothecin, 7- (4-methylpiperazinylmethylene) -10, 11-methylenedioxy-20 (S) -camptothecin, and 7- (2-N-isopropylamino) ethyl) - (S) 20S) -camptothecin and stereoisomers, salts and esters thereof.

As used herein (and unless otherwise specified) "DLS" refers to dynamic light scattering and "BDP" refers to bulk drug product.

In some embodiments, the liposomes of the invention encapsulate one or more agents that entrap the drug in the liposome (hereinafter referred to as entrapping agents).

"sustained release composition" as used in this specification includes irinotecan compositions, which when administered once every two weeks corresponds to 70mg/m²The dose of irinotecan free base administered to humans provides 80 to 125% of the following pharmacokinetic parameters: cmax 37.2(8.8) μ g irinotecan (as anhydrous free base)/mL and AUC_0-∞1364(1048) h · μ g irinotecan/mL (for irinotecan); or (for SN-38), Cmax5.4(3.4) μ g SN-38 (as anhydrous free base)/mL; AUC_0-∞620(329)h·ng SN-38/mL。

Unless otherwise indicated, the liposome formulation may comprise (e.g., spherical or substantially spherical) vesicles having at least one lipid bilayer, and may optionally include multi-compartment and/or single compartment vesicles, and vesicles that encapsulate and/or do not encapsulate a pharmaceutically active compound (e.g., camptothecin) and/or a capture agent. For example, unless otherwise indicated, pharmaceutical liposomal formulations comprising camptothecin liposomes can optionally include liposomes that do not comprise a camptothecin compound, including mixtures of unilamellar and multilamellar liposomes with or without a camptothecin compound and/or a capture agent.

Drawings

Fig. 1A shows a schematic diagram of irinotecan liposomes encapsulating an aqueous space containing irinotecan in a gel state or a precipitated state as sucrose octasulfate salt.

Figure 1B shows the equatorial cross-section of irinotecan liposomes of figure 1A.

Figure 2A is a graph showing stability ratio versus relative amount of lyso-PC (mol%) after 9 months of storage at 4 ℃ for a liquid irinotecan liposome composition having a specified pH value after preparation but before storage.

Figure 2B is a graph showing stability ratio versus relative amount of lyso-PC (mol%) after 6 months of storage at 4 ℃ for a liquid irinotecan liposome composition having a specified pH value after preparation but before storage.

Figure 2C is a graph showing stability ratio versus relative amount of lyso-PC (mol%) after 6 months of storage at 4 ℃ for a liquid irinotecan liposome composition having a specified pH value after preparation but before storage.

Figure 3A is a graph showing the relative amount of lyso-PC (mol%) versus the number of months of storage at 4 ℃ for two liposomal irinotecan compositions having a stability ratio of 1047 and a pH of 6.5.

Figure 3B is a graph showing the relative amounts (mol%) of lyso-PC versus the number of months of storage at 4 ℃ for two liposomal compositions of irinotecan with stability ratios of 992 and 942, respectively, and a pH of 6.5 after preparation but before storage.

Figure 3C is a graph showing the stability ratio of 785 versus the relative amount of lyso-PC (mol%) of the irinotecan liposome composition having a pH of 6.5 after preparation but before storage versus the number of months of storage at 4 ℃.

FIG. 3D is a graph showing a stability ratio of about 724 using TEA having a sulfate group concentration of 0.65M₈SOS preparation and relative amount of lyso-PC (mol%) for the two liposomal irinotecan compositions at pH6.5 after preparation but before storage versus months of storage at 4 ℃.

Figure 4A is a graph showing the relationship of the relative amount of lyso-PC (mol%) versus the number of months of storage at 4 ℃ for three liposomal compositions of irinotecan having a stability ratio of about 1047 and a pH of 7.25 after preparation but prior to storage. Liposome sample 5 (open square) was prepared at a concentration of irinotecan hydrochloride fraction equivalent to that provided by 5mg/mL irinotecan hydrochloride trihydrate, while liposome sample 13 (filled triangle) was also prepared at 20mg/mL irinotecan hydrochloride trihydrate. The liposomes in sample 13 were prepared in the same manner as in sample 5, but the liposome components per ml (i.e., phospholipids, cholesterol, irinotecan, and sulfatide) in the final liposome composition increased four-fold compared to sample 5.

Figure 4B is a graph showing the stability ratio of about 1047 and the relative amount of lyso-PC (mol%) of the two irinotecan liposome compositions after preparation but before storage at pH values of 7.25 and 7.5 versus the number of months of storage at 4 ℃.

Figure 4℃ is a graph showing the stability ratio of about 785 and the relative amounts of lyso-PC (mol%) of two irinotecan liposome compositions after preparation but before storage at pH values of 7.25 and 7.5 versus the number of months of storage at 4 ℃.

Figure 5 is a graph showing the lyso-PC concentration (mg/mL) versus the number of months of storage at 4 ℃ for three liposomal irinotecan compositions having a stability ratio of 1046-1064 and a pH of 7.3 after preparation but prior to storage.

Figure 6 is a graph showing the lyso-PC concentration (mg/mL) versus the number of months of storage at 4 ℃ for three liposomal irinotecan compositions having a stability ratio of 1046-1064 and a pH of 7.3 after preparation but prior to storage.

Figure 7 is a graph showing the estimated rate of formation of lyso-PC (mg/mL/month) during storage at 4 ℃ for irinotecan liposome compositions having varying amounts of substituted ammonium (protonated TEA).

Figure 8 is a graph of the gram equivalents of irinotecan and the sulfatide in the precipitate formed by combining irinotecan hydrochloride trihydrate and triethylammonium sulfatide in different ratios in aqueous solution (i.e., a gram equivalent ratio of 1:9 to 9: 1). The x-axis shows the relative gram-equivalent total amount of triethylammonium thionate (SOS) in the sample compared to the gram-equivalent of irinotecan anhydrous free base.

Figure 9 shows a graph plotting the average particle size of 12 different irinotecan sucrose octasulfate liposome product lots during storage at 4 ℃ for 12-36 months, with linear regression of the data obtained for each sample.

Figure 10 is a graph of the particle size polydispersity index (PDI) for the irinotecan sucrose octasulfate product lot shown in figure 9, with linear regression performed on the data obtained for each sample.

Figure 11A is a graph of pH of 13 different irinotecan sucrose octasulfate product lots during storage at 4 ℃ for 12-36 months with linear regression of the data obtained for each sample.

Figure 11B is a graph of the pH of 16 different irinotecan sucrose octasulfate product lots during 12 months of storage at 4 ℃ with linear regression of the data obtained for each sample.

Figure 12 is a graph of the concentration of lyso-PC (mg/mL) in two irinotecan liposome compositions over a 36 month period, along with a best-fit linear regression of the corresponding data points obtained from each irinotecan liposome sample.

Figure 13A is a representative chromatogram of method a at full scale.

Fig. 13B is a representative chromatogram of method a at an enlarged scale.

Detailed Description

A stable camptothecin composition can include liposomes encapsulating one or more camptothecin compounds. Liposomes can be used to administer drugs, including chemotherapeutic drugs. The present invention provides stable phospholipids, such as liposomal irinotecan, comprising camptothecin compound compositions that yield lesser amounts of lysophospholipids, such as lyso-PC.

The camptothecin liposomes can encapsulate the camptothecin and the capture agent within a lipid composition (e.g., a vesicle comprising a phospholipid). For example, figure 1A shows a schematic depicting irinotecan liposomes about 110nm in diameter and having a lipid membrane encapsulating irinotecan. The lipid film in this schematic contains the phospholipid ester MPEG-2000-DSPE. The MPEG-2000-DSPE lipids are located in the inner and outer lipid layers of the bilayer membrane, so that the PEG portion of the MPEG-2000-DSPE lipids is located in or on the outer surface of the liposomes, respectively. Figure 1B shows a cross-section of a particular embodiment of the liposomes generally described in figure 1A, wherein the monolayer lipid bilayer membrane comprises DSPC, cholesterol and MPEG-2000-DSPE and encapsulates irinotecan sucrose octasulfate.

It has now been found that novel stable liposomal compositions comprising irinotecan containing esterphospholipids with low levels of lyso-PC after prolonged storage at 2-8 ℃, e.g. at 4 ℃, comprising liposomes encapsulating irinotecan Sucrose Octasulfate (SOS) (irinotecan-SOS liposomes) and significantly reducing the formation of lyso-PC during refrigerated storage can be prepared. The present invention is based, in part, on a number of unexpected observations. First, the irinotecan-SOS liposome compositions surprisingly have significantly less lyso-PC during refrigerated storage when the amount of encapsulated irinotecan is increased relative to the amount of co-encapsulated SOS capture agent. Second, when the pH of the aqueous medium comprising irinotecan-SOS liposomes is greater than 6.5 after preparation but prior to storage, the irinotecan-SOS liposome composition surprisingly has less lyso-PC during refrigerated storage. Third, the irinotecan-SOS liposome composition surprisingly has less lyso-PC when the amount of residual liposome trapping agent ammonium/substituted ammonium cation measured in the composition is below 100 ppm.

Liposomal camptothecin composition component lipids

It is known in the art that various lipids, particularly phospholipids, may be components of liposomes, such as phosphatidylethanolamine and phosphatidylserine, and that liposomes are prepared with other such phospholipids within the skill of the art. In some embodiments, the liposomes of the invention consist of 1, 2-distearoyl-sn-glycerol-3-phosphocholine (DSPC), cholesterol, and methoxy-capped polyethylene glycol (MW2000) -distearoyl phosphatidylethanolamine (MPEG-2000-DSPE). Preferred embodiments regarding the lipids present in the liposomal formulations disclosed herein are described below.

The liposome components may be selected to produce liposome bilayer membranes that form unilamellar and/or multilamellar vesicles to encapsulate and retain the active substance until the active substance is delivered to the tumor site. Preferably, the liposome vesicles are unilamellar. The liposome components, when combined, are selected for their properties so as to produce liposomes that are capable of active loading and retention of active substances while maintaining low protein binding in vivo and thus extending the circulating life of the liposomes.

DSPC is preferably the major lipid component (e.g., 74.4% of the total weight of all lipid components) in the bilayer encapsulating the irinotecan liposomes. The phase transition temperature (Tm) of DSPC was 55 ℃.

Cholesterol may preferably comprise about 24.3% of the total weight of all lipid components. Cholesterol may be incorporated in an amount effective to stabilize the liposome phospholipid membrane so that the liposome phospholipid membrane is not disrupted by plasma proteins, thereby reducing the degree of binding of plasma opsonins responsible for rapid clearance of the liposomes from circulation, and reducing the permeability of the solute/drug in combination with the bilayer-forming phospholipids.

MPEG-2000-DSPE may preferably comprise about 1.3% of the total weight of all lipid bilayer components. The amount of MPEG-2000-DSPE present on the surface of the irinotecan liposomes can be selected to provide a minimal steric barrier to prevent liposome aggregation. The MPEG-2000-DSPE coated liposomes of the present invention were shown to be stable in size and drug encapsulation.

In some embodiments, the lipid membrane of the liposomal formulation preferably consists of: 1, 2-distearoyl-sn-glycerol-3-phosphocholine (DSPC), cholesterol, and methoxy-terminated polyethylene glycol (MW2000) -distearoyl phosphatidylethanolamine (MPEG-2000-DSPE) in a ratio of about one polyethylene glycol (PEG) -modified phospholipid molecule per 200 non-PEG-phospholipid molecules.

In a preferred embodiment, the liposomes of the invention are made from a mixture of DSPC, cholesterol and MPEG-2000-DSPE combined in a 3:2:0.015 molar ratio. In a preferred embodiment, the liposomal formulation of the present invention comprises 1, 2-distearoyl-sn-glycerol-3-phosphocholine (DSPC) at a concentration of about 6.81mg/mL, cholesterol at a concentration of about 2.22mg/mL, and methoxy-terminated polyethylene glycol (MW2000) -distearoyl phosphatidylethanolamine (MPEG-2000-DSPE) at a concentration of about 0.12 mg/mL.

In a more preferred embodiment, the liposomal formulation of the present invention comprises 1, 2-distearoyl-sn-glycerol-3-phosphocholine (DSPC) at a concentration of 6.81mg/mL, cholesterol at a concentration of 2.22mg/mL, and methoxy-capped polyethylene glycol (MW2000) -distearoyl phosphatidylethanolamine (MPEG-2000-DSPE) at a concentration of 0.12 mg/mL.

Capture agent for camptothecin composition

In some embodiments, the liposomes of the invention encapsulate one or more agents that entrap the drug in the liposome (hereinafter referred to as entrapping agents). The capture agent preferably comprises a polyanion compound having a plurality of negatively charged groups, or a combination of two or more different such compounds. In non-limiting examples, the polyanionic capture agent is a divalent anion, a trivalent anion, a multivalent anion, a polymeric multivalent anion, a polyanionized polyol, or a polyanionized sugar. In the context of the present invention, the polyanionic capture agent may be a polyanionic polyol or saccharide, for example a polyol or saccharide in which the hydroxyl groups have been modified in whole or in part or have been substituted with anionic groups (anionized). In one non-limiting example, a polyanionized polyol or polyanionized saccharide can include a polyol portion or a saccharide portion to which an anionic group is attached. Preferably, at least one anionic group in the polyanionized saccharide or polyanionized polyol scavenger is more than 50% ionized in a pH range of pH 3-12, preferably pH 6.5-8, when in an aqueous medium, or alternatively, the anionic group has a pKa of 3 or less, preferably 2 or less. In a preferred embodiment, the capture agent contains a sulfate moiety with a pKa of 1.0 or less. In one non-limiting example, the polyanionic capture agent may have a charge density of at least two, three, or four negatively charged groups per unit, such as each ring or carbon atom in a carbon chain or each monosaccharide unit in a sugar.

In some embodiments of the invention, the release rate of the liposome composition can be increased by using a polyanionized sugar or polyanionized polyol in admixture with one or more other monovalent or multivalent anions (e.g., chloride, sulfate, phosphate, etc.) as a capture agent. In another non-limiting example of increasing the release rate of a sustained release composition, a mixture of different polyanionic sugars and/or polyanionic polyols of different degrees of polyanionization (degree of polyanionization) are used as capture agents.

In some embodiments, the degree of polyanionization inside a liposome of the invention is greater than 90%, or greater than 99%, or 0.1% to 99%, 10% to 90%, or 20% to 80% of the total anions in the liposome, e.g., in the liposome that entraps the camptothecin or camptothecin derivative compound.

In some embodiments, the capture agent is a sulfated sugar and/or a polyol. Exemplary sulfated sugars of the invention are sulfated sucrose, which include, but are not limited to, sucrose hexasulfate, sucrose heptasulfate, and sucrose octasulfate (see ochi.k., et al.,1980, chem.pharm.bull., v.28, p.638-641). Similarly, reaction with phosphorus oxychloride or diethylphosphoryl chloride in the presence of a base catalyst produces a polyphosphorylated polyol or sugar. Polyphosphorylated polyols are also isolated from natural sources. For example, inositol polyphosphates, such as inositol hexaphosphate (phytic acid), can be isolated from corn. Various sulfated, sulfonated, and phosphated sugars and polyols suitable for practicing the present invention are disclosed, for example, in U.S. patent No. 5,783,568, the entire contents of which are incorporated herein by reference. Complexation of the polyol and/or saccharide with more than one boronic acid molecule also results in a polyanionic (polyborated) product. The reaction of polyols and/or sugars with carbon disulphide in the presence of a base produces polyanionic (polydithiocarbonated, polyorthoxanthated) derivatives. The polyanionized polyol or sugar derivative can be isolated in the form of the free acid and neutralized with a suitable base, for example with an alkali metal hydroxide, ammonium hydroxide, or preferably with a substituted amine, for example an amine corresponding to the substituted ammonium of the present invention, in pure form or in the form of a substituted ammonium hydroxide to provide the polyanionic salts of the substituted ammonium of the present invention. Alternatively, the sodium, potassium, calcium, barium or magnesium salt of the polyanionized polyol/sugar may be isolated by any known method (e.g., by ion exchange) and converted to a suitable form, such as a substituted ammonium salt form. Non-limiting examples of sulfated sugar scavengers are sulfated sucrose compounds, including but not limited to sucrose hexasulfate, sucrose heptasulfate, and Sucrose Octasulfate (SOS). Exemplary polyol scavengers include inositol polyphosphates, such as inositol hexaphosphate (also known as phytic acid IHP) or the sulfated form of other disaccharides.

In a preferred embodiment of the invention, the capture agent is a sulfated polyanion, a non-limiting example of which is Sucrose Octasulfate (SOS). The sulfatide is also known as sucrose octasulfate or sodium sucrose sulfate (SOS). Methods for preparing the sulfatase in the form of various salts (e.g., ammonium, sodium or potassium salts) are well known in the art (e.g., U.S. patent 4,990,610, the entire contents of which are incorporated herein by reference). Sucrose octasulfate (also known as thionate) is a fully substituted sucrose sulfate, in its fully protonated form, having the structure of formula (II):

methods for preparing the sulfatase in the form of various salts (e.g., ammonium, sodium or potassium salts) are well known in the art (see, e.g., U.S. patent No. 4,990,610, which is incorporated herein by reference in its entirety). Likewise, sulfated forms of other disaccharides such as lactose and maltose are envisioned to produce lactose octasulfate and maltose octasulfate.

In some embodiments, the liposomal formulation of the present invention comprises a camptothecin compound, such as irinotecan or topotecan, and an anion scavenger, such as SOS. The liposomes of the invention preferably comprise a camptothecin compound in stoichiometric ratio with an anion scavenger. For example, a liposomal formulation of irinotecan may encapsulate irinotecan and sucrose octasulfate at a molar ratio of about 8: 1. Stable compositions of liposomes can encapsulate an irinotecan composition of formula (I) wherein x is about 8:

liposomal irinotecan can comprise a composition of formula (I) encapsulated in liposomes. Preferably, the composition of formula (I) is formed (e.g. precipitated) in liposomes comprising cholesterol and one or more phospholipids, including for example PEG-containing phospholipids. For example, a composition of formula (I) may be formed in liposomes as follows: (1) camptothecin compounds (e.g., irinotecan, topotecan, etc.) are reacted with (2) liposomes encapsulating a polysulfated anion scavenger (e.g., sucrose octasulfate) in forming stable liposomal irinotecan compositions. Preferably, the liposomal irinotecan composition has a pH greater than 6.5 (e.g., 7.0-7.5, including 7.25, 7.3, and 7.5).

A preferred stable camptothecin composition comprises liposomal irinotecan.

The stable camptothecin compositions include high density camptothecin compound liposomal formulations comprising irinotecan, or a salt thereof, at a concentration of irinotecan moiety equivalent to that provided by 4.5 to 5.5mg/mL irinotecan hydrochloride trihydrate (i.e., 3.9-4.8mg/mL irinotecan anhydrous free base), and comprises DSPC at a concentration of 6.13 to 7.49mg/mL (preferably about 6.81mg/mL), cholesterol at a concentration of 2-2.4mg/mL (preferably about 2.22mg/mL) and MPEG-2000-DSPE at a concentration of 0.11-0.13mg/mL (preferably about 0.12mg/mL), characterized in that a small amount of lyso-PC (if any) is present during refrigerated storage (2-8 ℃), also provided are suitable amounts of camptothecin compounds, preferably in a more potent lactone form. The present invention encompasses pharmaceutical liposomal compositions of camptothecin compounds that can be stored under refrigeration (i.e., at 2-8 ℃) for at least the first 6 months, preferably at least the first 9 months, after preparation without formation of lyso-PC at levels greater than 20 mol%. More preferably, the present invention provides compositions comprising an amount of irinotecan moiety equivalent to that provided by 4.7-5.3mg/mL irinotecan hydrochloride trihydrate (i.e., 4.1-4.6mg of the anhydrous free base of irinotecan moiety), which can be present as sucrose octasulfate encapsulated in liposomes, and comprising 6.4-7.2mg/mL of (DSPC), 2.09-2.35mg/mL of cholesterol, and about 0.113-0.127mg/mL of MPEG-2000-DSPE containing no more than 20 mol% lyso-PC when stored at 2-8 ℃ for 6 or 9 months, or no more than 2mg/mL of lyso-PC when stored at 2-8 ℃ for 21 months.

Calculate the gram Equivalent Ratio (ER) of irinotecan/sulfate compound

The irinotecan/sulfate compound gram-Equivalent Ratio (ER) for each irinotecan liposome formulation can be calculated as follows: the formula is used: ER ═ I/(SN), the liposome composition was determined for the molar amount of liposomal co-encapsulated irinotecan (I) and sulfate compound (S) per unit (e.g., 1mL), where N is the valence of the sulfate compound anion (e.g., N is 8 for sulfatide, and SO4 for free sulfate^2-And N is 2). For example, a liposomal irinotecan sulfate composition comprising 7.38mM irinotecan and 1.01mM sulfate (N ═ 8) would have an ER of 7.38/(1.01 × 8) ═ 0.913. Preferably, the sulfate compound (S) is sucrose octasulfate, which contains 8 sulfate moieties per mole of SOS. The liposome composition will have a pH of 7.1 to 7.5 and have one of the following ER ranges: preferably 0.85 to 1.2, 0.85-1.1, or most preferably 0.9 to 1.05, for example about 1.02. Alternatively, the liposome composition will have an amount of irinotecan moiety equivalent to the amount provided by 500g (± 10%) irinotecan anhydrous free base per mole phospholipid and having one of the following ER ranges: preferably from 0.85 to 1.1, most preferably from 0.9 to 1.05, for example about 1.02.

Stable pH of camptothecin compositions

The pH of the liposome composition can be adjusted or otherwise selected to provide the desired storage stability properties (e.g., by reducing lyso-PC formation in the liposomes during storage at 4 ℃ for 180 days), for example, by preparing the composition at about pH 6.5-8.0 or any suitable pH therebetween, including, for example, 7.0-8.0 and 7.25. In some embodiments, the pH is about 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. Liposomes of irinotecan were prepared having a specific pH value, with the irinotecan moiety being equivalent to that provided by irinotecan anhydrous free base concentration (mg/mL) and various concentrations of sucrose octasulfate, as provided in more detail herein described. More preferably, the pH after preparation and before storage is between 7.1 and 7.5, even more preferably between about 7.2 and 7.3, most preferably about 7.25. The pH can be adjusted by standard methods, for example using 1N HCl or 1N NaOH as appropriate.

In some embodiments of the invention, the pH of the liposomal irinotecan formulation after preparation but prior to storage is greater than 6.5, preferably 7.2 to 7.3. In some embodiments of the invention, the pH is from 7.2 to 7.5.

Stable camptothecin compositions compound gram equivalent ratio ('ER')

The stable liposomal camptothecin composition can have a pH of greater than 6.5 and comprise liposomes encapsulating irinotecan and a sulfate polyanionic capture agent having an irinotecan/sulfate compound gram equivalent ratio ("ER") of greater than 0.9 (e.g., 0.9-1.1). The ER of irinotecan SOS liposomal formulation can be calculated as follows: the formula is used: ER ═ I/(SN), the liposome composition was determined for the molar amount of liposomal co-encapsulated irinotecan (I) and sulfate compound (S) per unit (e.g., 1mL), where N is the valence of the sulfate compound anion (e.g., N is 8 for sulfatide, and SO for free sulfate)₄ ^2-N is 2), I is the concentration of encapsulated irinotecan in the liposomal irinotecan composition, and S is the sulfate group concentration of encapsulated sucrose octasulfate in the liposomal irinotecan composition. Preferably, the sulfate compound (S) is sucrose octasulfate, which contains 8 sulfate moieties per mole of SOS.

While it is preferred to directly determine the concentration of sulfate groups encapsulating sucrose octasulfate (S · N) in liposomal irinotecan compositions, S · N can be determined by: liposome phospholipid concentration (P, mol/L), SOS sulfate group concentration in the inner space of the liposome (SOS sulfate group concentration in the solution used to prepare the capture agent liposome; parameter B, see stability ratio definition in this application), and liposome internal (entrapped) volume, i.e. the volume sequestered within the inner space of the liposome vesicle, per unit liposome phospholipid (Ve, L/mol phospholipid):

S·N＝P·Ve·B

for example, for phosphatidylcholine-cholesterol liposomes extruded through 100-nm polycarbonate filters, the hold-up volume can be close to 1.7L/mol phospholipid (Mui, et al 1993, biophysis.j., vol 65, p.443-453). In this case, irinotecan (molecular weight 586.7) was quantitatively loaded into SOS-encapsulated liposomes with 471g/mol phospholipids and a SOS sulfate group concentration of 0.45M, which would give ER as:

(471/586.7)/(1.7·0.45)＝1.049

at a concentration of SOS of 0.65M sulfate groups, ER will be:

(471/586.7)/(1.7·0.65)＝0.726

similarly, irinotecan (molecular weight 586.7) dosed into SOS-encapsulated liposomes of 500g (. + -. 10%)/mol phospholipid and SOS sulfate group concentration 0.45M will give an ER of about 1.11, and at SOS concentrations of 0.65M sulfate group, an ER of about 0.77.

Preparation of stable camptothecin compositions

The stable camptothecin compositions can comprise liposomes of camptothecin. Liposomes have been used to administer drugs, including chemotherapeutic drugs. Various techniques involving liposomes encapsulating drugs and methods for their preparation are generally known in the art and are therefore not described in any further detail herein. See, for example, U.S. patent No. 8,147,867, which is incorporated by reference herein in its entirety.

In some embodiments, the liposome encapsulating one or more camptothecin compounds within a vesicle comprises at least one phospholipid. The camptothecin compound can be loaded or otherwise entrapped in the liposome in a multi-step process comprising (a) forming a capture agent liposome encapsulating an anionic capture agent and a cation within a liposome vesicle comprising a phospholipid, and (b) subsequently contacting the capture agent liposome with the camptothecin compound under conditions effective to load the camptothecin compound into the capture agent liposome and retain the camptothecin compound in the liposome with the capture agent to form the camptothecin liposome.

The camptothecin compound can be loaded into the capture agent liposome using a gradient on the liposome membrane, allowing the camptothecin compound to enter the capture agent liposome to form the camptothecin liposome. Preferably, the capture agent liposomes have a transmembrane concentration gradient of transmembrane cations such as ammonium or substituted ammonium, and the camptothecin compound is effective to cause an exchange of the ammonium/substituted ammonium salt in the capture agent liposomes when heated above the phase transition temperature of the liposome lipid components. Preferably, the concentration of the capture agent in the capture agent liposome is higher than its concentration in the medium surrounding the capture agent liposome. Furthermore, in addition to the gradient created by the ammonium/substituted ammonium cation, the capture agent liposome may also comprise one or more transmembrane gradients. For example, the liposomes comprised in the capture agent liposome composition may additionally or alternatively comprise a transmembrane pH gradient, an ionic gradient, an electrochemical potential gradient and/or a solubility gradient.

In some embodiments, the capture agent (e.g., an SOS and/or another sulfated polyol capture agent, including acceptable salts thereof) used to prepare the liposomes has a sulfate group concentration of 0.3-08, 0.4-.05, 0.45-0.5, 0.45-.0475, 0.45-0.5, 0.3, 0.4, 0.45, 0.475, 0.5, 0.6, 0.7, or 0.8M, e.g., ± 10% of these particular values. In a preferred embodiment, the capture agent used to prepare the liposomes is a concentration of SOS and sulfate groups of about 0.45M or about 0.475M. In a more preferred embodiment, the capture agent used to prepare the liposomes is SOS and sulfate groups at a concentration of 0.45M or 0.475M.

Preferably, the camptothecin compound is loaded into the capture agent liposome as follows: the camptothecin compound is incubated with the capture agent liposomes in an aqueous medium at a suitable temperature, for example a temperature above the initial phase transition temperature of the component phospholipid during loading, while decreasing to below the initial phase transition temperature of the component phospholipid after loading of the camptothecin compound, preferably at about room temperature. The incubation time is generally based on the nature of the component lipids, the camptothecin compound to be loaded into the liposomes, and the incubation temperature. Typically, incubation times of a few minutes (e.g., 30-60 minutes) to a few hours are sufficient.

Since high rejection efficiencies of greater than 85%, typically greater than 90%, are achieved, it is generally not necessary to remove non-rejected entities. However, if so desired, non-entrapped camptothecin compounds can be removed from the composition by various methods, such as size exclusion chromatography, dialysis, ultrafiltration, adsorption, and precipitation.

In some embodiments, the camptothecin liposome is an irinotecan liposome. Irinotecan liposomes can be prepared by a process comprising the steps of: (a) preparing liposomes comprising Triethylamine (TEA) as the triethylammonium salt of the thiosugar ester (TEA-SOS), and (b) subsequently contacting the TEA-SOS liposomes with irinotecan under conditions effective to enter irinotecan into the liposomes and allow a corresponding amount of TEA to leave the liposomes (thereby depleting or reducing the TEA concentration gradient on the resulting liposomes).

Ionic strength outside of liposomes during loading of camptothecin liposomes

In some embodiments of the invention, the loading of the camptothecin of the liposome is carried out in an aqueous solution having an ionic strength of less than equivalent to 50mM NaCl, or more preferably less than equivalent to 30mM NaCl. After loading, a more concentrated salt solution, such as NaCl solution, may be added to increase the ionic strength above that equivalent to 50mM NaCl, or more preferably, above that equivalent to 100mM NaCl, preferably equivalent to about 140-.

Trapping agent cation

When heated above the phase transition temperature of the lipidic component as described above, the cations of the invention may be encapsulated in the capture agent liposomes in an amount effective to provide loading of the camptothecin compound into the capture agent liposomes. The cation is selected such that it can leave the trapping agent liposome during loading of the camptothecin compound into the liposome. After the liposome loaded with the camptothecin compound is prepared, the cations outside the liposome can be removed.

In some embodiments of the invention, the cation in the liposome, together with the capture agent, is a substituted ammonium compound. In some embodiments of the invention, the substituted ammonium compound has a pKa of at least about 8.0. In some embodiments of the invention, the substituted ammonium compound has a pKa of at least about 8.0, at least about 8.5, at least about 9.0, at least 9.5, at least 10.0, at least 10.5, or at least 11.0, as determined in aqueous solution at ambient temperature. In some embodiments of the invention, the substituted ammonium compound has a pKa of about 8.0 to 12.0, about 8.5 to 11.5, or about 9.0 to 11. In a preferred embodiment, the pKa is about that of TEA, or about that of DEA.

Non-limiting examples of such substituted ammonium compounds are compounds of the formula: n (R)₁)(R₂)(R₃)(R₄)⁺Wherein R is₁、R₂、R₃And R₄Each independently hydrogen or an organic group having up to 18 total carbon atoms, wherein R₁、R₂、R₃And R₄At least one of which is an organic group which is a hydrocarbyl group having up to 8 carbon atoms which may be an alkyl, alkylene, heterocycloalkyl, cycloalkyl, aryl, alkenyl or cycloalkenyl group or hydroxy-substituted derivatives thereof, the hydrocarbon portion of which optionally includes one or more S, O or N atoms to form an ether, ester, thioether, amine or amide bond. The substituted ammonium may be a sterically hindered ammonium compound (e.g., having at least one organic group having a secondary or tertiary carbon atom directly attached to the ammonium nitrogen atom). Furthermore, R₁、R₂、R₃And R₄At least one of which must be hydrogen. Preferably, the substituted ammonium cation is triethylammonium (protonated TEA) or diethylammonium (protonated DEA).

When the camptothecin compound is loaded into the liposome encapsulating the anion trapping agent, the concentration of the substituted ammonium cation in the trapping agent liposome can be reduced under conditions effective to form a liposome of the camptothecin compound. The liposomes of the invention may comprise an anion scavenger and an ammonium or substituted ammonium cation which is subsequently removed and/or replaced by the camptothecin compound loaded into the liposome in a subsequent drug loading step.

In a preferred embodiment, the concentration of ammonium or substituted ammonium cations in the camptothecin compound liposomes is sufficiently low to provide a low amount of lyso-PC after prolonged refrigerated storage of the camptothecin liposome formulation comprising the phospholipid. For example, as described in example 3, including the data in fig. 7, a reduction in the amount of lyso-PC formation is observed in irinotecan SOS liposome formulations having less than about 100ppm of substituted ammonium cations, preferably 20 to 80ppm, preferably less than about 50ppm, even more preferably less than about 40ppm, and still more preferably less than 30 ppm.

In some embodiments, irinotecan SOS liposomes (e.g., samples 24-29; Table 10 of the examples) contain less than 100ppm, or about 15-100ppm, of substituted ammonium SOS trap counterions. In some embodiments, irinotecan SOS liposomes (e.g., samples 24-29; Table 10 of the examples) contain about 15-80ppm of substituted ammonium. In some embodiments, the irinotecan SOS liposome comprises about 40-80ppm of substituted ammonium. In some embodiments, irinotecan SOS liposomes (e.g., samples 24-29; Table 10 of the examples) contain about 80-100ppm of substituted ammonium. In a preferred embodiment, the substituted ammonium present at any of the above ppm concentrations is derived from TEA or DEA.

Stable camptothecin compositions stability ratio

When phospholipid-based liposomes comprising camptothecin are prepared by reacting (1) a camptothecin drug with (2) liposomes encapsulating a polysulfated anion trap, the stability of the resulting drug-loaded liposomes depends on the ratio of camptothecin, anion trap and liposome-forming phospholipid, as defined below with a stability ratio of at least about 950. The stability ratio depends on the initial concentration of sulfate groups in the capture agent-liposomes and the ratio of encapsulated camptothecin to phospholipid in the liposomes. The stability ratio ("SR") used in this application is defined as follows:

SR＝A/B，

wherein:

a.A is the amount of entrapped irinotecan moiety in the trapping agent liposome during loading, in grams equivalent to irinotecan anhydrous free base per mole of phospholipid in the composition; and

b.B is the concentration of sulfate groups in the solution of the thiosugar ester (or other capture agent) used to prepare the capture agent liposomes, expressed in mol/L (based on the concentration of sulfate groups).

For the determination of the stability ratio, the number of moles of phospholipids in the liposome formulation was determined by the test described in the examples. The amount of irinotecan moiety for liposome loading was calculated accordingly (a above).

With respect to the determination of the stability ratio, the concentration B (in mol/L) of sulfate groups in the solution of the sulfatase (or other capture agent) is calculated as the concentration (in mol/L) of the sulfatase (or other capture agent disclosed herein) in a solution added to the lipid (which is typically dissolved in ethanol, typically in a volume of 10% or less of the volume of the capture agent solution added to the lipid). Thus, for the thionate, the concentration B of sulfate groups is the concentration of the thionate multiplied by 8 (i.e., the number of sulfate groups in one thionate molecule), or multiplied by the number of sulfate groups of the particular capture agent used (see example 1).

In some embodiments of the invention, both the stability ratio and the pH are increased to greater than 6.5. Thus, in certain preferred embodiments of the invention, the stability ratio is 942-1130, the pH is 7.2-7.5, and irinotecan and the SOS trapping agent are present in the liposome composition at a molar ratio of about 8: 1. Preferably, the stability ratio is 942-1130, the pH is about 7.25, and the irinotecan composition and SOS trapping agent are present in the liposomes at a molar ratio of 8: 1. The amount of lyso-PL, in particular lyso-PC, in the liposome formulations encapsulating the other camptothecin compounds can be controlled in a similar manner.

For example, the novel stable liposomal formulation of irinotecan may have 80% or less lyso-PC compared to irinotecan SOS liposomes prepared according to other methods (e.g., 80% or less lyso-PC after 9 months of refrigerated storage compared to that observed in comparative sample 12). The (comparative) liposomal irinotecan of sample 12 with a stability ratio of about 724 was prepared as follows: at a molar ratio of 8:1 [ (TEA)₈SOS]And Triethylamine (TEA) and sucrose octasulfate ("SOS" or "thionate") at a sulfate group concentration of 0.65M, a lipid mixture of 1, 2-distearoyl-sn-glycerol-3-phosphocholine (DSPC), cholesterol, and methoxy-capped polyethylene glycol (MW2000) -distearoyl phosphatidylethanolamine (MPEG-2000-DSPE) was heated at a molar ratio of 3:2:0.015 to produce TEA-SOS trap liposomes. Removing (TEA) not entrapped in the TEA-SOS trap liposomes₈After SOS, irinotecan was loaded into the resulting formulation containing TEA-SOS capture liposomes using an irinotecan solution under the following conditions: resulting in the removal of TEA and loading into liposomes a total amount of irinotecan was provided by 500g (± 10%) irinotecan anhydrous free base per mole of phospholipid in the TEA-SOS trap liposome formulation. The pH of the irinotecan liposome composition was 6.5 (measured according to the "pH measurement" section of the examples section of this application), with a 4.3mg portion of irinotecan per mL of irinotecan liposome composition in the irinotecan liposome. These liposomal irinotecan compositions comprising phosphatidylcholine produced levels of lyso-PC in excess of 30 mol% (relative to the total amount of phosphatidylcholine in the liposomal irinotecan composition) during 3 months of refrigerated storage (2-8 ℃) (and produced greater than 35 mol% lyso-PC during 9 months).

Calculating the stability ratio and the amount of lyso-PC in the exemplary embodiment

A series of different irinotecan liposomal formulations were prepared according to the methods described in this application (additional experimental details of the preparation and characterization of each sample are included in the examples below). The amounts of lyso-PC determined for each of the irinotecan liposome formulations are summarized in table 1A (the lyso-PC assay performed after 9 months of refrigerated storage) and table 1B (the lyso-PC assay performed after 6 months of refrigerated storage for a subset of the samples listed in table 1A). Each liposomal formulation of irinotecan comprises unilamellar liposomes having a diameter of about 110 ± 20nm, preferably 110 ± 10nm, encapsulating irinotecan with a sucrose octasulfate trapping agent. Liposomes were formed from a mixture of DSPC, cholesterol and MPEG-2000-DSPE with a 3:2:0.015 molar ratio and then loaded with irinotecan at a concentration of about 471g of irinotecan moiety (irinotecan or its salt provided an amount of irinotecan moiety equivalent to 500g (± 10%) of anhydrous irinotecan HCl) per mole of phospholipid. Each irinotecan liposome formulation contained different amounts of SOS trapping agent and was formulated at different pH values. The amount of lyso-PC in each irinotecan liposome formulation was measured at different times, including all samples after 9 months of continuous refrigerated storage (at 4 ℃). All samples in Table 1A used protonated TEA counter for SOSIon loading (i.e., loading irinotecan to encapsulate various concentrations of TEA)₈Liposomes of SOS, as shown in table 1A).

Table 1A: irinotecan liposome stability ratio and lyso-PC (after 9 months at 4 ℃ C.)^a

^aMeasured according to method B, as described herein.

FIG. 2A shows a graph depicting the amount of lyso-PC measured in each of the samples of Table 1A after 9 months of storage at 4 ℃. Sample 12 is labeled as reference in table 1A and fig. 2A. Both samples with stability ratios greater than about 900 and pH greater than 6.5 (e.g., 7.25 and 7.5) contained less than 20 mol% lyso-PC after 9 months of refrigerated storage at 4 ℃. Figure 2C is a graph of stability ratio as a function of relative amount of lyso-PC (mol%) after 6 months of storage of the liquid irinotecan liposome composition at 4 ℃ (data in table 6). The data points represented by open circles correspond to irinotecan samples having a pH greater than 6.5(7.25 or 7.5) measured after preparation but before storage. The data points represented by the diamonds correspond to irinotecan samples at pH6.5 measured after preparation but before storage. During the preparation of each sample, the stability ratio was calculated as defined herein. The mol% lyso-PC was measured after the first 6 months of storage after preparation of each sample.

Table 1B: irinotecan liposome stability ratio and lyso-PC (after 6 months at 4 ℃ C.)^b

^bMeasured according to method B, as described herein.

Figure 2B shows a graph depicting the amount of lyso-PCC measured in each sample of table 1B after 6 months of storage at 4 ℃. Both samples with stability ratios greater than about 989 and pH greater than 6.5 (e.g., 7.25 and 7.5) contained less than 20 mol% lyso-PC after 6 months of refrigerated storage at 4 ℃.

Figures 3A-3D are graphs showing mol% lyso-PC in irinotecan liposomal formulations selected from tables 1A and 1B, pH to 6.5. Each sample was assayed for lyso-PC after 0,1, 3, 6, 9 and/or 12 months of storage at 4 ℃. These figures include a linear regression line for the data as an estimate of the rate of increase of lyso-PC (mol%) in each sample over time. Slope, y-intercept and R of each plot²A summary of the values is shown in table 1C below.

TABLE 1C Mol% lyso-PC as a function of the refrigerated storage time (months) at pH6.5

In some embodiments, the stability of an irinotecan liposome formulation comprising irinotecan SOS encapsulated in liposomes having a diameter of about 100nm (e.g., 100 ± 20nm) is significantly increased in irinotecan liposomes having a stability ratio greater than 942. The effect of the stability ratio on the formation of lyso-PC in liposomal formulations was evaluated by maintaining the drug loading ratio of a 500g (± 10%) irinotecan fraction (based on anhydrous free base, as explained above) relative to total phospholipid unchanged, but varying the concentration of SOS trapping agent. Table 2 provides a summary of the amount of mol% lyso-PC detected in the irinotecan liposome formulation of table 1 formulated at the same pH as (comparative) sample 12(6.5) but at different concentrations of SOS trapping agent (i.e., at different stability ratios). Table 2 illustrates that for irinotecan liposomes comprising an SOS trapping agent and irinotecan having a stability ratio of greater than 942, the formation of lyso-PC during refrigerated storage is reduced. Reducing the amount of SOS trapping agent by up to 30% (i.e., increasing the stability ratio) relative to a reference irinotecan liposomal formulation resulted in a slight increase in the amount of lyso-PC by about 1% after 9 months of refrigerated storage. However, increasing the amount of SOS trapping agent in irinotecan liposomal formulations such that the stability ratio is greater than 942 resulted in a significant and unexpected decrease in the amount (mol%) of lyso-PC present after 9 months of refrigerated storage at 4 ℃. For example, a subsequent increase in the stability ratio by 5% compared to sample 3, such that the stability ratio is greater than 942 (i.e. a stability ratio of 992 for sample 2) resulted in a surprising decrease in the amount (mol%) of lyso-PC by 34%, equivalent to a decrease in the amount (mol%) of lyso-PC by 33% compared to sample 12 (as measured by 9 months of refrigerated storage at 4 ℃). Overall, a reduction of hemolysis-PC (mol%) of about 28-51% was achieved by increasing the stability ratio of irinotecan liposomes such that the stability ratio is greater than 942 compared to reference sample 12 after 9 months of refrigerated storage at 4 ℃. In some embodiments, the stability ratio of the irinotecan SOS liposome composition is greater than 942. In a preferred embodiment, the stability ratio of the irinotecan SOS liposome formulation is 942-1130 or greater (e.g., a stability ratio of 992-1047).

Table 2: stability ratio of irinotecan liposomes and haemolytic-PC (after 9 months at pH6.5, 4 ℃ C.)

Table 2 illustrates the criticality of irinotecan liposomes comprising an SOS trapping agent and irinotecan at a stabilizing pH of 6.5 at a stability ratio of greater than 942 (preferably greater than 950, most preferably greater than 992) to reduce lyso-PC formation in the liposomes during refrigerated storage. In general, by preparing the irinotecan liposome composition such that the pH is 6 and the stability ratio is greater than 950 (e.g., 950-. The reduction (i.e. increase in the stability ratio) of the concentration of SOS trapping agent used to prepare the trapping agent liposomes was up to 30% relative to the corresponding concentration of SOS trapping agent used to prepare the reference irinotecan liposome formulation (comparative samples 3 and 12), resulting in a slight increase of the amount of lyso-PC by about 1% after 9 months of refrigerated storage. However, increasing the amount of SOS trapping agent used to form the trapping agent liposomes prior to loading irinotecan to form an irinotecan liposome formulation with a stability ratio of 992 or greater resulted in a significant and unexpected decrease in lyso-PC formation after the first 9 months of refrigerated storage of the resulting irinotecan liposomes after preparation. For example, the data in table 2 shows that an increase in the stability ratio of 5% such that the stability ratio is greater than 942 results in a 34% decrease in lysopc after 9 months of storage at 4 ℃ (sample 2 compared to sample 3). An increase in the stability ratio from 992 (sample 2) to 1047 (6% increase in SR) resulted in a 26% decrease in lyso-PC produced after 9 months of storage at 4 ℃ (sample 6 compared to sample 2) and an 8% increase in lyso-PC produced after 9 months of storage at 4 ℃ (sample 1 compared to sample 2). Therefore, irinotecan SOS liposome compositions having a stability ratio greater than 1000, including irinotecan SOS liposome formulations having a stability ratio of 1000-1200 or greater (e.g., a stability ratio of 1053-111), are preferred.

In some embodiments of the invention, the stability of irinotecan SOS-containing liposomal formulations of irinotecan encapsulated in liposomes having a diameter of about 100 ± 20nm, preferably 100 ± 10nm, is significantly increased by raising the pH of the formulation after preparation but before storage such that the pH is greater than 6.5. The effect of pH on lyso-PC formation in liposomal formulations was evaluated by maintaining the loading ratio per mole of phospholipid of the 471g or 500g irinotecan fraction (based on anhydrous free base, as explained above) unchanged but changing the pH of the final pH of the irinotecan liposomal composition. Table 3 provides a summary of the amount of lyso-PC in the irinotecan liposome formulations of table 1 formulated at different pH values. Table 3A reports data from table 1 for irinotecan liposomal formulation by passing liposomes (encapsulating TEA)₈Concentration of SOS sulfate groups of 0.6M) was loaded with a total of 471g irinotecan moieties (based on anhydrous free base, as explained above) per mole of phospholipid (i.e., stability ratio of 471/0.6 or 785). The% change in lyso-PC formation was calculated for both sample 4 and sample 9 (both pH after preparation but before storage was 6.5). Table 3B reports data from table 1 for irinotecan liposomal formulation by passing liposomes (encapsulating TEA)₈SOS sulfate group concentration of 0.45M) was loaded with a total of 471g irinotecan moieties (based on anhydrous free base, as explained above) per mole of phospholipid (e.g., stability ratio of 471/0.45 or 1047). The calculations are for both sample 1 and sample 6 (both)The pH after preparation but before storage was 6.5).

Table 3A: pH and lyso-PC of irinotecan liposome formulation (471 g irinotecan fraction/mol phospholipid, 0.6M SOS sulfate group concentration after 9 months at 4 deg.C)

Table 3B: pH and lyso-PC of irinotecan liposome formulation (471 g irinotecan fraction/mol phospholipid, 0.45M SOS trap concentration after 9 months at 4 deg.C)

In the data in tables 3A and 3B above, increasing the pH from 6.5 to 7.25 or 7.5, the SOS liposome stability ratio for irinotecan was 785, and the amount of lyso-PC decreased by about 15-20% (table 3A); the amount of lyso-PC was reduced by about 20-70% in irinotecan SOS liposomes with a stability ratio of 1047 (table 3B). This was unexpected in view of previous reports showing that pH of 6.5 was The most suitable for minimizing phosphatidylcholine Hydrolysis (group, M et al, "Hydrolysis of particulate matter treated phosphate in aqueous lipid dispersions and The effects of cholesterol incorporation on Hydrolysis kinetics," The Journal of medicine and pHarmacology (1993) v 45, 6, pp 490-495).

Figures 4A-4℃ depict graphs showing the mol% lyso-PC measured in irinotecan liposome formulations having a pH of 7.25 or 7.5 selected from tables 1A and 1B after storage at 4 ℃ for 0,1, 3, 6, and/or 9 months. These figures include linear regression lines for the hemolysis-PC growth rate over time in each sample. Slope, y-intercept and R for each plot²A summary of the values is shown in table 4 below. Lower amounts of lyso-PC were observed in irinotecan liposome formulation samples having a stability ratio greater than 942 (e.g., 1047) and a pH of 7.25 or 7.5 (e.g., comparing samples 5,7, and 13 in fig. 4A and 4C to sample 10 at pH 7.25, orpH 7.5 comparing sample 8 in fig. 4B with sample 11 in fig. 4C). Furthermore, more lyso-PC was measured in irinotecan liposomal formulations with stability ratios below 942 after 9 months (e.g., 785 in samples 10 and 11, both with greater than 20 mol% lyso-PC after 6 months, even at pH greater than 6.5).

Table 4: mol% lyso-PC at pH >6.5 as a function of refrigerated storage time (months)

Table 5: mol% lyso-PC after 6 and 9 months of cold storage of SR >942

Additional camptothecin compositions

The camptothecin composition can be a sustained release composition comprising one or more camptothecin compounds and one or more phospholipids that produces a reduced amount of lysophospholipid after refrigerated storage (i.e., 2-8 ℃) after preparation of the camptothecin composition (e.g., starting when the camptothecin composition is sealed in a sterile container for administration of the drug.

The stable sustained release composition may include a matrix composition comprising the camptothecin compound and a phospholipid or other component that can be hydrolyzed to form a lysophospholipid. The matrix composition may be configured as a liposome encapsulating one or more camptothecin compounds within a vesicle comprising a phospholipid and other components, such as cholesterol and a lipid covalently attached to PEG.

In some embodiments of the invention, the matrix composition is stabilized, for example, as follows: preparing a matrix composition with an amount of an anion scavenger and an amount of a camptothecin compound, and having a specific pH in a medium comprising the matrix composition, thereby effective to reduce the amount of lysophospholipid formation in the matrix composition.

In some embodiments of the invention, the sustained release composition is a nanoparticle comprising triethylammonium sulfatide (SOS) and irinotecan releasably associated with a composition comprising a lipid and/or a biocompatible polymer, such as a cyclodextrin, a biodegradable polymer such as PGA (polyglycolic acid), and/or PLGA (poly (lactic-co-glycolic acid)).

In other embodiments, the sustained release formulation is a matrix composition comprising a releasably associated compound, such as topotecan, etirinotecan, and/or irinotecan (e.g., nanoparticles or polymers that releasably capture or retain a camptothecin or camptothecin derivative compound). The matrix composition may comprise a biocompatible polymer such as polyethylene glycol (PEG) or a functionally equivalent material. In a preferred embodiment, the biocompatible polymer is polyethylene glycol (MW 2000). In a more preferred embodiment, the biocompatible polymer is methoxy-terminated polyethylene glycol (MW 2000).

In some embodiments, the sustained release formulation can comprise a camptothecin compound conjugated to a biocompatible polymer such as a cyclodextrin or a cyclodextrin analog (e.g., sulfated cyclodextrin). For example, sustained release formulations can comprise a cyclodextrin-containing polymer that is chemically bonded to a camptothecin compound (e.g., irinotecan and/or SN-38). The cyclodextrin-camptothecin conjugated compound can be administered in a pharmaceutically acceptable dose. Examples of camptothecin-cyclodextrin conjugates include cyclodextrin-containing polymer conjugates and related intermediates.

In some embodiments of the invention, the sustained release composition comprising a lipid and/or a biocompatible polymer comprises a lipid matrix and/or a complexing agent, such as a cyclodextrin-containing composition, formulated to retain the camptothecin compound during storage and then release the compound into the patient.

In some embodiments of the invention, the matrix composition comprises a phospholipid, such as a phosphatidylcholine derivative, that is stable to reduce lyso-PC formation during refrigerated storage.

Preferably, the sustained release composition is prepared by a multi-step process comprising the steps of: (a) forming a matrix composition comprising a capture agent, and (b) contacting the matrix with a camptothecin compound under the following conditions: the camptothecin compound is effectively stably retained in the resulting sustained-release composition, which comprises a capture agent and the camptothecin compound associated with a matrix composition, in a manner that allows for the desired release of the camptothecin compound in the patient after administration to the patient.

In a preferred embodiment, the sustained release composition of the invention comprises an irinotecan portion of irinotecan or a salt thereof at a concentration equivalent to that provided by 4.3mg/mL irinotecan anhydrous free base per mL, while also comprising less than about 1mg/mL (or less than about 20 mol%) of lyso-PC upon refrigerated storage at 4 ℃ for 6 months. In a preferred embodiment, the sustained release composition of the invention comprises an irinotecan portion of irinotecan or a salt thereof at a concentration equivalent to that provided by 4.3mg/mL irinotecan anhydrous free base per mL, while also comprising less than about 2mg/mL (or less than about 30 mol%) of lyso-PC upon refrigerated storage at 2-8 ℃, even more preferably at about 4 ℃ for 12 months.

The sustained release composition may comprise liposomes. Liposomes generally comprise vesicles containing one or more lipid bilayers encapsulating an aqueous interior. Liposome compositions generally include liposomes in a medium, such as an aqueous fluid external to the liposomes. Liposomal lipids can include amphiphilic lipid components that spontaneously form bilayer membranes upon contact with aqueous media, such as phospholipids, e.g., phosphatidylcholine. Liposomes can also include a membrane hardening component, such as a sterol, e.g., cholesterol. In some cases, liposomes also include lipids conjugated to hydrophilic polymers, such as polyethylene glycol (PEG) liquid derivatives, which can reduce the tendency of the liposomes to aggregate, and also have other beneficial effects. One such PEG-lipid is N- (methoxy-PEG) -oxycarbonyl-distearoyl-phosphatidylethanolamine, wherein the PEG moiety has a molecular weight of about 2000, or MPEG-2000-DSPE. Liposomes generally have sizes in the micron or submicron range and alter their pharmaceutical properties in various beneficial ways due to their ability to carry drug substances, including anticancer drugs, such as irinotecanThe quality is widely accepted. Methods for preparing and characterizing pharmaceutical Liposome compositions are known in the art (see, e.g., Lasic D. liposomes: From physics to applications, Elsevier, Amsterdam 1993; G. Gregoriadis (ed.)), Liposome Technology,3^rdedition, vol.1-3, CRC Press, Boca Raton, 2006; hong et al, U.S. patent 8,147,867, the entire contents of which are incorporated herein by reference for all purposes).

In some embodiments, the liposomes are prepared as described in one or more examples or other embodiments herein, but the concentration of the final liposome composition is increased such that the formulation comprises an irinotecan moiety at a concentration equivalent to an irinotecan hydrochloride trihydrate concentration of about 10, 15, 20, 25, 30, 35, 40, 45, or 50 mg/mL. In some embodiments, the concentration of the irinotecan moiety is equivalent to 5-10, 10-20, 20-30, 30-40, or 40-50mg/mL of irinotecan hydrochloride trihydrate. In some embodiments, the liposome compositions mentioned in this section are used to treat brain tumors or any other disorder in a mammal, as described in U.S. patent No. 8,658,203, which is incorporated by reference herein in its entirety.

The liposomal formulation encapsulating irinotecan may be an injectable formulation comprising liposomes (including injectable formulations that may be subsequently diluted with a pharmaceutically acceptable diluent prior to administration to a patient). In some embodiments, an amount of irinotecan, or a salt thereof, is incorporated into liposomes containing one or more capture agents, wherein the irinotecan is present at a concentration of irinotecan moieties equivalent to 200g, 300g, 400g, 500g, 600g, or 700g, in grams of irinotecan anhydrous free base, per mole of phospholipid. In some embodiments, the irinotecan is present at a concentration of irinotecan moiety during the loading equivalent to 200 to 300g, 400 to 550g, 450 to 600g, or 600 to 700g per mole of phospholipid, based on grams of irinotecan anhydrous free base. Preferably, about 500g (± 10%) of the fraction is loaded into irinotecan liposomes per mole of liposome phospholipid, including 471g of irinotecan fraction per mole of total irinotecan liposome phospholipid. Specific examples in this application include measuring stable irinotecan liposomes containing 471g of irinotecan moieties per mole of total liposomal phospholipids, and measuring irinotecan liposomes containing 500g of irinotecan moieties per mole of total liposomal phospholipids.

In some embodiments, the concentration of irinotecan moiety equivalent to the concentration provided by irinotecan anhydrous free base in the liposomal formulation is about 2.5, about 3.0, about 3.5, about 4.0, about 4.3, about 4.5, about 5.0, about 5.5, or about 6.0 mg/mL. In some embodiments, the concentration of the portion of irinotecan equivalent to the concentration provided by the anhydrous free base of irinotecan in the liposomal formulation is 2.5-3.5, 3.5-4.5, 4.5-5.5, or 5.5-6.5 mg/mL. Most preferably it is 4.5-5.5 mg/mL. In preferred embodiments, the concentration of the irinotecan moiety in the liposomal formulation is about 4.3mg/mL irinotecan anhydrous free base per mL, and in a more preferred embodiment, it is 4.3mg/mL irinotecan anhydrous free base per mL. The liposome formulation may be a vial containing about 43mg irinotecan anhydrous free base in a liposome formulation having a volume of about 10mL, which may be subsequently diluted (e.g., to 500mL of a pharmaceutically acceptable diluent) and then administered intravenously to a patient.

Accordingly, some embodiments of the present invention provide a method of preparing a liposomal formulation of irinotecan comprising stable irinotecan liposomes encapsulating irinotecan Sucrose Octasulfate (SOS) in unilamellar lipid bilayer vesicles consisting of 1, 2-distearoyl-sn-glycerol-3-phosphocholine (DSPC), cholesterol, and methoxy-terminated polyethylene glycol (MW2000) -distearoyl phosphatidylethanolamine (MPEG-2000-DSPE), the method comprising the steps of: (a) contacting a solution comprising irinotecan with a sulfate concentration of 0.4-0.5M (from TEA) under conditions effective to load 500g (+ -10%) of irinotecan moiety per mole of phospholipid into the capture agent liposomes₈SOS provides) contacting capture liposomes encapsulating Triethylammonium (TEA) cations and Sucrose Octasulfate (SOS) capture agents without irinotecan to form irinotecan SOS liposomes, and (b) contacting the irinotecan SOS liposomes with 2- [4- (2-hydroxyethyl) piperazin-1-yl]Ethanesulfonic acid (HEPES) to give liposomal formulations of irinotecan having a pH of 7.25-7.50, resulting in storage at 4 ℃ of 3An irinotecan liposome formulation stabilized over a month to form less than 10 mol% lysophosphatidylcholine (lyso-PC) (relative to the total amount of phosphatidylcholine in the irinotecan liposome).

Storage stable irinotecan liposomes can be prepared in multiple steps, which include forming liposomes comprising TEA and then loading irinotecan into the liposomes as the TEA leaves the liposomes. The first step may include forming liposomes comprising TEA-sulfat by hydrating and dispersing the liposomal lipids in a TEA-sulfat solution. This can be done, for example, as follows: dissolving lipids including DSPC and cholesterol in heated ethanol and at a temperature greater than the transition temperature (T) of the liposome lipids_m) E.g., 60 c or more, to disperse the dissolved and heated lipid solution in the aqueous TEA-sulfate solution. The lipid dispersion may be formed into liposomes having an average size of 75-125nm (e.g., 80-120nm, or in some embodiments, 90-115nm) as follows: extruded through a track etched polycarbonate membrane with a defined pore size, e.g. 100 nm. The TEA-sulfat may comprise at least 8 molar equivalents of TEA per molar equivalent of sulfate to give a solution that may have a sulfate concentration of about 0.40-0.50M and a pH (e.g., about 6.5): the pH is selected to prevent unacceptable degradation of the liposome phospholipids during the dispersion and extrusion steps (e.g., the pH is selected to minimize degradation of the liposome phospholipids during these steps). Then, the non-entrapped TEA-SOS can be removed from the liposome dispersion, e.g., by dialysis, gel chromatography, ion exchange, or ultrafiltration, prior to irinotecan encapsulation. These liposomes can be stabilized by loading sufficient irinotecan into the liposomes to reduce the amount of TEA in the resulting liposome composition to the following levels: the level results in less than a given maximum level of lyso-PC formation, measured for example in mg/mL/month, or the percentage of PC converted to lyso-PC per unit time, e.g. mol% lyso-PC/month, after 180 days at 4 ℃ or more commonly at 5 ± 3 ℃. Next, TEA exchanged from the liposomes into the external medium during loading, along with any unentrapped irinotecan, is typically removed from the liposomes by any suitable known method (e.g., by gel chromatography, dialysis, diafiltration, ion exchange, or ultrafiltration). The liposome external medium can be exchanged for an injectable isotonic fluid (e.g., isotonic sodium chloride solution) buffered at the desired pH.

In some embodiments, when the amount of TEA is less than about 25ppm or less than about 20ppm, a liposomal composition of irinotecan comprising about 3.9-4.7mg/mL of irinotecan and less than 20% lyso-PC after 180 days at 4 ℃ can be obtained. Increasing the pH of the irinotecan liposome composition outside of the liposomes can also storage stabilize irinotecan thiolate liposomes comprising greater than 25ppm TEA, resulting in irinotecan liposomes forming less than 20% of additional lyso-PC after 180 days at 4 ℃. For example, a liposome composition of irinotecan comprising about 4-5mg irinotecan/mL and 100ppm TEA outside of the liposome and having a pH of about 7-8 can also form less than 20% lyso-PC after 180 days at 4 ℃. In another embodiment, the liposome composition comprises from about 3.9 to 4.7mg/mL irinotecan and the liposomes of the external vehicle have a pH of from 7 to 8, wherein the amount of residual TEA is less than about 25ppm (or preferably, less than 20ppm), and the amount of lyso-PC accumulated within the liposome composition within 180 days at 4 ℃ can be 10 mol% or less.

The present invention therefore provides a liposomal composition of irinotecan comprising irinotecan thionate encapsulated in phospholipid liposomes having a lyso-PC stability ratio of at least 990 (e.g., 990-1100 or about 1111).

The invention also provides a liposomal composition of irinotecan, the composition comprising a 4.3mg/mL (+ -10%) fraction equivalent to that provided by irinotecan anhydrous free base and a concentration of 0.4-0.5M sulfate encapsulated in vesicles comprising DSPC and cholesterol in a 3:2 molar ratio and a phospholipid in the vesicles at a ratio of 400-600g irinotecan/mol.

The invention also provides a liposomal composition of irinotecan, collectively comprising about 4.3mg irinotecan moieties per mL, wherein at least 98% of the irinotecan and Sucrose Octasulfate (SOS) are encapsulated within the liposomal composition at an irinotecan to SOS molar ratio of about 8:1, the liposomes having an average size of 75-125 nm. The size of the stable high density irinotecan liposomes is preferably about 110nm (+ -20 nm), more preferably 110nm (+ -10 nm) (measured after liposome loading). Preferably, at least about 95% of the irinotecan in the pharmaceutical composition is encapsulated in liposomes. The liposomes preferably comprise DSPC and cholesterol in a 3:2 molar ratio.

The present invention may also provide a method of preparing a medicament comprising stable irinotecan liposomes encapsulating irinotecan Sucrose Octasulfate (SOS) in unilamellar vesicles of unilamellar lipids, said vesicles consisting of 1, 2-distearoyl-sn-glycerol-3-phosphocholine (DSPC), cholesterol and methoxy-terminated polyethylene glycol (MW2000) -distearoyl phosphatidylethanolamine (MPEG-2000-DSPE), comprising the steps of: (a) contacting irinotecan with the trapping agent liposomes, the trapping agent liposomes acting as TEA, under conditions effective to load the irinotecan moiety into the trapping agent liposomes and allow the TEA cations to be released from the trapping agent liposomes₈SOS encapsulating Triethylammonium (TEA) cation and Sucrose Octasulfate (SOS) trapping agent in sulfate concentration of 0.4-0.5M without irinotecan to form irinotecan SOS liposome, (b) combining irinotecan SOS liposome with 2- [4- (2-hydroxyethyl) piperazin-1-yl]Ethanesulfonic acid (HEPES) to obtain an irinotecan liposome formulation having a pH of 7.25-7.50, to obtain an irinotecan liposome formulation stabilized to form less than 10 mol% of lysophosphatidylcholine (lyso-PC) during storage at 4 ℃ for 3 months, and (c) formulating a combination of irinotecan SOS liposomes and HEPES as a medicament.

In some embodiments of these methods, the irinotecan SOS liposomes in the irinotecan liposome formulation collectively comprise less than 100ppm TEA. In some embodiments, the unilamellar lipid bilayer vesicles consist of 6.81mg/mL 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 2.22mg/mL cholesterol, and 0.12mg/mL methoxy-capped polyethylene glycol (MW2000) -distearoyl phosphatidylethanolamine (MPEG-2000-DSPE). In some embodiments, the liposomal formulation of irinotecan comprises 500g (± 10%) irinotecan per mole of total stabilized liposome phospholipid of irinotecan, at least 98% of the irinotecan in the liposomal formulation of irinotecan being encapsulated in irinotecan liposomes. In some embodiments, the liposomal formulation of irinotecan further comprises 4.05mg/mL 2- [4- (2-hydroxyethyl) piperazin-1-yl ] ethanesulfonic acid (HEPES). In some embodiments, the liposomal formulation of irinotecan further comprises 8.42mg/mL of sodium chloride. In some embodiments, the liposomal formulation of irinotecan has a concentration of irinotecan moiety equivalent to the concentration provided by about 4.3mg/mL irinotecan anhydrous free base. In some embodiments, the stable liposome of irinotecan encapsulates irinotecan and SOS in a compound of formula (I) wherein x is 8.

In some embodiments, the composition comprises less than 2 mol% lyso-PC after 3 months of storage at 2-8 ℃. In some embodiments, the composition comprises less than 5 mol% lyso-PC after 3 months of storage at 2-8 ℃. In some embodiments, the liposome composition comprises less than 10 mol% lyso-PC after 6 months of storage at 2-8 ℃. In some embodiments, the composition comprises less than 10 mol% lyso-PC after 9 months of storage at 2-8 ℃. In some embodiments, the composition comprises less than 5 mol% lyso-PC after 6 months of storage at 2-8 ℃. In some embodiments, the composition comprises less than 5 mol% lyso-PC after 9 months of storage at 2-8 ℃. In some embodiments, the composition comprises less than 2 mol% lyso-PC after 6 months of storage at 2-8 ℃. In some embodiments, the composition comprises less than 2 mol% lyso-PC after 9 months of storage at 2-8 ℃. In some embodiments, the composition comprises less than 10 mol% lyso-PC after 12 months of storage at 2-8 ℃. In some embodiments, the composition comprises less than 5 mol% lyso-PC after 12 months of storage at 2-8 ℃. In some embodiments, the composition comprises less than 2 mol% lyso-PC after 12 months of storage at 2-8 ℃. In some embodiments, the composition comprises less than 10 mol% lyso-PC after 24 months of storage at 2-8 ℃. In some embodiments, the composition comprises less than 5 mol% lyso-PC after 24 months of storage at 2-8 ℃. In some embodiments, the composition comprises less than 2 mol% lyso-PC after 24 months of storage at 2-8 ℃. In some embodiments, the composition comprises less than 100ppm substituted ammonium. In some embodiments, the composition comprises 20 to 80ppm of a substituted ammonium compound that is protonated TEA or DEA.

In other embodiments, stable camptothecin compositions are provided as kits for the formulation of camptothecin compositions comprising one or more component vials. For example, a kit for a liposomal irinotecan formulation may include the following (stored in separate containers or separate parts of the same container):

irinotecan solution (e.g., irinotecan HCl for injection);

liposomes encapsulating a capture agent (e.g., capture agent liposomes formed from a sucrose octasulfate solution); and

instructions for combining the irinotecan solution and the trapping agent liposomes to form a liposomal irinotecan composition comprising a therapeutically effective amount of irinotecan encapsulated in liposomal irinotecan liposomes (e.g., 500g (± 10%) irinotecan per mole of total phospholipids in the trapping agent liposomes and 4.3mg total irinotecan per mL of liposomal irinotecan composition).

Therapeutic uses of camptothecin compositions

Camptothecin compositions, including the liposomes of irinotecan and other compositions and formulations disclosed herein, can be used in therapeutic and therapeutic methods, and/or for the preparation of medicaments for treating diseases such as cancer. In some embodiments, the treatment comprises administering a camptothecin composition for the treatment of cancer. For example, the cancer is selected from the group consisting of: basal cell carcinoma, medulloblastoma, hepatocellular carcinoma, rhabdomyosarcoma, lung carcinoma, bone tumor, pancreatic carcinoma, skin cancer, head and neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastric cancer, colon cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, endometrial cancer, cervical cancer, vaginal cancer, vulval cancer, hodgkin's disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, chronic or acute leukemia, lymphocytic lymphomas, cancer of the bladder, cancer of the kidney or ureter, cancer of the kidney, carcinoma of the renal pelvis, tumors of the central nervous system, primary central nervous system lymphoma, spinal axis tumors, brain stem glioma and pituitary adenoma, or a combination of one or more of these cancers. In some embodiments, the cancer is pancreatic cancer, optionally pancreatic adenocarcinoma, e.g., metastatic pancreatic cancer, e.g., symptoms of disease progression that have occurred following gemcitabine-based treatment. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is small cell lung cancer. In some embodiments, the cancer is biliary tract cancer.

When used as a treatment, the liposome composition can be used in a treatment regimen with one or more other compounds or compositions. Administration of the liposome composition with one or more other compounds or compositions may be simultaneous, separate or sequential. The one or more other compounds or compositions may be additional therapeutic agents, such as additional anti-cancer agents, or may be compounds designed to ameliorate negative side effects of the therapeutic agents. In some embodiments, the liposome composition is administered with leucovorin. In some embodiments, the liposome composition is administered with 5-fluorouracil (5-FU). In some embodiments, the liposome composition is administered with leucovorin and 5-fluorouracil (5-FU). This triple therapy may be used to treat pancreatic cancer, as discussed in the preceding paragraphs. 5-FU can be administered at a dose of 2400mg/m²The dose of the formyltetrahydrofolic acid which can be administered is 200mg/m²(formula I) or 400mg/m²(l + d-derotation). In some embodiments, the composition is also administered in a gemcitabine treatment regimen.

In some embodiments, where the liposomal composition is used to treat ovarian cancer, the liposomal composition is administered with a PARP (poly adenosine diphosphate-ribose polymerase) inhibitor.

In some embodiments, the sustained release matrix can be a nanoparticle (e.g., silica or polymer) or a polymer aggregate (e.g., PEG polymer) configured to retain the capture agent. During drug loading, the matrix can be contacted with the camptothecin compound under conditions effective to retain both the camptothecin compound and the capture agent, forming a stable sustained release formulation.

In some embodiments, the stable camptothecin composition is an irinotecan SOS liposomal formulation formulated for intraparenchymal administration to a patient during convection-enhanced delivery therapy. The concentration of irinotecan moiety equivalent to the concentration provided by the irinotecan anhydrous free base in the final liposomal formulation is about 17, about 20, about 25, about 30, about 35, or about 40 mg/mL. In some embodiments, the concentration of the irinotecan moiety equivalent to the concentration provided by the irinotecan anhydrous free base in the final liposomal formulation is 17-20, 17-25, 17-30, 17-35, or 17-40 mg/mL. Most preferably, the total concentration of irinotecan moieties equivalent to the concentration provided by irinotecan anhydrous free base (e.g., irinotecan sucrose octasulfate) in the irinotecan liposome formulation is 17mg/mL or 35 mg/mL. The liposomal formulation can encapsulate irinotecan sucrose octasulfate liposomes in a sterile container in a liposomal formulation at a concentration of irinotecan moiety equivalent to the concentration provided by the anhydrous free base of irinotecan of about 17mg/mL or about 35mg/mL or 17-35mg/mL for topical administration to a patient (e.g., into the brain of a patient diagnosed with glioma, into a site within the brain as part of a convection-enhanced delivery therapy). The concentration of 17-35mg/mL of irinotecan liposome can be equivalently expressed as the amount of irinotecan anhydrous free base present in 20-40mg of irinotecan hydrochloride trihydrate per mL of irinotecan liposome formulation. For example, a liposomal irinotecan formulation may be administered to a patient's brain at the following doses (e.g., via one or more catheters surgically placed at an intratumoral location): the provision of the total irinotecan moiety is equivalent to that provided by 17mg, 26mg, 52mg or 70mg of total irinotecan anhydrous free base. The total volume of irinotecan for delivery of the irinotecan liposome formulation to the intratumoral location in the brain of a patient over a period of about 2-4 hours (e.g., 2-3 hours, 3-4 hours, or 2-4 hours) can be about 1-2mL (e.g., 1.0, 1.5, or 2.0 mL).

Irinotecan liposomes preferably comprise irinotecan sulfate encapsulated within vesicles formed from lipids comprising DSPC and cholesterol in a 3:2 molar ratio. The vesicles may also comprise polyethylene glycol (PEG) -derived phospholipids, such as MPEG-2000-DSPE. The amount of MPEG-2000-DSPE may be less than 1 mol% of the liposome lipid (e.g., about 0.3 mol% in vesicles consisting of 3:2:0.015 molar ratio of DSPC, cholesterol, and MPEG-2000-DSPE). PEG can partition on both the interior and exterior of liposomal lipid vesicles encapsulating irinotecan. The encapsulated irinotecan is preferably in the form of a sucrose sulfate (thionate), for example, the salt of irinotecan thionate (CAS registry number 1361317-83-0). Preferably, at least 95% and most preferably at least about 98% of the irinotecan liposome composition is encapsulated within the liposome vesicles, with a total concentration of irinotecan moiety of about 3.87-4.73mg irinotecan (anhydrous free base) per mL irinotecan liposome composition. The pH of the liposomal composition of irinotecan outside the liposomes is preferably about 6.5-8.0, or about 6.6-8.0, 6.7-8.0, 6.8-8.0, 6.9-8.0, or 7.0-8.0, preferably about 7.2-7.6. In some embodiments, the pH is about 7.2-7.5. In some embodiments, the pH is about 7.25. In other embodiments, the pH is about 7.25-7.5. In other embodiments, the pH is about 7.4-7.5.

Combined embodiments

Features of embodiments numbered herein may be combined with features of other embodiments disclosed herein, including embodiments relating to compositions and embodiments relating to formulations.

The above-described methods have the same features as embodiments of the compositions and formulations described elsewhere in this specification, as they relate to the preparation of these compositions and formulations. The features disclosed in relation to the compositions and formulations may also be combined with the methods disclosed in the preceding paragraphs. Thus, the features of the preceding subsection, as well as portions of the embodiments numbered elsewhere in this application, such as below, may be combined with features disclosed in the methods of the various paragraphs of this subsection.

For example, the following are examples of various combinations of embodiments disclosed and/or exemplified in this application:

irinotecan liposome compositions comprising an irinotecan fraction of about 3.9-4.7mg/mL and less than 20% lyso-PC after 180 days storage at 4 ℃.

The lyso-PC stability ratio of the irinotecan liposome composition comprising irinotecan sulfate ester encapsulated in phospholipid liposomes is at least 990 (e.g., 990-.

Irinotecan liposome composition comprising a 4.3mg/mL (+ -10%) irinotecan fraction and a 0.4-0.5M concentration of sulfate encapsulated in vesicles comprising DSPC and cholesterol in a 3:2 molar ratio and a ratio of 450-550g irinotecan/mol total phospholipids in the vesicles.

Irinotecan liposome composition, collectively comprising about 4.3mg irinotecan fraction/mL, wherein at least 98% irinotecan and Sucrose Octasulfate (SOS) are encapsulated within the liposome composition at an irinotecan to SOS molar ratio of about 8:1, the liposomes having an average size of 75-125 nm.

The composition of any one of the preceding embodiments, wherein the irinotecan liposomes are obtained by a process comprising the steps of: irinotecan is contacted with Triethylammonium (TEA) sulfatase encapsulated within phospholipid liposomes.

The composition of the previous embodiment, wherein the concentration of TEA-SOS is about 0.40-0.50M.

A composition according to any of the preceding embodiments, wherein the liposomes are about 110nm (± 10%) in size.

A composition according to any one of the preceding embodiments comprising about 433g irinotecan moiety per mol phospholipid.

The composition of any one of the preceding embodiments, wherein the irinotecan liposome composition comprises less than about 100ppm triethylamine.

The composition of any one of the preceding embodiments, wherein the irinotecan liposome composition is a solution of liposomes in a liquid, wherein the pH of the liquid outside the irinotecan liposomes is about 7.0-8.0, such as 7.25-7.5, such as 7.25, optionally wherein the liquid outside the irinotecan liposomes is a pharmaceutically acceptable injectable fluid.

A composition according to any one of the preceding embodiments comprising a portion of irinotecan in an amount equivalent to that provided by 4.5-5.5mg/ml irinotecan hydrochloride trihydrate.

The composition of any one of the preceding embodiments, wherein at least about 95% of the irinotecan liposome composition is encapsulated within the liposomes.

The composition of any one of the preceding embodiments, wherein the liposomes comprise DSPC and cholesterol in a molar ratio of 3:2, for example wherein the liposomes comprise DSPC, cholesterol and MPEG (2000) -DSPE in a molar ratio of 3:2: 0.015.

The composition of any one of the preceding embodiments, having a stability ratio of 990-.

The composition of any of the preceding embodiments has a gram equivalent ratio of liposome-encapsulated irinotecan/sulfatide of at least 0.9, at least 0.95, at least 0.98, at least 0.99, or substantially 1.0.

The composition of any one of the preceding embodiments, wherein the liposomal phospholipid comprises no more than 20 mol% lyso-PC after 180 days of storage at about 4 ℃.

The composition of any of the preceding embodiments, wherein the irinotecan liposome composition further comprises a pharmaceutically acceptable injectable fluid having a pH of about 7.0-8.0 outside of the irinotecan liposome, comprising 4.3mg/mL irinotecan (calculated as the free base), optionally obtained by a process comprising the steps of: irinotecan is contacted with Triethylammonium (TEA) sulfatase encapsulated in phospholipid liposomes optionally having a concentration of encapsulated TEA sulfatase of about 0.40-0.50N.

The composition of any one of the preceding embodiments, wherein the composition comprises about 433g irinotecan moiety per mol phospholipid, and no greater than about 100ppm triethylammonium encapsulated in phospholipid liposomes.

The composition of any of the preceding embodiments having a gram equivalent ratio of encapsulated irinotecan/sulfatide of at least 0.9.

The composition of any of the preceding embodiments, wherein at least 90%, such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% (in other words, substantially all) of the encapsulated irinotecan thionate is a stoichiometric salt in precipitated or gel form, comprising eight molecules of irinotecan per molecule of thionate.

The composition of any of the preceding embodiments wherein at least 98%, such as at least 99%, of the irinotecan thionate encapsulated is a stoichiometric salt in the form of a precipitate or gel comprising eight molecules of irinotecan per molecule of thionate.

Irinotecan liposome composition of any of the preceding embodiments having no more than about 100ppm Triethylammonium (TEA).

Irinotecan liposome composition of any of the preceding embodiments having no more than about 20ppm Triethylammonium (TEA).

Irinotecan liposome composition of any of the preceding embodiments, having a total volume of about 10 mL.

Irinotecan liposome composition of any of the preceding embodiments, comprising 6.81mg/mL 1, 2-distearoyl-sn-glycerol-3-phosphocholine (DSPC), 2.22mg/mL cholesterol, and 0.12mg/mL methoxy-capped polyethylene glycol (MW2000) -distearoyl phosphatidylethanolamine (MPEG-2000-DSPE).

The liposomal irinotecan composition of any of the preceding embodiments, comprising polyethylene glycol both inside and outside of the liposome of irinotecan.

A stable injectable unit dose liposomal composition of irinotecan formulated for administration to a patient, the composition comprising a dose of irinotecan sufficient to deliver 70mg of irinotecan per m²A patient body surface area, wherein:

o at least 99% irinotecan encapsulated in vesicles comprising phospholipid and cholesterol, wherein up to 20 mol% of the phospholipid is lyso-PC, the remainder being DSPC, wherein the vesicles are in an injectable fluid having a pH of 7.0-8.0; or

An injectable unit dose liposome composition is a unit dose of the liposome composition of any of the above embodiments.

An injectable irinotecan liposomal unit dosage form comprising:

omicron at least about 98% irinotecan in a unit dosage form encapsulated in a liposome comprising a phospholipid comprising no greater than about 20 mol% lyso-PC; and

omicron the liposome composition according to any of the above embodiments.

A unit dosage form as disclosed in the above embodiment, wherein irinotecan is encapsulated in a vesicle surrounded by a lipid membrane consisting essentially of 1, 2-distearoyl-sn-glycerol-3-phosphocholine (DSPC), cholesterol and methoxy-capped polyethylene glycol (MW2000) -distearoyl phosphatidylethanolamine (MPEG-2000-DSPE).

The unit dosage form of embodiment 29 or 30, wherein the unit dosage form comprises at least about 6.81mg/mL 1, 2-distearoyl-sn-glycerol-3-phosphocholine (DSPC), about 2.22mg/mL cholesterol, and about 0.12mg/mL methoxy-capped polyethylene glycol (MW2000) -distearoyl phosphatidylethanolamine (MPEG-2000-DSPE) L.

The unit dosage form of any of embodiments 29-31, wherein the unit dosage form further comprises 2- [4- (2-hydroxyethyl) piperazin-1-yl ] ethanesulfonic acid (HEPES) as a buffer and sodium chloride as an isotonicity agent.

A liposomal composition according to any one of embodiments 1-27, or a unit dose according to any one of embodiments 29-32, for use in therapy.

A liposome composition or unit dose as disclosed in embodiments herein for use in the treatment of cancer.

The present embodiments disclose the use of a liposome composition or unit dose, wherein the cancer is selected from the group consisting of: basal cell carcinoma, medulloblastoma carcinoma, liver cancer, rhabdomyosarcoma, lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, hodgkin's disease, esophageal cancer, small bowel cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphomas, bladder cancer, cancer of the kidney or ureter, renal cell carcinoma, renal pelvis cancer, tumors of the central nervous system, primary lymphoma of the central nervous system, spinal axis tumors, brain stem glioma and pituitary adenoma, or a combination of one or more of these cancers.

An accordingly preferred embodiment of the present invention provides a method of treating cancer, wherein the cancer is pancreatic cancer, optionally pancreatic adenocarcinoma, e.g. metastatic pancreatic cancer, e.g. a condition in which disease progression has occurred following gemcitabine-based treatment.

An accordingly embodiment, wherein the cancer is colon cancer.

A liposomal composition or unit dose according to any one of the above embodiments, wherein the liposomal composition or unit dose is for use with leucovorin and/or 5-fluorouracil, optionally wherein administration of the liposomal composition or unit dose, leucovorin and/or 5-fluorouracil is simultaneous, separate or sequential.

The liposomal composition or unit dose according to any of the above embodiments, wherein the liposomes are such as to provide an equivalent of 80mg/m²A dose of irinotecan hydrochloride trihydrate.

A method of treating metastatic pancreatic cancer following disease progression following gemcitabine-based treatment in a patient in need thereof, the method comprising intravenously administering to the patient an injectable irinotecan liposomal unit dosage form of any one of the embodiments of the present application, comprising at least about 98% irinotecan in a unit dosage form encapsulated in liposomes comprising a phospholipid, comprising an amount of less than about 20% lyso-PC to provide an equivalent of 80mg/m, or a unit dose according to any of the embodiments described above²Irinotecan hydrochloride trihydrate.

A storage stable liposomal irinotecan composition having a pH of 7.00-7.50 and comprising a dispersion of irinotecan liposomes encapsulating irinotecan sucrose octasulfate in unilamellar vesicles, the vesicles are composed of cholesterol and the phospholipids 1, 2-distearoyl-sn-glycerol-3-phosphocholine (DSPC) and methoxy-capped polyethylene glycol (MW2000) -distearoyl phosphatidylethanolamine (MPEG-2000-DSPE), the liposomal irinotecan composition comprising irinotecan moieties at concentrations equivalent to, 500mg irinotecan per mmol of total liposomal phospholipid and 4.3mg irinotecan per mL of said liposomal irinotecan composition, in grams of irinotecan anhydrous free base, the storage-stable liposomal irinotecan composition stabilizes to form less than 1mg/mL of lyso-PC during storage at 4 ℃ for 6 months.

Liposomal irinotecan composition of the above embodiment, prepared by a process comprising:

(a) forming a dispersion of lipids in a solution consisting of DEA having a sulfate concentration of 0.4 to 0.5M₈SOS preparation and pH 5 to 7, the lipids in the dispersion being about 3:2:0.015 molar ratio of DSPC, cholesterol, and MPEG-2000-DSPE, respectively;

(b) extruding a lipid dispersion at 60-70 ℃ through at least one 0.1 μm membrane to form liposomes;

(c) substantially removing DEA-derived material outside the liposomes₈SOS and/or DEA₈An ion of SOS;

(d) contacting liposomes at a temperature of 60-70 ℃ with a solution prepared using irinotecan free base or an irinotecan salt, thereby forming a formulation of liposomes encapsulating irinotecan;

(e) substantially removing TEA-derived material outside the liposomes₈SOS and/or DEA₈(ii) SOS and irinotecan-containing substances; and

(f) the pH of the composition is adjusted to 7.0-7.5.

Liposomal irinotecan composition of any of the above embodiments, wherein the lipid dispersion is extruded through at least two superposed 0.1 μm polycarbonate membranes.

Liposomal irinotecan composition of any of the above embodiments, wherein the liposomes have a mean size of 110nm, the mean size being determined by dynamic light scattering, wherein the size is determined by the cumulant method.

Liposomal irinotecan composition of any of the above embodiments having a total irinotecan moiety content equivalent to 4.3mg/ml irinotecan anhydrous free base.

Liposomal irinotecan composition of any one of the embodiments above, wherein:

in step (a), the liposome is composed of DEA with sulfate concentration of 0.43-0.47M₈SOS formation; and

in step (d), a solution prepared using irinotecan free base or irinotecan salt has an irinotecan partial content equivalent to 500g (± 10%) irinotecan anhydrous free base per mole of DSPC; and

in step (f), the pH of the composition is adjusted to 7.2 to 7.3.

A liposome composition of any one of the preceding embodiments, which contains less than 1 mol% lysophosphatidylcholine (lyso-PC) prior to storage at about 4 ℃, and which contains 20 mol% or less (relative to total liposome phospholipids) lyso-PC after storage at about 4 ℃ for 180 days.

An accordingly preferred embodiment of the liposome composition of any one of the above embodiments, which comprises 20 mol% or less (relative to total liposome phospholipids) lysophosphatidylcholine (lyso-PC) after storage at about 4 ℃ for 6, 9 or 12 months.

Liposomal irinotecan composition of any of the above embodiments, collectively comprising 6.1 to 7.5mg DSPC/ml, 2 to 2.4mg cholesterol/ml and 0.11 to 0.13mg MPEG-2000-DSPE/ml, all in aqueous isotonic buffer.

Liposomal irinotecan composition of any of the above embodiments, wherein the liposomal irinotecan comprises irinotecan liposomes in an isotonic HEPES aqueous buffer at a concentration of 2 to 20 mM.

Liposomal irinotecan composition of any of the above embodiments, further comprising sodium chloride at a concentration of 130-160 mM.

Liposomal irinotecan composition of any of the above embodiments, wherein the irinotecan encapsulated in the liposomes is in a gel state or a precipitated state as sucrose octasulfate salt.

Liposomal irinotecan composition of any of the above embodiments, wherein the irinotecan liposomes have a diameter of 95-115nm, as measured by quasi-elastic light scattering.

Liposomal irinotecan composition of any of the above embodiments, which collectively comprises 6.81mg DSPC/mL, 2.22mg cholesterol/mL, and 0.12mg MPEG-2000-DSPE/mL, 4.05mg/mL HEPES aqueous buffer, and 8.42mg sodium chloride/mL.

Liposomal irinotecan composition of any of the above embodiments having a pH of 7.25, wherein the irinotecan liposomes are 110nm in diameter, as measured by quasi-elastic light scattering.

Liposomal irinotecan composition of any of the above embodiments, which forms less than 1mg/mL of lysophosphatidylcholine (lyso-PC) after 6 months of storage at about 4 ℃.

Liposomal irinotecan composition of any of the above embodiments, prepared by a process comprising:

(a) DEA is formed with lipids at a sulfate concentration of about 0.45M₈A dispersion in an SOS solution, said solution having a pH of about 6.5, the lipids in said dispersion consisting of 1, 2-distearoyl-sn-glycerol-3-phosphocholine (DSPC), cholesterol, and methoxy-capped polyethylene glycol (MW2000) -distearoyl phosphatidylethanolamine (MPEG-2000-DSPE), respectively, in a molar ratio of 3:2: 0.015;

(b) extruding a lipid dispersion at 60-70 ℃ through at least one 0.1 μm membrane to form liposomes;

(c) removing DEA-derived material outside the liposomes₈An ion of SOS;

(d) contacting liposomes at a temperature of 60-70 ℃ with a solution prepared using irinotecan hydrochloride trihydrate to form a liposome formulation encapsulating about 500g (± 10%) irinotecan per mole of total liposomal phospholipids;

(e) removing TEA-derived materials from the exterior of the liposomes₈(ii) SOS and irinotecan-containing substances; and

(f) the pH of the composition was adjusted to about 7.3.

Liposomal irinotecan composition of any of the above embodiments, which collectively comprises less than 100ppm of DEA.

Liposomal irinotecan composition of any of the above embodiments, which collectively comprises less than 100ppm of DEA.

The liposomal irinotecan composition of any one of the preceding embodiments, wherein at least 98% of the irinotecan is encapsulated in the irinotecan liposomes after 6 months of storage at about 4 ℃.

An irinotecan liposome formulation comprising stable irinotecan liposomes encapsulating irinotecan Sucrose Octasulfate (SOS) in unilamellar lipid bilayer vesicles having a diameter of about 110nm, consisting of 1, 2-distearoyl-sn-glycerol-3-phosphocholine (DSPC), cholesterol, and methoxy-capped polyethylene glycol (MW2000) -distearoyl phosphatidylethanolamine (MPEG-2000-DSPE), wherein the stable irinotecan liposomes are obtained by a process comprising the steps of:

(a) contacting irinotecan with the trapping agent liposomes as TEA under conditions effective to load 500g (+ -10%) of irinotecan moieties per mole of total liposomal phospholipids into the trapping agent liposomes and allow the release of DEA cations from the trapping agent liposomes₈SOS encapsulates Diethylammonium (DEA) cations and Sucrose Octasulfate (SOS) trapping agent without irinotecan at a concentration of 0.4-0.5M (based on sulfate group concentration) to form irinotecan SOS liposomes, and

(b) combining irinotecan SOS liposomes with 2- [4- (2-hydroxyethyl) piperazin-1-yl ] ethanesulfonic acid (HEPES) to give an irinotecan liposome formulation having a pH of 7.25-7.50, thereby giving an irinotecan liposome formulation that is stabilized to form less than 10 mol% lysophosphatidylcholine (lyso-PC) (relative to the total amount of phosphatidylcholine in the irinotecan liposomes) during storage at 4 ℃ for 3 months.

The irinotecan liposome formulation of any of the preceding embodiments, wherein the irinotecan SOS liposomes in the irinotecan liposome formulation collectively comprise less than 100ppm TEA.

An irinotecan liposome formulation of any one of the above embodiments, wherein the unilamellar vesicles of monocompartment lipids consist of 6.81mg/mL 1, 2-distearoyl-sn-glycerol-3-phosphocholine (DSPC), 2.22mg/mL cholesterol, and 0.12mg/mL methoxy-capped polyethylene glycol (MW2000) -distearoyl phosphatidylethanolamine (MPEG-2000-DSPE).

An irinotecan liposome formulation of any one of the above embodiments, collectively comprising 500mg of irinotecan per mole of total stabilized irinotecan liposome phospholipid, at least 98% of the irinotecan in the irinotecan liposome formulation being encapsulated within irinotecan liposomes.

The irinotecan liposome formulation of any of the above embodiments, wherein the irinotecan liposome formulation further comprises about 4.05mg/mL 2- [4- (2-hydroxyethyl) piperazin-1-yl ] ethanesulfonic acid (HEPES) at a pH of about 7.25-7.50.

The irinotecan liposome formulation of any of the above embodiments, wherein the irinotecan liposome formulation further comprises about 8.42mg/mL of sodium chloride.

An irinotecan liposome formulation of any of the above embodiments having a total of about 4.3mg irinotecan per mL irinotecan liposome formulation.

The composition of any one of the preceding embodiments, wherein the irinotecan liposomes are obtained by a process comprising the steps of: irinotecan is contacted with ammonium encapsulated in phospholipid liposomes.

An irinotecan liposome formulation comprising stable irinotecan liposomes encapsulating irinotecan Sucrose Octasulfate (SOS) in unilamellar lipid bilayer vesicles having a diameter of about 110nm, consisting of 1, 2-distearoyl-sn-glycerol-3-phosphocholine (DSPC), cholesterol, and methoxy-capped polyethylene glycol (MW2000) -distearoyl phosphatidylethanolamine (MPEG-2000-DSPE), wherein the stable irinotecan liposomes are obtained by a process comprising the steps of:

(a) contacting irinotecan with a capture agent liposome encapsulating an ammonium cation and a Sucrose Octasulfate (SOS) capture agent under conditions effective to load 500g (+ -10%) of irinotecan moiety per mole of total liposomal phospholipids into the capture agent liposome and to allow release of the ammonium cation from the capture agent liposome to form irinotecan SOS liposome, and

(b) combining irinotecan SOS liposomes with 2- [4- (2-hydroxyethyl) piperazin-1-yl ] ethanesulfonic acid (HEPES) to give an irinotecan liposome formulation having a pH of 7.25-7.50, thereby giving an irinotecan liposome formulation that is stabilized to form less than 10 mol% lysophosphatidylcholine (lyso-PC) (relative to the total amount of phosphatidylcholine in the irinotecan liposomes) during storage at 4 ℃ for 3 months.

SN38 liposomal formulation comprising stable liposomes comprising irinotecan and/or SN-38 in liposomes comprising 1, 2-distearoyl-SN-glycerol-3-phosphocholine (DSPC), cholesterol and methoxy-capped polyethylene glycol (MW2000) -distearoyl phosphatidylethanolamine (MPEG-2000-DSPE), the liposomal formulation being stabilized during storage at 4 ℃ for 3 months to form less than 10 mol% of lysophosphatidylcholine (lyso-PC) (with respect to the total amount of phosphatidylcholine in the liposomes).

The irinotecan liposome formulation of any of the preceding embodiments, wherein the stable irinotecan liposome encapsulates 30-100ppm of TEA or DEA, irinotecan and SOS, where x is 8, in the composition of formula (I).

In one embodiment, the irinotecan liposome composition disclosed herein is a stable irinotecan liposome composition comprising an irinotecan sulfate encapsulated in phospholipid liposomes having a lyso-PC stability ratio of at least 990 (e.g., 990-:

(i) the size of the liposomes was about 110nm (+ -10%),

(ii) the composition comprises about 433g or at least about 433g irinotecan moiety per mol phospholipid,

(iii) the composition comprises less than about 100ppm triethylamine,

(iv) the composition comprises a pharmaceutically acceptable injectable fluid having a pH of about 7.25 outside the liposomes of irinotecan,

(v) the liposomes comprised DSPC and cholesterol in a 3:2 molar ratio,

(vi) the composition has a gram equivalent ratio of liposomal encapsulated irinotecan/sulfatide of at least 0.9, at least 0.95, at least 0.98, or substantially 1.0; and

(vii) at least 90%, such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% (in other words, substantially all) of the encapsulated irinotecan sulfatide is a stoichiometric salt in the form of a precipitate or gel comprising eight molecules of irinotecan per molecule of sulfatide.

In one embodiment, the irinotecan liposome composition disclosed herein is an irinotecan liposome composition stabilized comprising an irinotecan sulfate encapsulated in phospholipid liposomes having a lyso-PC stability ratio of at least 990 (e.g., 990-:

(i) the size of the liposomes was about 110nm (+ -10%),

(ii) the composition comprises about 433g or at least about 433g irinotecan moiety per mol phospholipid,

(iii) the composition comprises less than about 100ppm triethylamine,

(iv) the composition comprises a pharmaceutically acceptable injectable fluid having a pH of about 7.25 outside the liposomes of irinotecan,

(v) the liposomes comprised DSPC and cholesterol in a 3:2 molar ratio,

(vi) the composition has a gram equivalent ratio of liposomal encapsulated irinotecan/sulfatide of at least 0.9, at least 0.95, at least 0.98, or substantially 1.0; and

(vii) at least 90%, such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% (in other words, substantially all) of the encapsulated irinotecan sulfatide is a stoichiometric salt in the form of a precipitate or gel comprising eight molecules of irinotecan per molecule of sulfatide.

Embodiment 1: a storage stable liposomal irinotecan composition having a pH of 7.00-7.50 and comprising a dispersion of irinotecan liposomes encapsulating irinotecan sucrose octasulfate in vesicles, the vesicles are composed of cholesterol and the phospholipids 1, 2-distearoyl-sn-glycerol-3-phosphocholine (DSPC) and methoxy-capped polyethylene glycol (MW2000) -distearoyl phosphatidylethanolamine (MPEG-2000-DSPE), the liposomal irinotecan composition comprising irinotecan moieties at concentrations equivalent to, 500mg (+ -10%) of irinotecan moiety per mmol of total liposomal phospholipid and 4.3mg of irinotecan moiety per mL of the liposomal irinotecan composition, in grams of irinotecan free anhydrous base, the storage stable liposomal irinotecan composition stabilizes to form less than 20 mol% lyso-PC during the first 6 months of storage at 4 ℃.

Embodiment 2: a storage stable liposomal irinotecan composition having a pH of 7.00-7.50 and comprising a dispersion of irinotecan liposomes encapsulating irinotecan sucrose octasulfate in unilamellar vesicles, the vesicles are composed of cholesterol and the phospholipids 1, 2-distearoyl-sn-glycerol-3-phosphocholine (DSPC) and methoxy-capped polyethylene glycol (MW2000) -distearoyl phosphatidylethanolamine (MPEG-2000-DSPE), the liposomal irinotecan composition comprising irinotecan moieties at concentrations equivalent to, 500mg (+ -10%) of irinotecan moiety per mmol of total liposomal phospholipid and 4.3mg of irinotecan moiety per mL of the liposomal irinotecan composition, in grams of irinotecan free anhydrous base, the storage stable liposomal irinotecan composition has an irinotecan/sulfate compound gram-equivalent ratio of 0.85-1.2.

Embodiment 3: the storage stable liposomal irinotecan composition stabilized to form less than 20 mol% lyso-PC during the first 6 months of storage at 4 ℃, the liposomal irinotecan composition prepared by a process comprising the steps of:

(a) formation of lipids in TEA from sulfate concentration of 0.4 to 0.5M₈SOS and/or DEA₈A dispersion of SOS preparation and a solution having a pH of 5 to 7, the lipids in the dispersion being about 3:2:0.015 molar ratio of DSPC, cholesterol, and MPEG-2000-DSPE, respectively;

(b) extruding a lipid dispersion at 60-70 ℃ through at least one 0.1 μm membrane to form liposomes;

(c) substantially removing TEA-derived material outside the liposomes₈SOS and/or DEA₈An ion of SOS;

(d) contacting liposomes at a temperature of 60-70 ℃ with a solution prepared using irinotecan free base or an irinotecan salt, thereby forming a formulation of liposomes encapsulating irinotecan;

(e) substantially removing TEA-derived material outside the liposomes₈SOS and/or DEA₈(ii) SOS and irinotecan-containing substances; and

(f) the pH of the composition is adjusted to 7.0-7.5.

Embodiment 4: the liposomal irinotecan composition of any one of embodiments 1-3, prepared by a process comprising the steps of:

(a) formation of lipids in TEA from sulfate concentration of 0.4 to 0.5M₈A dispersion of SOS preparation and a solution having a pH of 5 to 7, the lipids in the dispersion being about 3:2:0.015 molar ratio of DSPC, cholesterol, and MPEG-2000-DSPE, respectively;

(b) extruding a lipid dispersion at 60-70 ℃ through at least one 0.1 μm membrane to form liposomes;

(c) substantially removing TEA-derived material outside the liposomes₈An ion of SOS;

(d) contacting the liposomes with a solution prepared using irinotecan free base or an irinotecan salt at a temperature of 60-70 ℃, thereby forming a formulation of liposomes encapsulating irinotecan;

(e) substantially removing TEA-derived material outside the liposomes₈(ii) SOS and irinotecan-containing substances; and

(f) the pH of the composition is adjusted to 7.0-7.5.

Embodiment 5: the liposomal irinotecan composition of embodiment 4, wherein the lipid dispersion is extruded through at least two superposed 0.1 μm polycarbonate membranes.

Embodiment 6: the liposomal irinotecan composition of any of the preceding embodiments, wherein the liposomes have an average size of 110nm, the average size being determined by dynamic light scattering, wherein the size is determined by the cumulant method.

Embodiment 7: the liposomal irinotecan composition of any of the preceding embodiments having a total irinotecan moiety content equivalent to 4.3mg/ml irinotecan anhydrous free base.

Embodiment 8: the liposomal irinotecan composition of any one of embodiments 3-6, wherein:

in step (a), the liposome is composed of TEA with sulfate concentration of 0.43-0.47M₈SOS formation; and

in step (d), a solution prepared using irinotecan free base or irinotecan salt has an irinotecan partial content equivalent to 500g (± 10%) of irinotecan anhydrous free base per mole of DSPC; and

in step (f), the pH of the composition is adjusted to 7.2 to 7.3.

Embodiment 9: the liposome composition of any one of the preceding embodiments, which contains less than 1 mol% lysophosphatidylcholine (lyso-PC) prior to storage at about 4 ℃, and which contains 20 mol% or less (relative to total liposome phospholipids) lyso-PC after storage at about 4 ℃ for 180 days.

Embodiment 10: the liposome composition of embodiment 9, comprising 20 mol% or less (relative to total liposome phospholipids) lysophosphatidylcholine (lyso-PC) after 6, 9 or 12 months of storage at about 4 ℃.

Embodiment 11: the liposomal irinotecan composition of any of the preceding embodiments, collectively comprising 6.1 to 7.5mg DSPC/ml, 2 to 2.4mg cholesterol/ml, and 0.11 to 0.13mg MPEG-2000-DSPE/ml, all in an aqueous isotonic buffer.

Embodiment 12: the liposomal irinotecan composition of any of the preceding embodiments, wherein the liposomal irinotecan comprises irinotecan liposomes in an isotonic HEPES aqueous buffer at a concentration of 2 to 20 mM.

Embodiment 13: the liposomal irinotecan composition of any of the preceding embodiments, further comprising sodium chloride at a concentration of 130-160 mM.

Embodiment 14: the liposomal irinotecan composition of any of the preceding embodiments, wherein the irinotecan encapsulated in the liposomes is in a gel state or a precipitated state as sucrose octasulfate salt.

Embodiment 15: the liposomal irinotecan composition of any of the preceding embodiments, wherein the irinotecan liposomes have a diameter of 95-115nm, as measured by quasi-elastic light scattering.

Embodiment 16: the liposomal irinotecan composition of any of the preceding embodiments, which collectively comprises 6.81mg DSPC/mL, 2.22mg cholesterol/mL, and 0.12mg MPEG-2000-DSPE/mL, 4.05mg/mL HEPES aqueous buffer, and 8.42mg sodium chloride/mL.

Embodiment 17: the liposomal irinotecan composition of any of the preceding embodiments having a pH of 7.25, wherein the irinotecan liposomes are 110nm in diameter, as measured by quasielastic light scattering.

Embodiment 18: the liposomal irinotecan composition of any of the preceding embodiments, which forms less than 1mg/mL of lysophosphatidylcholine (lyso-PC) after 6 months of storage at about 4 ℃.

Embodiment 19: the liposomal irinotecan composition of any of the preceding embodiments, prepared by a process comprising the steps of:

(a) formation of lipids in TEA₈A dispersion of a solution having a concentration of SOS sulfate of about 0.45M, said solution having a pH of about 6.5, the lipids in said dispersion consisting of 1, 2-distearoyl-sn-glycerol-3-phosphocholine (DSPC), cholesterol, and methoxy-capped polyethylene glycol (MW2000) -distearoyl phosphatidylethanolamine (MPEG-2000-DSPE), respectively, in a molar ratio of 3:2: 0.015;

(b) extruding a lipid dispersion at 60-70 ℃ through at least one 0.1 μm membrane to form liposomes;

(c) removing TEA-derived materials from the exterior of the liposomes₈An ion of SOS;

(d) contacting the liposomes with a solution prepared using irinotecan hydrochloride trihydrate at a temperature of 60-70 ℃ to form a liposome formulation encapsulating about 500g (± 10%) irinotecan per mole of total liposomal phospholipids;

(e) removing TEA-derived materials from the exterior of the liposomes₈(ii) SOS and irinotecan-containing substances; and

(f) the pH of the composition was adjusted to about 7.3.

Embodiment 20: the liposomal irinotecan composition of any of the preceding embodiments, which collectively comprises less than 100ppm of TEA.

Embodiment 21: the liposomal irinotecan composition of any of the preceding embodiments, which collectively comprises 30-100ppm of TEA or DEA.

Embodiment 22: the liposomal irinotecan composition of any one of the preceding embodiments, wherein at least 98% of the irinotecan is encapsulated in the irinotecan liposome after 6 months of storage at about 4 ℃.

Embodiment 23: the liposomal irinotecan composition of any of the preceding embodiments, comprising in the irinotecan liposome an irinotecan composition of formula (I) wherein x is 8:

examples

The synthesis and characterization of several irinotecan liposomal formulations is described in the examples below. Unless otherwise indicated in the examples, these irinotecan liposomes can be obtained by the following multistep process. The invention therefore also provides a process for the preparation of liposomes of irinotecan, and variants and combinations thereof, which process complies with the preparation process described in this section and in the examples.

First, liposome-forming lipids are dissolved in heated ethanol. These lipids include DSPC, cholesterol and MPEG-2000-DSPE. Unless otherwise indicated, DSPC, cholesterol and MPEG-2000-DSPE are present in a molar ratio of 3:2: 0.015. The resulting ethanol-lipid composition was dispersed in an aqueous medium comprising substituted ammonium and polyanion under the following conditions: effective to form substantially unilamellar liposomes of suitable size (e.g., 80-120nm or 95-115nm, etc.) comprising substituted ammonium ions and a polyanionic capture agent (SOS). Liposome dispersions can be formed, for example, as follows: mixing the ethanolic lipid solution with an aqueous solution comprising substituted ammonium ions and polyanions at a temperature above the lipid transition temperature (e.g. 60-70 ℃), and then extruding the resulting lipid suspension (multilamellar liposomes) under pressure through one or more rows of undercutsEngraved, for example, polycarbonate membrane filters having a defined pore size, for example 50nm, 80nm, 100nm or 200 nm. Preferably the substituted ammonium is protonated Triethylamine (TEA) or Diethylamine (DEA) and the polyanion is Sucrose Octasulfate (SOS), preferably in stoichiometric combinations (e.g. TEA)₈SOS)。TEA₈The concentration of SOS can be selected based on the amount of irinotecan loaded into the liposomes (e.g., to substantially or completely deplete the concentration loading gradient on the liposomes, and/or to provide liposomes comprising about a 1:8 molar ratio of SOS to irinotecan). For example, for the preparation of irinotecan SOS liposomes with 471g or 500g of irinotecan moieties per mol of phospholipid, TEA is preferably used₈The concentration of SOS is about 0.4-0.5M sulfate groups (e.g., 0.45M or 0.475M sulfate groups, or 0.45M or 0.475M SOS). All or substantially all of the non-entrapped TEA or SOS is then removed (e.g., by gel filtration, dialysis, or ultrafiltration/diafiltration).

The resulting trapping agent liposomes (e.g., encapsulating a substituted ammonium compound, such as TEA) are then subjected to conditions effective to load irinotecan into the trapping agent liposomes (i.e., under conditions that allow irinotecan to enter the liposomes to exchange with TEA exiting the liposomes)₈SOS or DEA₈SOS) with irinotecan solution. Irinotecan-loaded solutions (e.g., 15mg/ml of anhydrous irinotecan-HCl, which can be prepared using corresponding amounts of irinotecan-HCl trihydrate) preferably contain an osmotic agent (e.g., 5% glucose) and have a pH of 6.5 (unless otherwise stated, the pH values referred to in this specification are determined at room temperature). Drug loading is facilitated by increasing the temperature of the composition above the transition temperature of the liposome lipids (e.g., to 60-70 ℃) to accelerate transmembrane exchange of the substituted ammonium compound (e.g., TEA) and irinotecan. In some embodiments, irinotecan sulfatide is in a gel state or a precipitated state in the liposome.

Irinotecan is preferably loaded by continuing transmembrane exchange of the liposomes with a substituted ammonium compound (e.g., TEA or DEA) until all or substantially all of the substituted ammonium compound (e.g., TEA) is removed from the liposomes, thereby depleting the concentration gradient of all or substantially all of the transmembrane liposomes. Preferably, the irinotecan liposome loading process continuesThis is continued until the gram equivalent ratio of irinotecan to SOS is at least 0.9, at least 0.95, 0.98, 0.99, or 1.0 (or a range of about 0.9-1.0, 0.95-1.0, 0.98-1.0, or 0.99-1.0). Preferably, the irinotecan liposome loading process is continued until at least 90%, at least 95%, at least 98%, or at least 99% or more of the TEA is removed from the interior of the liposome. In some embodiments of the invention, TEA is used in this way₈SOS prepared irinotecan SOS liposome compositions contain less than 100ppm TEA. In some embodiments of the invention, TEA is used in this way₈SOS prepared irinotecan SOS liposome compositions contain 20-100ppm, 20-80ppm, 40-80ppm, or 40-100ppm TEA.

The extracellularly irinotecan and substituted ammonium compounds (e.g., TEA or DEA) can be removed to yield the final irinotecan liposomal product. This removal can be facilitated by a variety of methods, non-limiting examples of which include gel (size exclusion) chromatography, dialysis, ion exchange, and ultrafiltration/diafiltration. The liposomal external medium is replaced with an injectable pharmaceutical fluid, such as buffered (pH 7.1 to 7.5, preferably pH 7.2 to 7.3) isotonic saline. Finally, the liposome composition is sterilized, e.g., by 0.2 micron filtration, dispensed into single dose vials, labeled, and stored, e.g., refrigerated at 2-8 ℃, until use. The liposomal external medium can be replaced with a pharmaceutical fluid while removing residual liposomal irinotecan and ammonium/substituted ammonium ions (e.g., TEA).

Quantitative capture agents

For the purposes of the present invention, a liposome trapping agent and a substituted ammonium compound counterion (e.g., TEA)₈SOS) was calculated based on concentration quantification for liposome preparation and based on the number of sulfate groups of the capture reagent. For example, 0.1M TEA₈SOS will be referred to herein as 0.8M/L sulfate because there are eight sulfate groups per SOS molecule. In the case where different capture agents are used, this calculation will be adjusted depending on the number of anionic groups (e.g., sulfate groups) per molecule of capture agent.

Quantification of lyso-PC in irinotecan liposomal formulations

The numbers of lyso-PCs tested in the irinotecan sucrose octasulfate liposome formulation the data in figures 11B and 12 were obtained by the HPLC method described in example 9 ("method a").

Using a different preparative (TLC) method (herein, "method B"), lyso-PC measurements were obtained from samples 1-23 herein, and lysophospholipids were determined by the following TLC method followed by phosphate analysis rather than the HPLC method (method a) discussed immediately above. lyso-PC was measured by method B following the following procedure. Aliquots of liposome samples containing approximately 500nmol of Phospholipid (PL) (e.g., 0.05mL of a 10mM PL liposome solution) were desalted using a PD-10 column (GE Healthcare) equilibrated with water. The sample was eluted from the column with water, divided into three portions each containing about 150nmol of PL, and then dried under vacuum using a centrifugal Concentrator (Savant Speed Vac Concentrator, Model # SVC 100X). The dried lipids were dissolved in 30 μ l chloroform/methanol (5/1, vol/vol) and applied to the non-adsorption area of a normal phase silica gel TLC plate (Uniplate by Analtech, cat. No. 44921) using a glass syringe. TLC was run by mobile phase consisting of chloroform/methanol/30% ammonium hydroxide/water (60/40/2.5/3.75, v/v/v/v) and showed lipids using iodine vapor. PL was measured as follows: spots on TLC corresponding to phospholipids and lysophospholipids were divided into separate 12 x 75mm borosilicate tubes for subsequent phosphate analysis.

Quantification of the molar amount of liposomal co-encapsulated irinotecan and sulfate compounds is provided in the examples.

Material

For samples 1-5 and 13 in preparative example 1 and samples 12 and 14-18 in example 2, USP GMP grade irinotecan hydrochloride ((+) -7-ethyl-10-hydroxycamptothecin 10- [1,4 '-bipiperidine ] -1' -carboxylate, monohydrochloride, trihydrate, CAS registry No. 100286-90-6) was purchased from SinoPharm (Taipei, Taiwan); 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and methoxy-terminated polyethylene glycol (MW-2000) -distearoyl phosphatidylethanolamine ((MPEG-2000-DSPE) from Avanti Polar Lipids (Alabaster, AL, USA); ultra-pure cholesterol (Chol) from Calbiochem (La Jolla, CA, USA); and sucrose octasulfate from Euticals (Lodi, Italy).

For samples 6-11 of preparative example 1, irinotecan hydrochloride trihydrate was obtained from pharmaengine (taiwan); 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and methoxy-terminated polyethylene glycol (MW-2000) -distearoyl phosphatidylethanolamine ((MPEG-2000-DSPE) from Avanti Polar Lipids (Alabaster, AL, USA); ultra-pure cholesterol (Chol) from Calbiochem (La Jolla, CA, USA); and sucrose octasulfate from Euticals (Lodi, Italy).

For samples 19-23 in preparative example 8, Vinorelbine (VNB) was obtained as a 10mg/mL solution of vinorelbine tartrate (Glaxo-SmithKline) from the pharmacy, and topotecan (TPT) powder was obtained as a gift from the Taiwan Liposome Company (Taipei, Taiwan).

All other analytically pure or purer chemicals were obtained from common suppliers.

The method comprises the following steps: samples 1-5 and 13 (example 1) and samples 6-11 and 19-23 (example 2) and samples 12 and 14-18 (example 3) were prepared using the following methods, unless otherwise noted below.

Triethylammonium sucrose octasulfate formulations

Triethylammonium sucrose octasulfate (TEA)₈SOS) and diethylammonium sucrose octasulfate (DEA)₈SOS) was prepared from the sodium salt of sucrose octasulfate using ion exchange chromatography. Briefly, 15g of sucrose octasulfate (sodium salt) was dissolved in water to give a sulfate concentration of 2.64M. The acidic form of sucrose octasulfate was prepared using Dowex 50W-8X-200 cation exchange resin. The defined resin was washed twice with 2 volumes of 1N NaOH and then with ddH₂O (double distilled water) to neutral pH, twice with 2 volumes of 1N HCl and finally with ddH₂The O wash is neutral and then repeated. The column was poured onto a resin of 450mL volume and washed with 3 volumes of 3N HCl, then ddH₂And O washing until the conductivity reaches less than 1 mu S/cm. Sucrose octasulfate (sodium salt) solution (about 10% column capacity) was loaded onto the column and treated with ddH₂And (4) eluting with O. Column eluent is usedThe elution of sucrose octasulfate from the column was monitored with a conductivity detector. The acidic sucrose octasulfate is then titrated with triethylamine or diethylamine to a pH of 6-7 and the sulfate content is determined using a method modified from B.Sorbo et al, Methods in Enzymology,143:3-6,1984 (see sulfate determination). The solution was finally diluted to a sulfate concentration corresponding to 0.65M sulfate. The pH is generally in the range of 6 to 7. Residual sodium was measured using a sodium electrode, and no more than any solution of 1 mol% residual sodium was used.

Determination of sulfate group

The sulfate content of the sucrose octasulfate solution was determined by nephelometry. The solution consisted of the following: (1) 15g PEG 6000 and 1.02g barium acetate in 100mL water; (2) 142mg sodium sulfate in 1mL water; (3) barium working solution: 0.1mL of sodium sulfate solution was added dropwise to 100mL of barium solution while stirring. This solution should be equilibrated for 1 hour before use and can be stored for no more than one week; (4)0.4M trisodium citrate solution; (118mg trisodium citrate/mL water); and (5) 10mM sulfate standard diluted from 1N sulfuric acid in water. Standards and solutions were made to a final volume of 100 μ l using borosilicate tubes. Standards were prepared in the range of 0.2-1. mu. mol sulfate (20-100. mu.l of 10mM standard). For the 0.6M sulfate solution sample, 1/100 and a dilution (0.6. mu. mol) with a volume of 100. mu.l were used. Each 100. mu.l sample/standard was treated with 100. mu.l 70% perchloric acid and heated at 110 ℃ and 120 ℃ for 12 minutes. After cooling, 0.8mL of 0.4M trisodium citrate solution was added, followed by vortexing. A volume of 0.25mL of the barium working solution from the stirring was transferred to each tube and immediately vortexed. All samples/standards were allowed to equilibrate for 1 hour, then vortexed and absorbance at 600nm measured. Using SO₄Determination of unknown SO from a linear standard curve of concentration versus OD600₄And (4) concentration.

Sucrose octasulfate assay by HPLC

The concentration of sucrose octasulfate (mg/mL) in a sample can be calculated based on the area of the sucrose octasulfate peak produced by a standard of known concentration. The calculated sucrose octasulfate concentration is then used to calculate the sulfate concentration (mM) in the sample.

The samples to be analyzed were chromatographed by HPLC using Phenomenex, Bondclone 10. mu.NH₂300X 3.90mm, PN 00H-3128-C0, or Waters μ Bondapak NH₂ 10μm(3.9 mm. times.300 mm), Part No. WAT084040 using a 0.60M ammonium sulfate mobile phase, pH3.0 eluting at 1.00mL/min at 40 ℃ column temperature. The sample is detected by a refractive index detector, also at 40 ℃, for example using Agilent's HPLC and refractive index detector. USP sucrose octasulfate potassium heptahydrate was used as reference standard; CAS 76578-81-9, accession number 1551150.

SOS assay standards and assay control samples were integrated using baseline versus baseline integration. The EA-SOS samples were then integrated using the baseline for baseline integration. This can be done by manually starting the baseline before the void volume trough to the end of the SOS tail, and then dropping a line at the beginning of the TEA peak and at a low point between the two peaks. Note that: if a single baseline starting before the void volume valley to the end of the SOS tail crosses the low point between the TEA and SOS peaks, two separate lines can be used that approximate the approach to the baseline relative to the baseline. The TEA-SOS sample showed a TEA peak at a relative retention time of about 0.45 relative to the retention time of the SOS peak.

Drug analysis

HPLC analysis of irinotecan was performed on a Dionex system using C₁₈Reverse phase silica gel column (Supelco C)₁₈Column, internal diameter 250 mm. times.4 mm, particle size 5 μm), preceded by Supelco C₁₈And (4) protecting the column. A sample injection volume of 50. mu.l was used and the column was eluted at a constant rate of 1.0mL/min using a mobile phase consisting of 0.21M aqueous triethylammonium ammonium acetate (pH 5.5) and acetonitrile (73:27, v: v). Irinotecan and SN-38 typically elute at 5.1 minutes and 7.7 minutes, respectively. Irinotecan was detected by absorbance at 375nm using a diode array detector and SN-38 was detected by fluorescence (370nm excitation and 535nm emission).

Phosphate determination

The following phosphate determination method was used to analyze samples 1-23. A modified Bartlett phosphate assay can be used to measure Phospholipids (PL). Phosphate standards in the range of 10-40nmol were placed in 12X 75mm borosilicate tubes and treated correctly as samples. Sulfuric acid (100. mu.l of 6M H)₂SO₄) Added to each tube placed in a heating block and heated to 180 ℃ for 45 minutes. Hydrogen peroxide (20. mu.l of a 30% solution) was added to each tube, followed by heating at 150 ℃ for 30 minutes. Ammonium molybdate (0.95mL of a 2.2g/l solution) and ascorbic acid (50. mu.l of a 10% aqueous solution) were then added to each tube. After vortexing, the tubes were developed in boiling water for 15 minutes and then cooled to room temperature. For lysolipid analysis using Thin Layer Chromatography (TLC), silica was precipitated by centrifugation at 1000rpm for 5 minutes, and the blue color in the supernatant was measured by reading the absorbance at 823 nm. Samples without silica may exclude a centrifugation step.

Drug retention and stability

The stability (in terms of drug retention) of liposomal irinotecan was determined by separating liposomal irinotecan from extracellularly-irinotecan using a PD-10(Sephadex G-25) size exclusion column. Drug leakage was determined by comparing the ratio of irinotecan (HPLC) to PL (described in the phospholipid assay) before and after isolation of irinotecan outside the liposome. The degradation of irinotecan was determined by observing additional peaks in the chromatogram after HPLC analysis. The irinotecan-to-phospholipid ratio and the drug encapsulation efficiency were calculated using the following formulas 1 and 2, respectively.

(1) Irinotecan to phospholipid ratio[ irinotecan](mg/mL)×1000

(g irinotecan/mol PL) [ phospholipid ] (mM)

(2) Encapsulation efficiency (%) ═(ratio of irinotecan to phospholipid) AC

(ratio of irinotecan to phospholipid) BC

Where (ratio of irinotecan to phospholipid) AC is the ratio of drug to phospholipid after purification on a G-25 size exclusion column, (ratio of irinotecan to phospholipid) BC is the ratio of drug to phospholipid before purification on the column.

Measurement of Encapsulated and free irinotecan in Liposome compositions

The liposome encapsulation and free (unencapsulated) irinotecan in the irinotecan sulfatide liposome compositions of examples 3 and 4 were determined using the column (cartridge) adsorption method. Oasis 60mg 3cc HLB column (Waters) was conditioned sequentially as follows: 2mL of methanol, 1mL of HEPES-buffered saline (HBS; 5mM HEPES, 140mM NaCl, pH6.5) and 0.5mL of human serum albumin in 10% physiological saline, followed by 1mL of HBS. The liposomal irinotecan sulfatide composition was diluted to about 2.2mg/mL irinotecan with physiological saline and a 0.5mL aliquot was applied to the cassette. The eluate was collected, the column was washed with two portions of HBS (1.5mL, 3mL), and the wash and eluate were combined to make a liposome fraction. The column was additionally washed with 1.5mL HBS and eluted with two 3mL methanol-HCl portions (90 vol% methanol, 10 vol% 18mM HCl). The eluates are combined to prepare a free drug fraction. The liposome drug fraction was transferred to a 25mL volumetric flask, the free drug fraction was transferred to a 10mL volumetric flask, methanol-HCl was added to the scale, mixed well, and the flask of liposome fraction was heated at 60 ℃ for 10 minutes to dissolve the drug. After cooling, the solution was filtered and irinotecan in both fractions was quantified using reverse phase HPLC on a Phenomenex Luna C18(2) column, eluted with 20mM potassium phosphate ph3.0 methanol mixture (volume ratio 60:40) etc. and UV detected at 254 nm. Drug peaks were integrated and the amount of irinotecan in the sample was calculated by comparison to a linear standard curve obtained using irinotecan hydrochloride trihydrate USP reference standard under the same conditions. Drug encapsulation efficiency was calculated as the percentage of encapsulated drug relative to the total amount of free and encapsulated drug in the sample.

pH measurement

The pH was always measured at ambient temperature (i.e., 20-25 ℃) using a potentiometric standard glass electrode method. By placing a glass electrode in the liposome formulation and taking a pH reading, the pH of the liposome formulation is measured accordingly.

TEA/DEA ppm of the sample analyzed

Sample analysis was performed by headspace Gas Chromatography (GC) separation as follows: gradient temperature elution was performed using a capillary GC column (50 m.times.0.32 mm. times.5. mu. mRestek Rtx-5 (5% phenyl-95% dimethylpolysiloxane)), followed by Flame Ionization Detection (FID). The sample formulation and the standard formulation were analyzed and the resulting peak area responses were compared. The amount of residual amine (e.g., TEA or Diethylamine (DEA)) is quantified using external standards. In the case of TEA, the standard is 99% or more. Other reagents include triethylene glycol (TEG), sodium hydroxide, and Deionized (DI) water.

The GC conditions were: carrier gas: helium gas; column flow rate: 20cm/sec (1.24 mL/min); the split ratio is as follows: 0:1 (adjustable as long as all system applicability criteria are met); injection mode: split 0: 1; lining: 2mm straight grooves (suggested but not required); injection port temperature: 140 ℃, detector temperature: 260 ℃ (FID); initial column oven temperature: 40 ℃; column oven procedure:

run time of 54 minutes

Headspace parameters: platform temperature: 90 ℃; sample circulating temperature: 100 ℃; transmission line temperature: 100 ℃; the balance time is as follows: 60 minutes; injection time: 1 minute; vial pressure: 10 psi; pressurizing time: 0.2 minutes; shaking: (iii) performing (medium); sample introduction amount: 1.0mL headspace; GC cycle time: 60 minutes (recommended but not required).

If TEA is not detected, then "not detected" is reported; if TEA results <30ppm, reported as < QL (30 ppm); the results for TEA were > 30ppm and reported as integers.

Determination of liposome size

Liposome particle size measurement Using Dynamic Light Scattering (DLS) Using Malvern Zeta Sizer Nano ZS^TMOr similar apparatus in aqueous buffer (e.g., 10mM NaCl, pH 6.7) at 23-25 deg.C using a cumulative methodAnd (6) measuring. The z-average particle size and polydispersity index (PDI) are reported. Instrument performance was verified using the nanosphere NIST provenance standard for 100nm polymers (Thermo Scientific 3000 series nanosphere size standard P/N3100A or equivalent analytical certificate including Hydrodynamic Diameter). As used herein, "DLS" refers to dynamic light scattering and "BDP" refers to bulk drugs.

Example 1: effect of SOS Capture agent concentration and pH on storage stability of Liposomal irinotecan formulations

The objective of this study was to determine in particular any change in physical and chemical stability of liposomes encapsulating irinotecan and a Sucrose Octasulfate (SOS) trapping agent when stored for a certain period of time at about 4 ℃. For this study, the liposomal SOS capture agent concentration was reduced while the proportion of phospholipids per total moles of 471 grams of irinotecan moiety was maintained.

A series of irinotecan SOS liposomal formulations were prepared in a multi-step process using different concentrations of SOS trapping agent and adjusting the pH of the final liposomal formulation to different pH values. Irinotecan SOS liposome formulations each contained an irinotecan moiety at a concentration equivalent to 5mg/mL irinotecan hydrochloride trihydrate. The irinotecan SOS liposomal formulations of samples 1-5 and 13 were prepared by the multi-step method of example 1.

The amounts of DSPC, cholesterol (Chol) and PEG-DSPE weighed correspond to a 3:2:0.015 molar ratio (e.g., 1264mg/412.5mg/22.44mg), respectively. The lipids were dissolved in chloroform/methanol (4/1, v/v), mixed well and divided into 4 aliquots (a-D). Each sample was evaporated to dryness at 60 ℃ using a rotary evaporator. Residual chloroform was removed from the lipids by placing under vacuum (180 μ torr) at room temperature for 12 hours. Dissolving dried lipid in ethanol at 60 deg.C, adding preheated TEA with appropriate concentration₈SOS, to a final alcohol content of 10%. The lipid concentration was about 75 mM. Lipid dispersions were extruded through 2 stacked 0.1 μm polycarbonate membranes (Nuclepore) at about 65 ℃ using a Lipex thermo barrel extruder (Northern Lipids, Canada)^TM)10 times to produce liposomes with typical average diameters of 95-115nm (determined by quasielastic light scattering; see section "determination of liposome size"). pH of the extruded liposomes is based onAdjustments are needed to correct for pH variations during extrusion. Liposomes are purified by a combination of ion exchange chromatography and size exclusion chromatography. First, Dowex^TMThe IRA910 resin was treated with 1N NaOH, followed by 3 washes with deionized water, then 3N HCl 3 times, then multiple washes with water. Liposomes were passed through the prepared resin and the conductivity of the eluted fractions was measured by using a flow cell conductivity meter (Pharmacia, Uppsala, Sweden). If the conductivity is less than 15. mu.S/cm, the fraction is considered acceptable for further purification. The liposome eluate was then applied to a Sephadex G-75(Pharmacia) column equilibrated with deionized water, and the conductivity of the collected liposome fraction was measured (typically less than 1. mu.S/cm). Transmembrane isotonicity is achieved as follows: a40% glucose solution was added to a final concentration of 5% (w/w), and a buffer (Hepes) was added from a stock solution (0.5M, pH6.5) to a final concentration of 10 mM.

Stock solutions of irinotecan were prepared as follows: irinotecan-HCl trihydrate powder was dissolved in deionized water to give anhydrous irinotecan-HCl of 15mg/mL, taking into account the water content and impurity content obtained in the certificate of analysis of each batch. Drug loading begins as follows: irinotecan HCl anhydrous (corresponding to 471g irinotecan anhydrous free base) was added in an amount per mole of liposomal phospholipid and heated to 60 ± 0.1 ℃ for 30 minutes in a hot water bath. The solution was removed from the water bath and rapidly cooled by immersion in ice-cold water. The extra-liposomal drug was removed by size exclusion chromatography using a Sephadex G75 column equilibrated and eluted with Hepes buffered saline (10mM Hepes, 145mM NaCl, pH 6.5). Samples were analyzed for irinotecan by HPLC and for phosphate by the Bartlett method (see section "phosphate assay"). For storage, samples were divided into 4mL aliquots and pH adjusted using 1N HCl or 1N NaOH, sterile filtered under sterile conditions, and filled into sterile clear glass vials with argon gasThe lining screw cap was sealed and placed in a 4 ℃ thermostatically controlled ice box. At defined time points, remove from each sampleAliquots were aliquoted and tested for appearance, liposome size, drug/lipid ratio, and drug and lipid chemical stability.

For example 1, liposome size distribution was determined by dynamic light scattering in diluted samples using a Coulter Nano-Sizer at a 90 degree angle and expressed as mean. + -. standard deviation (nm) from the cumulative method.

The liposomal formulations of irinotecan of samples 1-5 and 13 were further obtained as follows. The freshly extruded liposomes comprised two groups, each incorporating TEA at the following concentrations₈SOS as capture agent: (A)0.45M sulfate group (112.0. + -. 16nm), (B)0.475M sulfate group (105.0. + -. 16nm), (C)0.5M sulfate group (97. + -.30 nm), and (D)0.6M sulfate group (113. + -.10 nm). In the description of example 1, samples 1-5 and 13 were loaded at an initial ratio of 471g irinotecan anhydrous free base per mole of total liposomal phospholipid and purified as described above (equivalent to 500g irinotecan HCl anhydrous). Samples 1, 5 and 13 were derived from the extruded sample (a); sample 2 is from extruded sample (B); samples 3 and 4 were from extruded samples (C) and (D), respectively. After purification, pH adjustment was performed using 1N HCl or 1N NaOH, followed by sterilization and filling of the vials. Data from samples 1-5 are shown in table 7 (example 1) and data from sample 13 are shown in table 8 (example 2).

The liposomal formulation of irinotecan of samples 6-11 was further obtained as follows. The freshly extruded liposomes comprised two groups, each incorporating TEA at the following concentrations₈SOS as capture agent: (A)0.45M sulfate group (116. + -. 10nm) and (B)0.6M sulfate group (115.0. + -. 9.0 nm). Samples 6-8 were derived from extruded sample (A) and samples 9-11 were derived from extruded sample (B). After purification, the pH is adjusted, if necessary, by the appropriate addition of 1N HCl or 1N NaOH. Sample 12 was prepared as described in example 2 and is included in table 7 for comparison purposes.

For certain irinotecan liposome compositions, irinotecan liposomes having an in vitro pH, irinotecan free base concentration (mg/mL), and various concentrations of sucrose octasulfate are listed in table 6 below (stored at 4 ℃ for 6 months) and table 7 below, and are prepared according to the more details described herein.

Figures 4A-4C are graphs showing mol% lyso-PC in irinotecan liposomal formulations with a pH greater than 6.5 (i.e., 7.25 or 7.5 shown in each figure) selected from table 7. hemolysis-PC was determined after the first 1 month, the first 3 months, the first 6 months and/or the first 9 months of storage of each sample at 4 ℃ by method b (tlc) as disclosed herein. These figures include a linear regression line of the data for each sample as an estimate of the rate of increase (mol%) of lyso-PC in each sample over time. Surprisingly, increasing the pH of the irinotecan liposome formulation so that it is greater than 6.5 (e.g., 7.25 and 7.5) reduces the amount of lyso-PC measured during refrigerated storage at 4 ℃ compared to irinotecan liposomes formed at comparable stability ratios. This tendency is evident at various concentrations of liposomal irinotecan. For example, with respect to liposomal irinotecan compositions prepared at an intensity of about 4.3mg irinotecan fraction/mL, the measured values for mol% lyso-PC levels in samples 5 and 7 were significantly lower at all data points (after 1 month, 6 months and 9 months prior to storage at 4 ℃ after preparation) compared to the mol% lyso-PC levels in sample 1 measured at pH6.5 (data in table 7). Similarly, for liposomal irinotecan compositions prepared at an intensity of about 18.8mg irinotecan fraction/mL, the mol% hemolytic-PC levels measured in sample 13 were significantly lower at all data points (after 1 month prior to storage at 4 ℃ after preparation and after 9 months prior) compared to the mol% hemolytic PC levels measured for sample 12 or sample 14 at pH6.5 (data in table 8).

TABLE 6 measurement of hemolysis-PC after 6 months of refrigerated storage

Additional results of the comparative stability study in example 1 are provided in table 7 below. mol% lyso-PC was determined after 1, 3, 6, 9 and/or 12 months of storage of the liposome formulation at 4 ℃ as shown in table 7. For each sample, table 7 provides the SOS concentration used to prepare the liposomes, expressed as the molar concentration of sulfate groups (one SOS molecule containing 8 sulfate groups). Unless otherwise indicated, all irinotecan liposomes in table 7 were prepared using a ratio of irinotecan moieties (as explained above, based on anhydrous free base) to total phospholipids of 471g of irinotecan moieties (equivalent to the amount of irinotecan moieties in 500g of anhydrous irinotecan HCl salt) per mole of total liposome phospholipids, respectively. Table 7 also contains the stability ratio of each sample, calculated as the ratio of 471g irinotecan moieties (based on anhydrous free base) per mole of phospholipid divided by the concentration of sulfate groups used to prepare the liposomes in mol/L. The liposomes of the samples described in table 7 each had a measured size (volume weighted average) of about 89-112nm and an irinotecan encapsulation efficiency of at least 87.6%. Encapsulation efficiency was determined according to the subsection "drug retention and stability".

Table 7: irinotecan liposome formulations (liposomal vesicles formed from 3:2:0.015 molar ratio of DSPC, cholesterol (Chol) and PEG-DSPE) with various stability ratios and pH^c

^cMeasured according to method B, as described herein.

The results of this storage stability study show that the concentration of SOS trapping agent (measured as the molar concentration of sulfate) used to prepare the liposomes decreased while the ratio of irinotecan anhydrous free base (in g) to total liposomal phospholipids (in mol) remained unchanged, resulting in greater storage stability of irinotecan SOS liposomes as measured by the detected amount of lyso-PC after cold storage of the irinotecan liposome formulation at 4 ℃ for 6 months and 9 months. In a liposome preparation prepared to pH6.5 (see "pH measurement" method described herein), reducing the SOS trapping agent concentration during liposome preparation resulted in a reduction in the detected amount of lyso-PC after storage of the liposome preparation at 4 ℃.

Without being bound by theory, it is believed that the interior space of the liposomes is acidified once purified by the extra-liposomal capture agent during the preparation process. This may be due to: TEA in the removal of liposomes₈The SOS is then redistributed from the amine component of the liposome internal to the external trapping agent salt of the liposome, where each occurrence dissociates hydrogen ions within the liposome. Drugs capable of protonation, such as irinotecan, are added, also partitioned between the outer and inner space of the liposome. Protonation of the drug distributed within the liposome and binding of the protonated drug to the thionate affects the loading of the drug within the liposome and results in a decrease in the intraliposomal concentration of both TEA and hydrogen ions, thereby reducing the degree of acidification within the liposome. In the case of irinotecan liposomes, it was hypothesized that at a drug loading of 500g irinotecan hydrochloride (i.e., 471mg irinotecan) per mol of liposomal phospholipid (where the sulfate concentration of the SOS is 0.6M), TEA depletion in the excess liposomes was incomplete. While not the basis for retaining the drug in the liposomes, this may provide an acidic liposome interior, which may help degrade the drug and lipid components of the liposomes, as seen in samples 7 and 13. In contrast, samples 8 and 5 had the same drug loading of 500g irinotecan hydrochloride (i.e., 471mg irinotecan moiety) per mole, but had lower SOS concentrations of 0.45M sulfate and 0.475M sulfate, respectively. In these particular cases, the level of lysolipid measurement is lower. Finally, it is clear that the most stable liposome formulations combine a higher drug/capture agent ratio with a higher external pH (i.e. pH 7.25).

The irinotecan liposomes of samples 1-11 retain good colloidal stability at 4 ℃ for up to 9 months as judged by the absence of precipitation and a relatively narrow and reproducible particle size distribution, where the concentration of the irinotecan moiety corresponds to 4.71mg/mL irinotecan anhydrous free base. Irinotecan is effectively and stably entrapped during long-term storage and has minimal leakage (< 10%) (see the "drug retention and stability" methods described herein).

Samples 1 and 2 had the same initial loading of about 471g irinotecan moieties (based on anhydrous free base, as explained above) per mole of phospholipid, but lower concentrations of SOS of 0.45M sulfate groups and 0.475M sulfate groups, respectively. Similarly, samples 6,7 and 8 had a lower SOS concentration of 0.45M sulfate but the same drug loading of 471g irinotecan moieties (based on anhydrous free base) per mol phospholipid as explained above, resulting in significantly lower lyso-lipid content (7-17% after 9 months).

The level of lyso-PC measured for the samples at pH6.5 increased regardless of drug loading or capture agent concentration during liposome preparation, with some samples (1,2 and 3) reaching up to 35 mol% phospholipid. Adjusting the pH to 7.25 makes the liposomes less prone to forming lyso-PC at levels up to 9.72% of total PC (e.g. comparing the levels of lyso-PC in samples 1 and 13). The samples containing higher concentration ratios of drug to capture agent and higher pH values formed less lyso-lipids, which contained 7-8 mol% of-lipids after 9 months, as seen in samples 7 and 8. The combination of a higher drug capture agent ratio and a higher pH (e.g., as compared to sample 12) reduces lyso-lipid formation. The most stable liposome formulation combines a higher drug/capture agent ratio (i.e. stability ratio greater than 942 defined with respect to the amount of irinotecan free base) and a higher external pH greater than 6.5 (e.g. compare samples 1 and 13).

In addition, the irinotecan liposome formulations 1-11 had a% SN38 measurement of no greater than about 0.05% SN38 (i.e., the relative amount of SN38 to irinotecan and SN 38) within 9 months, while the sample 12 irinotecan liposome formulation had an SN38 measurement of 0.20-0.50% over the same time period (as determined by the "drug analysis" method described herein). In each of samples 1-5 and 13, irinotecan was stably entrapped by the liposomes, with low leakage rates (less than 13%; determined by the "drug retention and stability" method described herein) and low conversion to the active cytotoxin SN-38, i.e., less than 0.1%, and less than 0.05% in the higher pH (7.25) stored samples.

Example 2: increasing the concentration of irinotecan liposomes in liquid formulations

The purpose of this storage stability study was to determine any change in the physical and chemical stability of liposomal irinotecan SOS upon storage at 4 ℃. During this study, the concentration of Sucrose Octasulfate (SOS) capture reagent used to prepare liposomes was maintained at a sulfate group concentration of 0.65M while varying: (1) preparation of irinotecan liposomes (using TEA)₈SOS or DEA₈Initial counter ion of SOS trapping agent during SOS), (2) amount of irinotecan anhydrous free base (in g) to phospholipid (in mol) (about 471g or 707g of irinotecan moiety (based on anhydrous free base, as explained above) per mole of phospholipid), (3) concentration of irinotecan anhydrous free base in the liquid irinotecan formulation (4.7mg/mL or 18.8mg/mL of irinotecan encapsulated in the liquid irinotecan liposome formulation (based on the concentration of the equivalent irinotecan moiety from irinotecan hydrochloride trihydrate)), (4) adjustment of the pH of the irinotecan liposome formulation (pH 6.5 or 7.25), and (5) buffer of irinotecan liposome formulation (HEPES or histidine).

Formulation parameters studied included: liposome size, drug to phospholipid ratio in irinotecan liposomes, irinotecan drug encapsulation efficiency and general appearance, presence of irinotecan degradation products and lyso-PC (in mol%) formation.

A series of irinotecan SOS liposomal formulations were prepared in a multi-step process using different concentrations of SOS trapping agent relative to the encapsulated irinotecan, and the pH of the final liposomal formulation was adjusted to different pH values. The amounts of DSPC, cholesterol (Chol) and PEG-DSPE weighed out correspond to a 3:2:0.015 molar ratio (730.9mg/238.5mg/13.0mg), respectively. The lipids were dissolved in chloroform/methanol (4/1, v/v), mixed well and divided into 2 aliquots. Each sample was evaporated to dryness using a rotary evaporator at 60 ℃. Residual chloroform was removed from the lipids by placing under vacuum (180 μ torr) at room temperature for 12 hours. Dissolving dried lipid in ethanol at 60 deg.C, adding preheated TEA₈SOS or DEA₈SOS (concentration of sulfate groups of 0.65M) resulted in a final ethanol content of 10% (v/v) and samples designated A and B, respectively. The lipid concentration was about 75 mM. The lipid dispersion being extruded through 0.1 μmPolycarbonate membrane (Nuclepore)^TM)10 times to produce liposomes with typical mean diameters of 95-115 nm. The pH of the extruded liposomes was adjusted as needed (with 1N NaOH) to the pH of the selected formulation. Liposomes are purified by a combination of ion exchange chromatography and size exclusion chromatography. First, Dowex^TMThe IRA910 resin was treated with 1N NaOH, followed by 3 washes with deionized water, then 3N HCl 3 times, then multiple washes with water. The conductivity of the eluted fractions was measured by using a flow cell conductivity meter (Pharmacia, Uppsala, Sweden). If the conductivity is less than 15. mu.S/cm, the fraction is considered acceptable for further purification. The liposome eluate was then applied to a Sephadex G-75(Pharmacia) column equilibrated with deionized water, and the conductivity of the collected liposome fraction was measured (typically less than 1. mu.S/cm). Transmembrane isotonicity is achieved as follows: a40% glucose solution was added to a final concentration of 5% (w/w), and a buffer (Hepes) was added from a stock solution (0.5M, pH6.5) to a final concentration of 10 mM.

Stock solutions of irinotecan were prepared as follows: 326.8mg of irinotecan-HCl trihydrate powder was dissolved in 20.0mL of deionized water to give 15mg/mL of anhydrous irinotecan-HCl, taking into account the water content and impurity content obtained in the certificate of analysis of each batch. Drug loading begins as follows: irinotecan anhydrous free base was added at 500g/mol or 750g/mol phospholipid and heated to 60 + -0.1 deg.C for 30 minutes in a hot water bath. The solution was rapidly cooled after removal from the water bath by immersion in ice-cold water. The in vitro liposome drug was removed by size exclusion chromatography using a Sephadex G75 column equilibrated and eluted with Hepes buffered saline (10mM) (HBS) (pH 6.5 for sample a, histidine buffered saline pH 7.25 for sample B). Samples were analyzed for irinotecan by HPLC and for phosphate by the Bartlett method (see phosphate assay).

For storage, the samples were divided into 4mL aliquots and pH adjusted as needed using 1N HCl or 1N NaOH, sterile filtered under sterile conditions, and filled into sterile clear glass vials under argon withThe lining screw cap was sealed and placed in a 4 ℃ thermostatically controlled ice box. At the specified time points, aliquots were taken from each sample and tested for appearance, size, drug/lipid ratio, and drug and lipid chemical stability.

Liposome size was determined by dynamic light scattering in diluted samples using a Coulter Nano-Sizer at a 90 degree angle and expressed as the mean. + -. standard deviation (nm) obtained by the cumulative method.

The results from the comparative stability study are provided in Table 8 (for the use of TEA)₈Samples prepared using SOS trap starting material) and table 9 (for using DEA)₈Samples prepared from SOS trap feedstock).

Table 8: using TEA₈Liposome of irinotecan (10mM) prepared in Hepes buffer with SOS trapping agent^d

^dMeasured according to method B, as described herein.

Sample 13 (example 2, table 8) was stored at a concentration 4 times greater (20mg irinotecan/mL) than samples 1-5 (example 1) and still maintained good colloidal stability with no observable aggregation or precipitation.

Table 9: at pH 7.25 with DEA having a sulfate group concentration of 0.65M₈Irinotecan liposome prepared from SOS capture agent^e

^eMeasured according to method B, as described herein.

Freshly extruded liposome size encapsulation (A)0.6TEA of 5M sulfate₈SOS (113.0 + -23.8 nm) or (B) DEA of 0.65M sulfate group₈SOS (103.2. + -. 21.1nm) (the only exception being sample 13, which has 0.45M sulphate groups). Samples 12 and 14 from (a) and samples 15-18 from sample (B) were obtained, where samples 12,14, 15 and 16 were loaded with 471g irinotecan anhydrous free base (equivalent to 500g irinotecan HCl anhydrous) per mole of total lipid phospholipids and samples 16-18 were loaded with 750g irinotecan moieties (as explained above, based on anhydrous free base) per mole of phospholipids. After purification, the pH is adjusted to pH6.5 or 7.25 using 1N HCl or 1N NaOH as appropriate as described in tables 7 and 8. Sample 12 was prepared as described in example 1 and included in table 8 for comparison purposes.

The data show that liposomes retain good colloidal stability at 4 ℃ for up to one year as judged by the absence of precipitation and a relatively narrow and reproducible particle size distribution. Secondly, it is clear that colloidal stability is also good for more concentrated samples when stored at high pH and high drug to phospholipid ratios, indicating that liposomes are stable and can prevent aggregate formation when irinotecan hydrochloride trihydrate at partial concentrations equivalent to 20mg/mL and 40mg/mL of irinotecan.

In all cases, irinotecan was stably entrapped in the liposomes, with low leakage rates and low conversion to the active cytotoxin SN-38 (i.e., relative amounts of SN38 compared to irinotecan and SN 38); less than 0.5 mol% in all cases, except sample 12, less than 0.1 mol% SN-38. Data were obtained from the "drug retention and stability" method and the "drug analysis" method described herein.

An increase in lyso-PC levels was measured in samples prepared having been adjusted to a pH of 6.5 and at a ratio of 471g irinotecan fraction (using an equivalent amount of 500g irinotecan HCl anhydrous as explained above) per mole of phospholipid, reaching 36-37 mol% (relative to total phosphatidylcholine) for samples 12 and 14, whereas adjusting the pH to 7.25 made the liposomes less prone to forming lyso-lipids, with the level of lyso-PC approaching only 11 mol% (relative to total phosphatidylcholine) after one year for sample 15.

Adjusting the pH of the liposomes from 6.5 to 7.25 did not adversely affect colloidal stability or drug leakage.

Example 3: storage stability of irinotecan liposomes stabilized at varying amounts of TEA (SOS trap counterion)

Irinotecan liposomes are prepared by loading irinotecan into liposomes encapsulating Sucrose Octasulfate (SOS) and a substituted ammonium counterion (such as protonated TEA). The effect of varying the residual amount of substituted ammonium in loaded irinotecan SOS liposomes was evaluated as follows: a plurality of irinotecan SOS liposomes containing varying amounts of entrapped residual substituted ammonium ions were prepared, stored refrigerated at 4 ℃ for 6 months, and then the amount of lyso-PC (in mol%) in the irinotecan SOS liposomes was measured.

The data show that reducing the amount of substituted ammonium ions in irinotecan SOS liposomes results in lower levels of lyso-PC after 6 months of refrigerated storage at 4 ℃. In particular, irinotecan SOS liposomes having less than 100ppm (e.g., 20-100ppm TEA) substituted ammonium exhibit lower levels of lyso-PC formation after 6 months of refrigerated storage at 4 ℃.

Six batches (samples 24-29) of liposomal irinotecan thionate were prepared according to certain embodiments of the present invention, according to the protocol described herein, with stability ratios of 1046-1064, and molar ratios of DSPC, cholesterol, and MPEG-2000-DSPE in the lipid composition of 3:2:0.015, respectively.

The amount of lyso-PC in table 10 was determined by HPLC (method a of the present application).

Table 10: irinotecan liposome formulation at pH 7.3 (irinotecan SOS encapsulated in vesicles formed from DSPC, cholesterol (Chol) and PEG-DSPE at a molar ratio of 3:2: 0.015)

^fMeasured according to method a, as described herein.

^gMeasured according to method a, as described herein.

Liposomes (100-115nm) were obtained as follows: lipids dispersed in TEA-SOS solution (0.4-0.5M sulfate) were extruded through 100-nm polycarbonate membrane (nucleocore), TEA-SOS was purified from the extracellosomal by tangential flow diafiltration buffer exchange osmotic equilibration of glucose solution, loaded with irinotecan by raising the temperature to 68 ℃, stirred for 30 minutes, rapidly cooled, and TEA and any non-encapsulated drug from the extracellosomal were purified by tangential flow diafiltration buffer exchange buffered physiological sodium chloride solution. The irinotecan sulfatase liposome composition was filter-sterilized by passing through a 0.2- μm membrane filter, aseptically dispensed into sterile glass vials, and incubated under refrigerated conditions (5 ± 3 ℃). At about 0, 3, 6, 9 and in some cases 12 months of refrigerated storage time, duplicate vials of each batch were removed and analyzed for the cumulative amount of lyso-PC using an HPLC method using an evaporative scattering detector. The liposome composition is also characterized by particle size, irinotecan and liposome phospholipid concentrations, the pH of the liposome composition, irinotecan/sulfatide gram-equivalent ratio (Iri/SOS ratio), and residual triethylammonium (protonated TEA) as triethylamine. Average particle size (D) and polydispersity index (PDI) by using Malvern Zeta Sizer NanoZS^TMThe DLS method of (1). The concentration of irinotecan in the liposome composition was determined by HPLC. Total phospholipids were determined by blue phosphomolybdate spectrophotometry after digestion of liposomes in a sulfuric acid/hydrogen peroxide mixture.

The drug/lipid (DL) ratio is calculated by dividing the amount of drug in g (as anhydrous free base) by the molar amount of liposomal phospholipids in the liposomal formulation. The SOS trapped by the liposomes was quantified after the liposomes were passed through a Sephadex G-25 gel chromatography column (PD-10, GE Healthcare) eluted with physiological saline. To determine the irinotecan/SOS gram-equivalent ratio, 0.1mL aliquots of the eluted liposome fractions were mixed in triplicate with 0.05mL 70% perchloric acid, hydrolyzed at 95-100 ℃ for 1 hour, neutralized with 0.8mL 1M sodium acetate, filtered to remove insoluble lipid products, and the amount of sulfate ester-derived sulfate groups in the filtrate was determined by nephelometry using barium-PEG reagent essentially as described under method. Three aliquots of another set of identical liposome eluents were dissolved in 70% acidified (0.1M HCl) aqueous isopropanol and irinotecan was assayed spectrophotometrically at 365 nm. The irinotecan/sulfatide gram-equivalent ratio (Iri/SOS ratio) in each eluted liposome fraction was calculated as follows: the measured molarity of the drug is divided by the measured molarity of the sulfate group. The pH was measured as described in the section "pH measurement". TEA was quantified as follows: the headspace Gas Chromatography (GC) separation was performed by gradient temperature elution on a capillary GC column, followed by Flame Ionization Detection (FID). The results are expressed as ppm (parts per million) TEA. The level of TEA was determined by external quantification against a standard.

5. The data in 6,7, 10, 11A, 11B, and 12 were obtained from a sample of liposomal irinotecan prepared as follows: make 0.4-0.5M TEA₈The SOS trap liposomes are loaded with about 400-600mg (e.g., about 500g) irinotecan moieties per mole of total phospholipids (stability ratio ranging from about 1000-1200) and a pH after preparation of about 7.0-7.5 (e.g., about 7.25). The amount of respective lyso-PC in these liposomal irinotecan samples was measured using the HPLC method of example 9 at the time points shown in fig. 5-7.

The accumulation data of lyso-PC (in mg lyso-PC/mL liposome composition) was plotted against storage time as shown on fig. 5 (samples 24-26/batches 1-3) or fig. 6 (samples 27-29/batches 4-6). A linear relationship was observed where the lyso-PC accumulation varied from about 0.008 mg/mL/month to about 0.06 mg/mL/month, with higher ratios being characteristic of compositions with higher TEA amounts. The amount of lyso-PC accumulation when stored for 180 days (about 6 months) was determined from a linear approximation of the multipoint data (FIGS. 5A and 5B) and is expressed as mol% of PC, where the molecular weight of lyso-PC is equal to 523.7 g/mol. All six batches (samples 24-29; see Table 10) accumulated less than 20 mol% lyso-PC when stored refrigerated for 180 days. Batches with less than 20ppm TEA and Iri/SOS gram equivalent ratios greater than 0.98 showed the least hemolysis-PC accumulation (less than about 0.015 mg/mL/month, with hemolysis-PC at 180 days of 3.0 mol% or less); batches of TEA less than 80ppm accumulated lyso-PC at a rate of about 0.03 mg/mL/month or less and had less than 7 mol% lyso-PC over 180 days; the batch with 100ppm residual TEA accumulated lyso-PC at a rate of about 0.06 mg/mL/month and had about 10 mol% lyso-PC at 180 days.

Figure 7 is a graph showing the rate of hemolysis-PC accumulation (in mg/mL/month) versus TEA content (in ppm) at 5 ± 3 ℃ for a stable irinotecan sulfatide liposome composition stored, along with a linear regression line from the data. Five additional batches of liposomal irinotecan sulfatide were prepared similarly to example 3. The irinotecan fraction of the formulation stored (based on anhydrous free base, as explained above) was about 4.3mg/mL irinotecan anhydrous free base per mL, and the lyso-PC formation and TEA content described in example 3 were analyzed periodically. The hemolysis-PC accumulation rate was calculated as the slope of a linear regression line, which was obtained as follows: the hemolysis-PC data for each batch was fitted against the storage time and the average TEA readings for the BDP/DP pair batch were plotted against TEA content (fig. 6). As can be seen from the figure, the formulation had a TEA accumulation of hemolysis-PC of about 25ppm or less at a rate of less than 0.02 mg/mL/month (less than a 2.5 mol% increase in hemolysis-PC over a 180 day period); the formulations had less than about 70ppm TEA accumulated lyso-PC at a rate of less than 0.033 mg/mL/month (less than 4.3 mol% increase in lyso-PC over a period of 180 days), and all formulations had less than about 100ppm TEA and accumulated lyso-PC at a rate of less than 0.062 mg/mL/month (less than 8.0 mol% increase in lyso-PC over a period of 180 days).

Samples 24, 25 and 28 each had less than 20ppm (e.g., about 10-20ppm) substituted ammonium ion (protonated TEA) and the lowest amount of lyso-PC (2.2-3 mol% lyso-PC) was observed after 6 months of refrigerated storage at 4 ℃. Comparing samples 26 and 27, the amount of substituted amine scavenger counter ions (e.g. protonated TEA) remaining in irinotecan SOS liposomes was increased from about 39ppm to 79ppm (103% increase), with an unexpected decrease in the amount of lyso-PC observed after 180 days (from 6.9 mol% to 5.4 mol%, 22% decrease in lyso-PC). However, the amount of residual substituted ammonium ions (e.g. protonated TEA) in irinotecan SOS liposomes further increased from 79ppm (sample 27) to 100ppm (sample 29) (i.e. 27% increase) with an additional 87% increase in the amount of lyso-PC observed after 6 months of refrigerated storage at 4 ℃ (i.e. from 5.4 mol% in sample 27 to 10.1 mol% in sample 29).

Example 4 interaction of irinotecan with Thioglycolate

Figure 8 is a graph showing the gram-equivalent amounts of irinotecan and a thionate in precipitates formed by combining irinotecan hydrochloride and triethylammonium thionate in various ratios of thionate (SOS) in aqueous solution, as described in example 4.

When irinotecan hydrochloride solution is combined with liposomes containing triethylammonium sulfatide, hydrogen ions can be scavenged and irinotecan sulfatide salt can be formed. To study the reaction between irinotecan and triethylammonium sulfathionate, we prepared a 25mM (16.93mg/mL) irinotecan hydrochloride trihydrate USP aqueous solution and a 250meq/L (31.25mM) triethylammonium sulfatide (TEA-SOS) solution (essentially as described in the methods section). An aliquot of the irinotecan hydrochloride solution was diluted with water, heated to 65 ℃, and combined with an aliquot of the TEA-SOS solution to yield a range of irinotecan-SOS gram-equivalent ratios of 9:1 to 1:9, with the total gram-equivalent concentration of the two compounds added together equaling 25 meq/L. The samples were mixed rapidly by vortexing, incubated at 65 ℃ for 30 minutes, frozen in ice water, and allowed to equilibrate at 4-6 ℃ overnight. In all samples, precipitation was observed. The next day, the samples were centrifuged at 10000xg for 5 minutes and 14000xg for an additional 5 minutes, the clear supernatant was separated (relative to a loose mass of white to beige precipitate), and the non-precipitated irinotecan and SOS were analyzed, essentially as described in the examples, to determine the amount and composition of the precipitate. The results are plotted against the gram equivalent percent of SOS in the sample (figure 8). In the 20-80 equivalent% range of SOS, the plot of the two components consists of two linear branches that intersect at 50 equivalent%, indicating that irinotecan and the sulfatide form an insoluble salt with stoichiometry of the sulfate group of one molecule of irinotecan per sulfatide (i.e., eight molecules of irinotecan (IRI) per molecule of sulfatide (SOS)):

8IRI.HCl+TEA₈SOS→(IRI.H)₈SOS↓+TEACl

despite the significant differences in molecular size and shape of the protonated irinotecan molecules and the sulfatide anion, their salts surprisingly remained in close stoichiometric ratios, i.e., eight protonated irinotecan molecules for one sulfatide molecule, even at large excesses of either component (fig. 8). Therefore, irinotecan sulfatide can exist in the liposome in a precipitated state or gel-like form of poor solubility. The fact that the salt precipitation remains strictly stoichiometric allows the process to be advanced to the point where almost all or substantially all of the sulfate groups of the thiosugar ester are bound to the drug molecule. Consistent with the irinotecan-sulfatase gram-equivalent ratio measurements of example 6, the process of loading irinotecan to obtain stable liposomes of the present invention, in some embodiments, can comprise a liposome precipitate of stoichiometric drug salt until at least 90%, at least 95%, even at least 98%, and in some cases substantially all of the free liposomal sulfate ester is consumed from the liposomal aqueous phase by precipitation and/or gelation of its irinotecan salt.

Example 5: preparation and solubility determination of irinotecan sulfate

An amount of 1.64g irinotecan hydrochloride trihydrate was added to 160mL of water acidified with 0.008mL of 1n hcl and stirred with heating in a 65 ℃ water bath until the drug dissolved. 5mL of 0.46M (radical sulfate concentration) triethylammonium thionate were added with vigorous stirring and stirred for an additional 5 minutes. After overnight storage at 4-6 ℃, the pale yellow oily precipitate solidified to a brittle material. The material was ground with a glass rod to give a fluffy off-white precipitate and incubated under refrigeration for 25 days. The precipitate was separated by centrifugation and the supernatant solution was discarded. Resuspending the pellet in 5 volumes of deionized water and precipitating out by centrifugation; this washing step was repeated two additional times until the pH of the suspension was about 5.8. Finally, the pellet was resuspended in an equal volume of deionized water to give about 26mL or a product containing 46.0mg/mL irinotecan (free base) (84% theoretical yield). Aliquots were dissolved in 1N HCl and analyzed for irinotecan (by spectrophotometry in 270% aqueous isopropanol-0.1N HCl at 365 nm) and determined using barium sulfate turbidimetry for the sulfate after hydrolysis in dilute (1:4) perchloric acid. Irinotecan and SO were discovered₄Is 1.020. + -. 0.011. Equal parts of the irinotecan sulfatide suspension were added to deionized water to give final drug salt concentrations of 0.93, 1.85, and 3.71 mg/mL. The sample was incubated at 4-6 ℃ for 22 hours with stirring, solid material was removed by centrifugation at 14000g for 10 minutes and irinotecan in the supernatant was analyzed spectrophotometrically. The irinotecan concentrations in the solutions were found to be 58.9. + -. 0.90. mu.g/mL, 63.2. + -. 0.6. mu.g/mL and 63.4. + -. 1.3. mu.g/mL, respectively, i.e., their mean values correspond to a molar solubility of irinotecan sulfatide of 1.32X 10^-5M。

Example 6: various irinotecan liposomes

All experiments of this example were performed as follows: a 25mm extruder, hollow fiber or Tangential Flow Filtration (TFF) set-up for the initial diafiltration step, micro-scale drug loading and TFF set-up for final diafiltration, followed by EAV filtration were used. Due to the limited volume of drug loaded material, the final filtration after dilution uses 20cm in a biosafety cabinet²The EAV filter is completed instead of the two EBV filters.

Table 11:

^hmeasured according to method a, as described herein.

Referring to table 11, a series of different irinotecan liposomes with different amounts of lyso-PC were prepared. Unless otherwise indicated, irinotecan liposomes encapsulate irinotecan sucrose octasulfate in vesicles consisting of DSPC, cholesterol and MPEG2000DSPE in a 3:2:0.015 molar ratio.

Sample 30 (batch 1) was obtained as follows: liposomes were prepared as described in example 1 (except as shown in this example), and the extruded liposomes were then held at 72 ℃ for 8 hours after liposome extrusion, with pH adjusted to 6.2-6.9 at the end of 8 hours, resulting in a composition with about 45 mol% lyso-PC (i.e., about 1.7 mg/mL). The time for MLV preparation was considered to be time 0. This experiment was performed using an aliquot from baseline experiment 1. The liposomes in the composition from which sample 30 (batch 1) was prepared had a lower DSPC to cholesterol molar ratio (about 2:1 instead of 3:1 in the other samples). The resulting liposomal composition of irinotecan has high levels of lyso-PC (i.e., greater than 1mg/mL and greater than 40 mol% lyso-PC).

Samples 31a and 31b (batches 2a and 2b) were prepared using the method of example 1, with modifications made to test the effect of increasing the concentration of TEA-SOS solution in the liposomes prior to irinotecan loading and the effect of reducing the irinotecan loading ratio by 15% on the properties of the resulting irinotecan liposome composition. The material of sample 31a (2a) was obtained as follows: liposomes were formed with vesicles comprising DSPC and cholesterol (in the ratios provided in table 11) encapsulating TEA-SOS solution at a concentration of 0.5M sulfate groups to form multilamellar vesicles (MLVs), and these liposomes were contacted with irinotecan hydrochloride solution in an amount of 510g irinotecan anhydrous free base per mol PL to load the drug into the liposomes. The material of sample 31b (2b) was obtained as follows: the liposome composition of sample 31a (2a) was maintained at 40 ℃ for 1 week, and then the sample was analyzed again. Both of the resulting irinotecan liposome compositions of samples 31A and 31b (2A and 2b) contained very low levels of lyso-PC (i.e., less than about 0.06mg/mL or 4 mol% in sample 31A (2A) and about 0.175mg/mL in sample 31b (2 b)).

Samples 32a and 32b (batches 3a and 3b, respectively) were prepared using the method of example 1, with modifications selected to study the combined effect of formulation buffer pH and reduced irinotecan loading ratio. The material of sample 32a (3a) was obtained as follows: liposomes having vesicles comprising DSPC and cholesterol (in the ratios provided in table 10) encapsulating a solution of TEA-SOS solution to form MLVs, contacting these liposomes with irinotecan to load the drug into the liposomes, irinotecan sucrose octasulfate being formed in the liposomes at the irinotecan loading ratios shown in table 11 (lower irinotecan loading ratios than samples 33(4) and 34 (5)) in a buffer selected to provide a pH of about 6.50 (instead of a pH of about 7.25 in sample 30 (1)). The material of sample 32b (3b) was obtained as follows: the composition of sample 3a was maintained at 40 ℃ for 1 week and then the sample was analyzed again. Both the resulting irinotecan liposome compositions 32a (3a) and 32b (3b) contained low levels of 0.076mg/mL and 0.573mg/mL of lyso-PC, respectively.

Samples 33(4) and 34(5) were prepared according to the method described in example 1. The materials of samples 33(4) and 34(5) were obtained as follows: liposomes were formed with vesicles comprising DSPC and cholesterol (in the ratios provided in table 11) encapsulating a solution of TEA-SOS solution to form MLVs, these liposomes were contacted with irinotecan to load the drug into the liposomes, irinotecan sucrose octasulfate was formed in the liposomes at 500g irinotecan moiety (based on anhydrous free base)/mol phospholipid in a buffer chosen to provide a pH of about 7.25 (instead of a pH of about 6.5 in samples 3a and 3 b). Both the resulting irinotecan liposome compositions 3a and 3b contained low levels of 0.24mg/mL and 0.79mg/mL of lyso-PC, respectively.

Figure 12 is a graph showing the amount of hemolysis-PC measured in sample 33(4) (circles, bottom line) and sample 34(5) ("+" data points, top line). The lyso-PC formation rate was higher in sample 34(5) than in sample 33 (4).

The linear fit of the data points in FIG. 12 is as follows:

sample 33 (4): hemolytic-PC, mg/mL-0.0513596 +0.0084714 cumulative age

Sample 34 (5): hemolytic-PC, mg/mL-0.1766736 +0.0279783 cumulative age

At 22 months, the total lyso-PC concentrations of irinotecan liposomes in formulation samples 33 and 34 were 0.24mg/mL and 0.79mg/mL, respectively.

Example 7: irinotecan liposome injection

A preferred embodiment of a storage stable liposomal formulation of irinotecanProducts for sale (yi)Rituximab liposome injection) (Merrimack Pharmaceuticals, inc., Cambridge, MA).The product is a topoisomerase inhibitor formulated with irinotecan hydrochloride trihydrate into a liposome dispersion for intravenous use. Indication ofA product for use in combination with fluorouracil and leucovorin in the treatment of patients with metastatic pancreatic cancer following disease progression following gemcitabine-based treatment.

The recommended dosage of the product is 70mg/m²It was administered by intravenous infusion for 90 minutes, once every two weeks. Will be provided withThe product is administered in combination with leucovorin and fluorouracil for the treatment of certain forms of pancreatic cancer. In these pancreatic cancer patients known to be homozygous for the UGT1A1 × 28 alleleThe recommended starting dose of the product is 50mg/m²It was administered by intravenous infusion for 90 minutes. Will be provided withThe dosage of the product is increased to 70mg/m²Which is tolerated in subsequent cycles. For patients with serum bilirubin above the upper limit of normal values, there is no recommended doseAnd (5) producing the product.

Will be provided withProducts such asAdministered to the patient. First, the volume is calculatedThe product is removed from the vial. Then the amount ofThe product was diluted in 500mL of 5% glucose injection, USP or 0.9% sodium chloride injection, USP and mixed by slow inversion. The dilution should be protected from light. Then when stored at room temperature within 4 hours of preparation or when refrigerated [2 ℃ to 8 ℃ (36 ° F to 46 ° F) within 24 hours of preparation]Dilutions were applied during storage. The diluted solution is allowed to reach room temperature before application and should not be frozen. The diluent was then infused over 90 minutes without the use of an in-line filter and the unused portion discarded.

Will be provided withThe product (topoisomerase inhibitor) was formulated with irinotecan hydrochloride trihydrate into a liposome dispersion for intravenous use. The chemical name of irinotecan hydrochloride trihydrate is (S) -4, 11-diethyl-3, 4,12, 14-tetrahydro-4-hydroxy-3, 14-dioxo-1H-pyrano [3 ', 4': 6,7]-indoxazino [1,2-b]Quinolin-9-yl- [1,4' -bipiperidine]-1' -carboxylic acid ester, monohydrochloride, trihydrate. Experimental formula is C₃₃H₃₈N₄O₆HCl 3H2O, molecular weight 677.19 g/mol. The molecular structure is as follows:

the product is provided as a sterile, white to yellowish opaque isotonic liposome dispersion. Each 10mL single dose vial contained an equivalent amount of 43mg irinotecan free base, at a concentration of 4.3mg/mL irinotecan anhydrous free base per mL (i.e.,4.3mg irinotecan fraction/mL). Liposomes are unilamellar lipid bilamellar vesicles, about 110nm in diameter, encapsulating an aqueous space containing irinotecan in either a gel or precipitated state as sucrose octasulfate salt. The vesicles consisted of 6.81mg/mL of 1, 2-distearoyl-sn-glycerol-3-phosphocholine (DSPC), 2.22mg/mL of cholesterol, and 0.12mg/mL of methoxy-capped polyethylene glycol (MW2000) -distearoyl phosphatidylethanolamine (MPEG-2000-DSPE). Each mL also contained 2- [4- (2-hydroxyethyl) piperazin-1-yl as a buffer]Ethanesulfonic acid (HEPES)4.05mg/mL and sodium chloride 8.42mg/mL as an isotonicity agent.

Irinotecan liposome injection is a topoisomerase 1 inhibitor encapsulated in lipid bilayer vesicles or liposomes. Topoisomerase 1 relieves torsional strain in DNA by inducing single strand breaks. Irinotecan and its active metabolite SN-38 bind reversibly to the topoisomerase 1-DNA complex and prevent re-ligation of single-strand breaks, leading to exposure-time dependent double-stranded DNA damage and cell death. In mice with human tumor xenografts, irinotecan liposomes were administered at 5-fold lower irinotecan HCl equivalent dose than irinotecan HCl, achieving similar intratumoral exposure to SN-38.

Is receivingThe plasma pharmacokinetics of total irinotecan and total SN-38 were evaluated in cancer patients of the product,the product is administered as a single dose or combination chemotherapy at a dose of 50 to 155mg/m²353 cancer patients used a population pharmacokinetic analysis.

At 70mg/m²The pharmacokinetic parameters of total irinotecan and total SN-38 after administration as a single agent or combination chemotherapy are presented below.

Table 12: summary of the mean work values (+ -standard deviation) for total irinotecan and total SN-38

C_max: maximum plasma concentration

AUC_0-∞: area under plasma concentration curve extrapolated to infinity time

t_1/2: terminal elimination half-life

CL: clearance rate

V_d: dispense volume

In the dosage range of 50 to 155mg/m²The Cmax and AUC of total irinotecan increase with dose. In addition, Cmax of total SN-38 increased proportionally with dose; however, the increase in AUC for total SN-38 with dose was less than the increase proportional to dose.

Direct measurements of irinotecan liposomes showed that 95% of irinotecan remained liposome encapsulated, and the ratio between total and encapsulated forms did not change with time from 0 to 169.5 hours after administration.

The product should be stored at 2 ℃ to 8 ℃ (36 ° F to 46 ° F), should be protected from light, and should not be frozen.

A plurality ofThe product formulations were left in a long-term stable state and analyzed after 12-36 months of storage at 2-8 ℃ (refrigerated conditions). The results are plotted in the graphs in fig. 9, 10, 11A and 11B, as described below. In one study, 12 were measuredParticle size (figure 9) and particle size distribution (figure 10) of the product formulation over 12-36 months. PDI remained well below 0.1, below about 0.05 for all samples. In another study, 13 differences were measuredThe product has 12-36 preparationspH within month (fig. 11A). The pH of all samples remained greater than 6.8 during the study. In another study, 16 differences were measuredAmount of lyso-PC within 12 months during refrigerated storage of the product formulation (fig. 11B). The amount of lyso-PC remained below 1mg/mL for all samples.

For measuring storage at different time pointsFor purposes of the concentration of irinotecan free base in the product embodiment, irinotecan free base is quantified as provided in the examples section. For measuring storage at different time pointsFor purposes of the lipid composition in the product embodiment, lipids are quantified using standard HPLC methods standard in the art.

For measuring storage at different time pointsFor purposes of the mean particle size (D) and polydispersity index (PDI) of the liposomes of the product embodiments, the DLS method is used in conjunction with a Malvern Zeta Sizer Nano ZSTM.

For measuring storage at different time pointsFor the purpose of the presence of lyso-PC in the product embodiment, the lyso-PC is quantified as described in the "examples" section. Furthermore, it is also envisioned in the context of the present invention that lyso-PC can be quantified by HPLC as described in the specification.

Example 8: topotecan and vinorelbine liposome

The objective of this storage stability study was to determine any change in the physical and chemical stability of topotecan (TPT) liposomes and Vinorelbine (VNB) liposomes prepared with sucrose octasulfate trapping agent when stored at 4 ℃. Specifically, the study examined whether the reduction of the Sucrose Octasulfate (SOS) capture agent concentration from 0.6M to 0.45M sulfate groups during liposome preparation, while maintaining the indicated topotecan or vinorelbine to phospholipid ratio below each mole of phospholipid, would have an effect on the amount of lyso-PC present in the liposome samples. Similarly, the effect of increasing the pH from 6.5 to 7.5 was examined to determine if this pH increase reduced the presence of lyso-PC in the liposome composition. TPT and VNB were encapsulated with SOS trapping agent in liposomes containing DSPC, cholesterol (Chol) and PEG-DSPE in a 3:2:0.015 molar ratio. Formulation parameters studied included: the pH of the solution (6.5-7.5), the concentration of sucrose octasulfate capture agent during liposome preparation (0.45-0.6M sulfate), the encapsulated drug (TPT or VNB), and the drug to lipid ratio (500 g TPT HCl per mole phospholipid during liposome loading; 350 to 450g VNB moiety per mole phospholipid during liposome loading for VNB). Various physicochemical properties of liposomes monitored during this stability study were: liposome size, drug to phospholipid ratio, drug encapsulation efficiency, general appearance, and lyso-lipid formation.

DSPC, cholesterol (Chol) and PEG-DSPE were weighed out in amounts corresponding to a molar ratio of 3:2:0.015 (790.15mg/257.8mg/14.0mg), respectively. The lipids were dissolved in chloroform/methanol (4/1, v/v), mixed well and divided into 2 equal parts (a and B). Each sample was evaporated to dryness at 60 ℃ using a rotary evaporator. Residual chloroform was removed from the lipids by placing under vacuum (180 μ torr) at room temperature for 12 hours. Dissolving the dried lipids in ethanol at 60 deg.C, and adding preheated TEA at an appropriate concentration₈SOS resulted in a final ethanol content of 10% (v/v). The total phospholipid concentration was about 75 mM. The lipid solution was extruded through a 0.1 μm polycarbonate membrane (Nuclecore)^TM)10 times to produce liposomes with typical mean diameters of 95-115 nm. The pH of the extruded liposomes was adjusted (with 1N NaOH) to pH6.5 as needed. Liposomes are purified by a combination of ion exchange chromatography and size exclusion chromatography. First, Dowex^TMThe IRA910 resin was treated with 1N NaOH, followed by 3 washes with deionized water, then 3 washes with 3N HCl, then multiple washes with water. Elution is carried outThe conductivity of the fractions was measured by using a flow cell conductivity meter (Pharmacia, Uppsala, Sweden). If the conductivity is less than 15. mu.S/cm, the fraction is considered acceptable for further purification. The liposome eluate was then applied to a Sephadex G-75(Pharmacia) column equilibrated with deionized water, and the conductivity of the collected liposome fraction was measured (typically less than 1. mu.S/cm). A40% glucose solution was added to achieve a final concentration of 5% (w/w), and buffer (Hepes) was added from the stock solution (0.5M, pH6.5) to a final concentration of 10 mM.

Stock solutions of topotecan hydrochloride were prepared by dissolving 50mg in 10mL of deionized water. In results table 13, the drug was added to the liposome solution at the indicated drug/lipid ratio for each formulation. For TPT loading, the pH was adjusted to pH 6.0 prior to loading. Vinorelbine was added directly from a commercial USP injection solution at the pharmacy and the pH of the resulting mixture was adjusted to 6.5 with 1N NaOH prior to heating. Drug loading begins as follows: the liposome/drug mixture was heated to 60 ℃ for 30 minutes. The solution was removed from the water bath and rapidly cooled by immersion in ice-cold water. The extra-liposomal drug was removed by size exclusion chromatography using a Sephadex G75 column equilibrated and eluted with Hepes Buffered Saline (HBS) pH 6.5. Samples were analyzed for irinotecan by HPLC and for phosphate by the Bartlett method (see phosphate assay).

For storage, the samples were divided into 4mL aliquots and pH adjusted as needed using 1N HCl or 1N NaOH, sterile filtered under sterile conditions, and filled into sterile clear glass vials under argon withThe lining screw cap was sealed and placed in a 4 ℃ thermostatically controlled ice box. At the specified time points, aliquots were taken from each sample and tested for appearance, size, drug/lipid ratio, and drug and lipid chemical stability. Liposome size was determined by dynamic light scattering in diluted samples using a Coulter Nano-Sizer at a 90 degree angle and expressed as the mean. + -. standard deviation (nm) obtained by the cumulative method.

The results from the comparative stability study are provided in table 13.

TABLE 13 use of TEA₈Topotecan and vinorelbine liposomes prepared with SOS trap (0.6N SOS sulfate group, stored at drug concentration of 2mg/mL)

ⁱMeasured according to method B, as described herein.

^j500g topotecan HCl per mole total phospholipids

No effect of the pH of the storage medium on the production of lysolipids in topotecan-loaded liposomes was observed in samples 19 and 20. Both formulations of samples 19 and 20 showed approximately 30 mol% of lysolipids after 9 months, but sample 19 was stored at pH6.5 and sample 20 was stored at pH 7.25.

The liposomal vinorelbine was more resistant to hydrolysis of the lipids than the two liposomal camptothecins, since the highest amount of lyso-lipids was measured in sample 21, which had 9.5 mol% of lyso-lipids after 9 months. Although not obvious, we can also detect the dependence on the stability ratio and the pH of the storage medium. A higher stability ratio results in a reduction in lipid hydrolysis (compare samples 21 and 23). A PH of 7.25 also reduced the amount of lipid hydrolysis observed (compare samples 21 and 22).

Example 9: HPLC method for measuring lyso-PC ("method A")

The amount of lyso-PC in the irinotecan sucrose octasulfate liposome formulation was tested to obtain the data in fig. 11B and fig. 12, as detected by evaporative light scattering using HPLC. A suitable HPLC method (referred to herein as "method a") is a quantitative method for measuring the amount of stearic acid, lyso-PC, cholesterol and DSPC (1, 2-distearoyl-sn-glycerol-3-phosphocholine) in a pharmaceutical product. The liposomes were broken down into their respective lipid components using a methanol-tetrahydrofuran solution. Lipid components were quantified using reverse phase high pressure liquid chromatography equipped with an evaporative light scattering detector.

Sample and Standard preparation

Preparation of standards：

Hemolytic PC

The five point standard curve was prepared as follows: appropriate amounts of lysopc were diluted with 85:15 methanol-tetrahydrofuran to target final concentrations of 4, 8,20, 32 and 40 μ g/mL.

Stearic acid

The five point standard curve was prepared as follows: an appropriate amount of stearic acid was diluted with 85:15 methanol-tetrahydrofuran to target final concentrations of 2, 4, 10, 16 and 20.4 μ g/mL.

Cholesterol

The five point standard curve was prepared as follows: appropriate amounts of cholesterol were diluted with 85:15 methanol-tetrahydrofuran to target final concentrations of 90, 144, 183.7, 224.9 and 266.6 μ g/mL.

DSPC

The five point standard curve was prepared as follows: appropriate amounts of DSPC were diluted with 85:15 methanol-tetrahydrofuran to target final concentrations of 220, 352, 449, 549.8 and 651.7 μ g/mL.

Assay control

Analytical controls were prepared as follows: stearic acid was diluted in diluent (85:15 methanol-tetrahydrofuran) to target final concentrations of 9.0. mu.g/mL and 18.0. mu.g/mL.

Sample preparation：

The samples were prepared as follows: each sample was diluted in 85:15 methanol-tetrahydrofuran solution to a target final DSPC concentration of 475. mu.g/mL.

Stability of solution

The test sample standards and analytical controls have shown acceptable stability in solution for up to 48 hours when stored at ambient temperature.

Instrument and instrument parameters

If desired, a suitable high pressure chromatography system equipped with an evaporative light scattering detector can vary the gain and filter settings throughout the run to ensure proper peak detection. The instrument operating parameters are listed in table 14.

Table 14: chromatographic conditions

Table 15: system applicability

Each lipid concentration was determined by analyzing the peak area of the sample against a standard curve. The second order polynomial equation (quadratic curve) trend line was used to calculate the lipid concentrations of lyso-PC and stearic acid. Linear trend lines were used to calculate lipid concentrations of DSPC and cholesterol.

Representative chromatograms are given in fig. 13A and 13B.

All references cited in this application are incorporated by reference in their entirety.

The present application also relates to the following:

a storage stable liposomal irinotecan composition having a pH of 7.00-7.50 and comprising a dispersion of irinotecan liposomes encapsulating irinotecan sucrose octasulfate in vesicles, the vesicles are composed of cholesterol and the phospholipids 1, 2-distearoyl-sn-glycerol-3-phosphocholine (DSPC) and methoxy-capped polyethylene glycol (MW2000) -distearoyl phosphatidylethanolamine (MPEG-2000-DSPE), the liposomal irinotecan composition comprising irinotecan moieties at concentrations equivalent to, 500mg (+ -10%) of irinotecan moiety per mmol of total liposomal phospholipid and 4.3mg of irinotecan moiety per mL of the liposomal irinotecan composition, in grams of irinotecan free anhydrous base, the storage stable liposomal irinotecan composition stabilizes to form less than 20 mol% lyso-PC during the first 6 months of storage at 4 ℃.

A storage stable liposomal irinotecan composition having a pH of 7.00-7.50 and comprising a dispersion of irinotecan liposomes encapsulating irinotecan sucrose octasulfate in unilamellar vesicles, the vesicles are composed of cholesterol and the phospholipids 1, 2-distearoyl-sn-glycerol-3-phosphocholine (DSPC) and methoxy-capped polyethylene glycol (MW2000) -distearoyl phosphatidylethanolamine (MPEG-2000-DSPE), the liposomal irinotecan composition comprising irinotecan moieties at concentrations equivalent to, 500mg (+ -10%) of irinotecan moiety per mmol of total liposomal phospholipid and 4.3mg of irinotecan moiety per mL of the liposomal irinotecan composition, in grams of irinotecan free anhydrous base, the storage stable liposomal irinotecan composition has an irinotecan/sulfate compound gram-equivalent ratio of 0.85-1.2.

A storage stable liposomal irinotecan composition stabilized to form less than 20 mol% lyso-PC during the first 6 months of storage at 4 ℃ prepared by a process comprising:

(a) forming a dispersion of lipids in a solution consisting of TEA having a sulfate concentration of 0.4 to 0.5M₈SOS and/or DEA₈SOS preparation and pH 5 to 7, the lipids in the dispersion being about 3:2:0.015 molar ratio of DSPC, cholesterol, and MPEG-2000-DSPE, respectively;

(b) extruding a lipid dispersion at 60-70 ℃ through at least one 0.1 μm membrane to form liposomes;

(c) substantially removing TEA-derived material outside the liposomes₈SOS and/or DEA₈An ion of SOS;

(d) contacting liposomes at a temperature of 60-70 ℃ with a solution prepared using irinotecan free base or an irinotecan salt, thereby forming a formulation of liposomes encapsulating irinotecan;

(e) substantially removing TEA-derived material outside the liposomes₈SOS and/or DEA₈(ii) SOS and irinotecan-containing substances; and

(f) the pH of the composition is adjusted to 7.0-7.5.

The liposomal irinotecan composition of any one of items 1-3, prepared by a process comprising the steps of:

(a) forming a dispersion of lipids in a solution consisting of TEA having a sulfate concentration of 0.4 to 0.5M₈SOS preparation and pH 5 to 7, the lipids in the dispersion being about 3:2:0.015 molar ratio of DSPC, cholesterol, and MPEG-2000-DSPE, respectively;

(b) extruding a lipid dispersion at 60-70 ℃ through at least one 0.1 μm membrane to form liposomes;

(c) substantially removing TEA-derived material outside the liposomes₈An ion of SOS;

(d) contacting liposomes at a temperature of 60-70 ℃ with a solution prepared using irinotecan free base or an irinotecan salt, thereby forming a formulation of liposomes encapsulating irinotecan;

(e) substantially removing TEA-derived material outside the liposomes₈(ii) SOS and irinotecan-containing substances; and

(f) the pH of the composition is adjusted to 7.0-7.5.

The liposomal irinotecan composition of item 5 item 4, wherein the lipid dispersion is extruded through at least two superposed 0.1 μm polycarbonate membranes.

The liposomal irinotecan composition of any one of the preceding claims, wherein the liposomes have a mean size of 110nm, as determined by dynamic light scattering, wherein the size is determined by the cumulant method.

The liposomal irinotecan composition of any one of the preceding claims having a total irinotecan moiety content equivalent to 4.3mg/ml irinotecan anhydrous free base.

The liposomal irinotecan composition of any one of items 3-6, wherein:

in step (a)The liposome is prepared from TEA with sulfate concentration of 0.43-0.47M₈SOS formation; and

in step (d), a solution prepared using irinotecan free base or irinotecan salt has an irinotecan partial content equivalent to 500g (± 10%) of irinotecan anhydrous free base per mole of DSPC; and

in step (f), the pH of the composition is adjusted to 7.2 to 7.3.

The liposome composition of any one of the preceding claims, which contains less than 1 mol% lysophosphatidylcholine (lyso-PC) prior to storage at about 4 ℃, and which contains 20 mol% or less (relative to total liposomal phospholipids) lyso-PC after 180 days of storage at about 4 ℃.

The liposome composition of item 9, which contains 20 mol% or less (relative to total liposome phospholipids) lysophosphatidylcholine (lyso-PC) after 6, 9, or 12 months of storage at about 4 ℃.

The liposomal irinotecan composition of any one of the preceding claims, all in aqueous isotonic buffer, collectively comprising 6.1 to 7.5mg DSPC/ml, 2 to 2.4mg cholesterol/ml, and 0.11 to 0.13mg MPEG-2000-DSPE/ml.

The liposomal irinotecan composition of any one of the preceding claims, wherein the liposomal irinotecan comprises irinotecan liposomes at a concentration of 2 to 20mM in isotonic HEPES aqueous buffer.

The liposomal irinotecan composition of any one of the preceding claims, further comprising sodium chloride at a concentration of 130 and 160 mM.

The liposomal irinotecan composition of any one of the preceding claims, wherein the irinotecan encapsulated in the liposomes is in a gel state or a precipitated state as sucrose octasulfate salt.

The liposomal irinotecan composition of any one of the preceding claims, wherein the irinotecan liposomes have a diameter of 95-115nm, as measured by quasi-elastic light scattering.

The liposomal irinotecan composition of any one of the preceding claims, which collectively comprises 6.81mg DSPC/mL, 2.22mg cholesterol/mL, and 0.12mg MPEG-2000-DSPE/mL, 4.05mg/mL HEPES aqueous buffer, and 8.42mg sodium chloride/mL.

The liposomal irinotecan composition of any one of the preceding claims having a pH of 7.25, wherein the irinotecan liposomes are 110nm in diameter, as measured by quasielastic light scattering.

The liposomal irinotecan composition of any one of the preceding claims which, upon storage at about 4 ℃ for 6 months, forms less than 1mg/mL of lysophosphatidylcholine (lyso-PC).

The liposomal irinotecan composition of any one of the preceding claims, prepared by a process comprising the steps of:

(a) formation of TEA with lipid concentration of about 0.45M in sulfate₈A dispersion in a solution of SOS, said solution having a pH of about 6.5, the lipids in said dispersion consisting of 1, 2-distearoyl-sn-glycerol-3-phosphocholine (DSPC), cholesterol and methoxy-terminated polyethylene glycol (MW2000) -distearoyl phosphatidylethanolamine (MPEG-2000-DSPE), respectively, in a molar ratio of 3:2: 0.015;

(b) extruding a lipid dispersion at 60-70 ℃ through at least one 0.1 μm membrane to form liposomes;

(c) removing TEA-derived materials from the exterior of the liposomes₈An ion of SOS;

(d) contacting liposomes at a temperature of 60-70 ℃ with a solution prepared using irinotecan hydrochloride trihydrate, thereby forming a formulation of liposomes encapsulating about 500g (± 10%) irinotecan per mole of total liposomal phospholipids;

(e) removing TEA-derived materials from the exterior of the liposomes₈(ii) SOS and irinotecan-containing substances; and

(f) the pH of the composition was adjusted to about 7.3.

The liposomal irinotecan composition of any one of the preceding claims, which collectively comprises less than 100ppm of TEA.

The liposomal irinotecan composition of any one of the preceding claims, which collectively comprises 30-100ppm of TEA or DEA.

The liposomal irinotecan composition of any one of the preceding claims, wherein at least 98% of the irinotecan is encapsulated in the irinotecan liposome after 6 months of storage at about 4 ℃.

The liposomal irinotecan composition of any one of the preceding claims comprising within the irinotecan liposome an irinotecan composition of formula (I) wherein x is 8:

Claims (20)

1. a storage-stable liposomal irinotecan composition comprising irinotecan Sucrose Octasulfate (SOS), cholesterol, and one or more phospholipids encapsulated in unilamellar liposomes, the composition having:

(i) a ratio of irinotecan to total phospholipids corresponding to a total of 500g + -10 wt.% irinotecan moieties per mole of total phospholipids;

(ii) a pre-storage pH of about 7.25 to about 7.5 at 20-25 ℃; and

(iii) less than 20 mol% lyso-phosphatidylcholine ("lyso-PC") after six months of storage of the composition at a temperature of 2 to 8 ℃ compared to total phospholipids prior to storage.

2. The composition of claim 1, wherein the one or more phospholipids comprise distearoylphosphatidylcholine and N- (methoxy-poly (ethylene glycol) -oxycarbonyl) -distearoylphosphatidylethanolamine.

3. The composition of claim 2, wherein the N- (methoxy-poly (ethylene glycol) -oxycarbonyl) -distearoylphosphatidylethanolamine is methoxy-terminated polyethylene glycol (MW2000) -distearoylphosphatidylethanolamine.

4. The composition of claim 3, wherein the composition contains less than 20 mol% lyso-PC after 9 months of storage at 2 to 8 ℃ compared to total phospholipids prior to storage.

5. The composition of claim 3, wherein the composition contains less than 10 mol% lyso-PC after 6 months of storage at 2 to 8 ℃ compared to total phospholipids prior to storage.

6. The composition of claim 3, wherein the composition contains less than 10 mol% lyso-PC after 9 months of storage at 2 to 8 ℃ compared to total phospholipids prior to storage.

7. The composition of claim 3, wherein the composition contains less than 10 mol% lyso-PC after 12 months of storage at 2 to 8 ℃ compared to total phospholipids prior to storage.

8. The composition of claim 3, wherein the composition contains less than 10 mol% lyso-PC after 24 months of storage at 2 to 8 ℃ compared to total phospholipids prior to storage.

9. The composition of claim 3, wherein the composition contains less than 5 mol% lyso-PC after 6 months of storage at 2 to 8 ℃ compared to total phospholipids prior to storage.

10. The composition of claim 3, wherein the composition contains less than 5 mol% lyso-PC after 9 months of storage at 2 to 8 ℃ compared to total phospholipids prior to storage.

11. The composition of claim 3, wherein the composition contains less than 5 mol% lyso-PC after 12 months of storage at 2 to 8 ℃ compared to total phospholipids prior to storage.

12. The composition of claim 3, wherein the composition contains less than 5 mol% lyso-PC after 24 months of storage at 2 to 8 ℃ compared to total phospholipids prior to storage.

13. The composition of claim 3, wherein the composition contains less than 2 mol% lyso-PC after 6 months of storage at 2 to 8 ℃ compared to total phospholipids prior to storage.

14. The composition of claim 3, wherein the composition contains less than 2 mol% lyso-PC after 9 months of storage at 2 to 8 ℃ compared to total phospholipids prior to storage.

15. The composition of claim 3, wherein the composition contains less than 2 mol% lyso-PC after 12 months of storage at 2 to 8 ℃ compared to total phospholipids prior to storage.

16. The composition of claim 3, wherein the composition contains less than 2 mol% lyso-PC after 24 months of storage at 2 to 8 ℃ compared to total phospholipids prior to storage.

17. The composition of claim 3, wherein the composition further comprises a total amount of triethylammonium or diethylammonium of less than 100 ppm.

18. The composition of claim 3, wherein the composition further comprises a total amount of 30 to 100ppm of triethylammonium or diethylammonium.

19. The composition of claim 3, wherein the composition further comprises a total of 4.05mg/mL 2- [4- (2-hydroxyethyl) piperazin-1-yl ] ethanesulfonic acid and a total of 8.42mg/mL sodium chloride.

20. The composition of claim 3, wherein the composition further comprises a histidine buffer.