Stabilized camptothecin pharmaceutical composition

Abstract

To provide stabilizing camptothecin pharmaceutical compositions.SOLUTION: Irinotecan phospholipid liposomes with improved storage stability are provided, with related methods of treatment and manufacture. The irinotecan liposomes can have reduced formation of lyso-phosphatidylcholine (lyso-PC) during storage, and prior to administration to a patient. The present invention relates to stabilizing pharmaceutical compositions comprising camptothecin compounds, including liposomal camptothecin pharmaceutical formulations stabilized to reduce formation of lyso-lipid formation during storage.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Priority claim This patent application claims the benefit of U.S. Provisional Patent Applications Nos. 62 / 242,835 (filed October 16, 2015), 62 / 242,873 (filed October 16, 2015), 62 / 244,061 (filed October 20, 2015), and 62 / 244,082 (filed October 20, 2015), each of which is incorporated by reference in its entirety.

[0002] Technical Field The present disclosure relates to stabilized pharmaceutical compositions comprising camptothecin compounds, including liposomal camptothecin pharmaceutical formulations that are stabilized to reduce the formation of lyso-lipid formation during storage. [Background technology]

[0003] background Camptothecin compounds (such as irinotecan or topotecan) can be used to treat tumors and / or cancers in the human body. For example, injectable liposomal pharmaceutical products for treating certain forms of cancer can be prepared as a dispersion of liposomes encapsulating the camptothecin compound. This liposomal camptothecin composition can encapsulate the camptothecin compound together with a polyanionic sequestering agent within liposomes containing cholesterol and one or more phospholipids ("PL"). However, in camptothecin liposomes containing one or more phospholipids, hydrolysis of the phospholipids and the active lactone structure in camptothecin can occur. Hydrolytic degradation of liposomal phospholipids, such as phosphatidylcholine ("PC"), can alter the release of camptothecin compounds, such as irinotecan, from liposomes. The first step in the hydrolysis of PL (such as PC) can result in the formation of lyso-PL (such as lysophosphatidylcholine ("lyso-PC"), which is a glycerylphosphocholine fatty acid monoester).

[0004] Liposomal camptothicin compositions are affected by pH in at least two ways. First, the pH of the liposomal camptothecin (e.g., The hydrolytic degradation of phospholipids in liposomal irinotecan tends to be pH-dependent, with pH 6.0 or 6.5 believed to minimize phosphatidylcholine hydrolysis. Conditions above pH 6.5 tend to (1) enhance the conversion of camptothecin compounds, such as irinotecan, to the less active carboxylate form and (2) increase the amount of lyso-PC in the liposomes. Second, camptothecin compounds undergo pH-dependent conversion between the less active carboxylate form (predominant at neutral and alkaline pH) and the more active lactone form, which predominates at acidic pH. For example, conversion of the carboxylate form of irinotecan to the lactone form occurs primarily between pH 6.0 (approximately 85% of irinotecan is in the more active lactone form) and pH 7.5 (only approximately 15% of irinotecan is in the more active lactone form). At pH 6.5, approximately 65% ​​of irinotecan is in the more active lactone form.

[0005] We unexpectedly found that the stability of phospholipid-containing liposomal camptothecin prepared at pH 6.5 was adversely affected by the formation of lyso-PC during storage under refrigerated conditions (2-8°C). For example, Sample 12, an irinotecan liposome composition (irinotecan octasulfate sucrose encapsulated in irinotecan liposomes containing DSPC, cholesterol, and MPEG-2000-DSPE in a molar ratio of 3:2:0.015, prepared at pH 6.5), subsequently generated levels of lyso-PC greater than 30 mol% (relative to the total amount of phosphatidylcholine in the irinotecan liposome composition) during the first 3 months of refrigerated storage (2-8°C) after manufacture (and greater than 35 mol% lyso-PC during the first 9 months).

[0006] Thus, there remains a need for stabilized camptothecin pharmaceutical compositions. For example, there is a need for improved liposomal formulations of irinotecan that are more stable and produce less lyso-PC during refrigerated storage at 2-8°C after manufacture. The present invention addresses this need. Summary of the Invention [Means for solving the problem]

[0007] Abstract The present invention provides novel camptothecin pharmaceutical compositions (e.g., liposomal irinotecan) with improved stability, including camptothecin liposomal compositions comprising ester-containing phospholipids that exhibit a reduced rate of lyso-phospholipid ("lyso-PL") (e.g., lyso-phosphatidylcholine, or "lyso-PC") formation. The present invention is based, in part, on the surprising realization that liposomal compositions of camptothecin compounds (e.g., irinotecan) can be prepared that result in reduced amounts of lyso-phospholipid after prolonged storage at 2-8°C. The preparation of such stabilized liposomal compositions is made possible by the unexpected finding that controlling specified parameters during liposome preparation (drug-to-phospholipid ratio relative to the amount of entrapment agent, pH of the liposomal preparation, and amount of counterion of the entrapment agent in the liposomal preparation) synergistically reduces lyso-phospholipid formation during storage of camptothecin liposomal preparations. The present invention provides significant information useful in the design and identification of improved, more robust liposomal compositions while reducing the costs associated with their development.

[0008] Stabilized camptothecin compositions comprising one or more phospholipids (including PEG-containing phospholipids) preferably form 20 mol% or less (relative to total liposomal phospholipids) of lyso-PL during the first 6 months of storage at 4° C. and / or 25 mol% or less of lyso-PL during the first 9 months of storage at 4° C. Stabilized irinotecan liposomes preferably form 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° C. after preparation of the camptothecin composition. A preferred stabilized camptothecin composition comprises irinotecan or a salt thereof (e.g., irinotecan octasulfate sucrose) in a liposomal irinotecan composition comprising cholesterol and one or more phospholipids (including PEG-containing phospholipids), wherein the stabilized camptothecin composition forms 20 mol% or less (based on total liposomal phospholipids) of lyso-PC during 6 months of storage at 4° C. and / or 25 mol% or less of lyso-PC during 9 months of storage at 4° C. (e.g., during the first 6 and / or 9 months of stability testing after manufacture). The stabilized irinotecan liposomes may form lyso-PC at a rate of less than about 2 mol% (e.g., 0.5-1.5 mol%) per month during storage at 4° C. (during the first 9 months of stability testing after manufacture). The stabilized phosphatidylcholine-containing irinotecan liposomal composition may generate less than 1 mg of lyso-PC during the first 9 months of stability testing at 2-8°C after manufacture.

[0009] In a first embodiment, the stabilized liposomal camptothecin composition comprises liposomes encapsulating irinotecan and a polyanionic sequestering agent of sulfate (e.g., irinotecan sucrose, or "SOS") having a pH greater than 6.5 (e.g., 7.0-7.5, including 7.25, 7.3, or 7.5) and a gram equivalent ratio ("ER") of irinotecan / sulfate compound greater than 0.9 (e.g., 0.9-1.1). The ER is determined for an irinotecan SOS liposomal preparation by determining the molar amounts of irinotecan (I) and sulfate compound (S) co-encapsulated by the liposome per unit of liposomal composition (e.g., 1 mL) using the following formula: ER = I / (SN), where N is the valency of the sulfate compound anion (e.g., for sucrose, N is 8, and the free sulfate S04 is 0.4). 2- where N is 2). Preferably, the sulfate compound (S) is sucrose octasulfate, which contains 8 sulfate moieties per mole of SOS.

[0010] In a second embodiment, stabilized liposomal camptothecin compositions are obtained using specific ratios of camptothecin, anionic sequestering agent, and liposome-forming phospholipids, preferably having a stability ratio ("SR") greater than about 950 (e.g., 950-1050), including irinotecan liposomes prepared with an SR greater than about 990 (e.g., 990-1,100, including 992-1,087). This embodiment provides a manufacturing standard that describes liposome stability, as reflected by the stability ratio, as described more fully below. This embodiment of the present invention is based, in part, on the discovery that when phospholipid-based camptothecin-containing liposomes are prepared by reacting (1) camptothecin compound(s) (e.g., irinotecan, topotecan, etc.) with (2) liposomes encapsulating a polysulfated anionic sequestering agent (e.g., sucrose octasulfate), the stability of the resulting drug-loaded liposomes depends on the initial concentration of sulfate groups in the sequestering agent-liposome and the ratio of camptothecin to phospholipid encapsulated in the liposome. This stability ratio is defined as follows: SR = A / B, where A is the amount of irinotecan moiety encapsulated in the entrapment agent liposomes during the drug loading process, in grams equivalent of anhydrous irinotecan free base per mole of phospholipid in the composition, and B is the concentration of sulfate groups in the sucrosophate (or other entrapment agent) solution used to make the entrapment agent liposomes, expressed in moles / L (based on the concentration of sulfate groups). The stability ratio surprisingly predicted a dramatic reduction in the formation of lyso-PC in phospholipid-based camptothecin-containing liposomes, even at pH 6.5. After 9 months of storage at 4°C, irinotecan liposomes prepared at a stability ratio of approximately 990 (Sample 2) produced approximately 24 mol% lyso-PC, while phosphatidylcholine-containing irinotecan liposomes prepared at a stability ratio of approximately 942 (Sample 3) produced approximately 36 mol% lyso-PC (i.e., under these conditions, an approximately 5% increase in stability ratio resulted in a 34% decrease in lyso-PC production).In contrast, improving the stability ratio of irinotecan liposomes by approximately 30%, from 724 (sample 12) to 942 (sample 3), resulted in the production of approximately 1% more lyso-PC after 9 months of storage at 4°C (e.g., compare 35.7 mol% lyso-PC in sample 3 with 35.4 mol% lyso-PC in sample 12).

[0011] In a third embodiment, novel stabilized compositions of liposomes encapsulating irinotecan that produce reduced amounts of lyso-phosphatidylcholine (lyso-PC) during storage at 2-8°C can include an irinotecan composition of formula (I), where x is 8. [ka] The liposomal irinotecan may comprise a composition of formula (I) encapsulated in a liposome. Preferably, the composition of formula (I) is formed (e.g., precipitated) within a liposome comprising cholesterol and one or more phospholipids (e.g., comprising a PEG-containing phospholipid). For example, the compound of formula (I) may be formed within a liposome by reacting (1) a camptothecin compound(s) (e.g., irinotecan, topotecan, etc.) with (2) a liposome encapsulating a polysulfated anionic scavenger (e.g., sucrose octasulfate) in the step of forming a stabilized liposomal irinotecan composition. Preferably, the liposomal irinotecan composition has a pH greater than 6.5 (e.g., 7.0 to 7.5, including 7.25, 7.3, and 7.5).

[0012] A preferred stabilized camptothecin composition comprises a liposomal irinotecan composition comprising irinotecan or a salt thereof (e.g., irinotecan octasulfate sucrose) encapsulated in an irinotecan liposome comprising cholesterol and the phospholipids 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and methoxy-terminated polyethylene glycol-distearoylphosphatidylethanolamine (e.g., MPEG-2000-DSPE) in an aqueous isotonic buffer, wherein the liposomal irinotecan composition is Contains (or forms) less than 10 mol% lyso-phosphatidylcholine (lyso-PC) after the first 3 months of storage at 8°C, contains (or forms) less than 20 mol% lyso-phosphatidylcholine (lyso-PC) after the first 6 months (i.e., 180 days) of storage at 2-8°C, and / or contains (or forms) less than 25 mol% lyso-phosphatidylcholine (lyso-PC) after the first 9 months of storage at 2-8°C (e.g., during the first 9 months of stability testing after manufacture).

[0013] Irinotecan liposomes preferably contain cholesterol, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and methoxy-terminated polyethylene glycol-distearoylphosphatidylethanolamine (e.g., MPEG-2000-DSPE) in a molar ratio of 3:2:0.015, and encapsulate 500 mg (±10%) of irinotecan per mmol of total liposomal phospholipid. Stabilized liposomal irinotecan compositions preferably contain irinotecan liposomes that provide a total of about 4.3 mg of irinotecan moieties per mL of liposomal irinotecan composition, with at least about 98% of irinotecan being encapsulated in the irinotecan liposomes (e.g., as irinotecan octasulfate sucrose, such as the compound of formula (I) above). Certain preferred liposomal compositions have a pH of 7.00 to 7.50 (e.g., 7.0, 7.25, 7.3, 7.5), and are composed of cholesterol and the phospholipids 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and methacrylate at a concentration of irinotecan moieties equivalent in grams of anhydrous irinotecan free base to 500 mg (±10%) per mmol of total liposomal phospholipid, and 4.3 mg of irinotecan per mL of liposomal irinotecan composition. A storage-stable liposomal irinotecan composition comprising a dispersion of irinotecan liposomes encapsulating sucrose irinotecan octasulfate in unilamellar bilayer vesicles composed of carboxy-terminated polyethylene glycol (MW2000)-distearoylphosphatidylethanolamine (MPEG-2000-DSPE), wherein the storage-stable liposomal irinotecan composition is stabilized to form less than 1 mg / mL of Lyso-PC during the first 6 months of storage at 4°C.For example, certain preferred pharmaceutical liposomal irinotecan compositions comprise irinotecan or a salt thereof (e.g., irinotecan octasulfate sucrose) encapsulated in irinotecan at 4.3 mg / mL of the irinotecan moiety, 6.81 mg / mL of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 2.22 mg / mL of cholesterol, and 0.12 mg / mL of methoxy-terminated polyethylene glycol (MW2000)-distearoylphosphine in an aqueous isotonic buffer. and lyso-phosphatidylethanolamine (MPEG-2000-DSPE), wherein the liposome composition contains less than 10 mol% lyso-phosphatidylcholine (lyso-PC) after 3 months of storage at 2 to 8°C, less than 20 mol% lyso-phosphatidylcholine (lyso-PC) after 6 months (or 180 days) of storage at 2 to 8°C, and / or less than 25 mol% lyso-phosphatidylcholine (lyso-PC) after 9 months of storage at 2 to 8°C.

[0014] In some embodiments, the liposome composition is prepared by contacting a solution containing irinotecan moieties with entrapment liposomes encapsulating triethylammonium (TEA) and irinotecan octasulfate sucrose (SOS) scavenger at a concentration of 0.4-0.5 M (relative to sulfate group concentration) as TEA8SOS (preferably, a TEA8SOS scavenger solution) under conditions effective to load 500 g (±10%) of irinotecan moieties / mol of phospholipid into the PL-containing entrapment liposomes and to permit release of TEA cations from the entrapment liposomes, thereby forming SOS liposomes. and (b) combining irinotecan SOS liposomes with 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) to obtain an irinotecan liposome composition having a pH of 7.25 to 7.50, which is stabilized to the extent that it forms less than 10 mol % (relative to the total amount of phosphatidylcholine in the irinotecan liposome composition) of lyso-phosphatidylcholine (Lyso-PC) during storage at 4°C for 3 months.

[0015] For example, the present invention provides an irinotecan liposome composition comprising a stabilized irinotecan liposome encapsulating irinotecan sucrose octasulfate (SOS) in a unilamellar lipid bilayer vesicle having a diameter of approximately 110 nm, the vesicle consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and methoxy-terminated polyethylene glycol (MW2000)-distearoylphosphatidylethanolamine (MPEG-2000-DSPE). The stabilized irinotecan liposome comprises: (a) a scavenger liposome encapsulating irinotecan as TEA8SOS at a concentration of 0.4 to 0.5 M (relative to sulfate group concentration) of TEA cation and SOS scavenger, with 500 g (±10%) of irinotecan moiety per 1 mol of phospholipid; and (b) contacting the irinotecan SOS liposomes with 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) under conditions effective to load the liposomes and release triethylammonium (TEA) cations from the entrapment liposomes to form sucrose octasulfate (Irinotecan SOS) liposomes; and (b) combining the Irinotecan SOS liposomes with 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) to obtain an Irinotecan liposome composition having a pH of 7.25 to 7.50, and which is stabilized to form less than 10 mol % (relative to the total amount of phosphatidylcholine in the Irinotecan liposome composition) of lyso-phosphatidylcholine (Lyso-PC) during storage at 4°C for 3 months.

[0016] The liposomal irinotecan composition is useful in treating patients diagnosed with various forms of cancer. For example, liposomal irinotecan can be administered to treat small cell lung cancer (SCLC) without other antitumor agents. In some embodiments, the liposomal irinotecan composition is administered in combination with other antitumor agents. For example, a liposomal irinotecan composition, 5-fluorouracil, and leucovorin (without other antitumor agents) can be administered to treat patients diagnosed with metastatic adenocarcinoma of the pancreas with disease progression after gemcitabine-based therapy. A liposomal irinotecan composition, 5-fluorouracil, leucovorin, and oxaliplatin (without other antitumor agents) can be administered to treat patients diagnosed with previously untreated pancreatic cancer. A liposomal irinotecan composition, 5-fluorouracil, leucovorin, and an EGFR inhibitor (e.g., an oligoclonal antibody EGFR inhibitor such as MM-151) can be administered to treat patients diagnosed with colorectal cancer.

[0017] Unless otherwise specified herein, the liposome compositions contain an amount of irinotecan (free base or salt form) in grams to moles of phospholipid in a ratio equivalent to that provided by either 471 g or 500 g (±10%) of irinotecan free base per mole of phospholipid.

[0018] As used herein (and unless otherwise specified), "irinotecan moiety" refers exclusively to irinotecan lactone, i.e., irinotecan lactone free base anhydrate.

[0019] As used herein (and unless otherwise specified), the term "camptothecin" includes camptothecin and camptothecin derivatives (irinotecan, topotecan, lurtotecan, ciratecan, etirinotecan pegol, TAS103, 9-aminocamptothecin, 7-ethylcamptothecin, 10-hydroxycamptothecin, 9-nitrocamptothecin, 10,11-methylenedioxycamptothecin, 9-amino-10,11-methylenedioxycamptothecin, 10,11-methylenedioxy- ... Camptothecin, 9-chloro-10,11-methylenedioxycamptothecin, (including 7-(4-methylpiperazinomethylene)-10,11-ethylenedioxy-20(S)-camptothecin, 7-(4-methylpiperazinomethylene)-10,11-methylenedioxy-20(S)-camptothecin and 7-(2-N-isopropylamino)ethyl)-(20S)-camptothecin, and stereoisomers, salts and esters thereof).

[0020] As used herein (and unless otherwise specified), "DLS" refers to dynamic light scattering and "BDP" refers to bulk drug product.

[0021] In some embodiments, the liposomes of the present invention encapsulate one or more agents (hereinafter referred to as entrapment agents) that entrap pharmaceutical agents within the liposome.

[0022] As used herein, a "sustained release composition" refers to a 70 mg / m irinotecan free base administered once every two weeks. 2 When administered to humans at a dose corresponding to the following pharmacokinetic parameters: Cmax 37.2 (8.8) μg irinotecan (as anhydrous free base) / mL and AUC 0-∞ 1364 (1048) h·μg irinotecan / mL (for irinotecan); or (for SN-38), Cmax 5.4 (3.4) μg SN-38 (as anhydrous free base) / mL; AUC 0-∞ Contains an irinotecan composition that yields 80–125% of SN-38 / mL of 620(329) h·ng.

[0023] Unless otherwise indicated, liposome preparations can include vesicles (e.g., spherical or substantially spherical) having at least one lipid bilayer, and may optionally include multilamellar and / or unilamellar vesicles, as well as vesicles that encapsulate and / or do not encapsulate a pharmaceutically active compound (e.g., camptothecin) and / or entrapment agent(s). For example, unless otherwise indicated, a pharmaceutical liposome preparation comprising camptothecin liposomes may optionally include liposomes that do not contain the camptothecin compound, including a mixture of unilamellar and multilamellar liposomes with or without the camptothecin compound(s) and / or entrapment agent(s). [Brief explanation of the drawings]

[0024] [Figure 1A] FIG. 1A shows a schematic diagram of an irinotecan liposome encapsulating an aqueous space containing irinotecan in a gel or precipitated state as a salt of sucrose octasulfate.

[0025] [Figure 1B] FIG. 1B shows an equatorial cross section of the irinotecan liposome in FIG. 1A.

[0026] [Figure 2A] FIG. 2A is a graph of stability ratio values ​​versus the relative amount of lyso-PC (mol%) for liquid irinotecan liposomal compositions after 9 months of storage at 4° C., the liposomal compositions having the indicated pH values ​​after manufacture but before storage.

[0027] [Figure 2B] FIG. 2B is a graph of stability ratio values ​​versus the relative amount of lyso-PC (mol%) for liquid irinotecan liposomal compositions after 6 months of storage at 4° C., the liposomal compositions having the indicated pH values ​​after manufacture but before storage.

[0028] [Figure 2C]FIG. 2C is a graph of stability ratio values ​​versus the relative amount of lyso-PC (mol%) for liquid irinotecan liposomal compositions after 6 months of storage at 4° C., the liposomal compositions having the indicated pH values ​​after manufacture but before storage.

[0029] [Figure 3A] FIG. 3A is a graph of the relative amount of lyso-PC (mol %) versus months of storage at 4° C. for two irinotecan liposomal compositions having a stability ratio of 1047 and a pH of 6.5.

[0030] [Figure 3B] FIG. 3B is a graph of the relative amount of lyso-PC (mol%) versus months of storage at 4° C. for two irinotecan liposomal compositions having stability ratios of 992 and 942, respectively, and a pH of 6.5 after manufacture but before storage.

[0031] [Figure 3C] FIG. 3C is a graph of the relative amount of lyso-PC (mol %) versus months of storage at 4° C. for an irinotecan liposomal composition having a stability ratio of 785 and a pH of 6.5 after manufacture but before storage.

[0032] [Figure 3D] FIG. 3D is a graph of the relative amount of lyso-PC (mol%) versus months of storage at 4° C. for two irinotecan liposomal compositions having a pH of 6.5 after manufacture but before storage, and having a stability ratio of approximately 724, prepared using TEA8SOS with a sulfate group concentration of 0.65 M.

[0033] [Figure 4A]Figure 4A is a graph of the relative amount of lyso-PC (mol%) versus months of storage at 4°C for three irinotecan liposome compositions having a stability ratio of approximately 1047 and a pH of 7.25 after manufacture but before storage. Liposome Sample 5 (open squares) was prepared with an irinotecan moiety concentration equivalent to that provided by 5 mg / mL of irinotecan hydrochloride trihydrate, while liposome Sample 13 (closed triangles) was similarly prepared with 20 mg / mL of irinotecan hydrochloride trihydrate. The liposomes in Sample 13 were prepared in the same manner as Sample 5, but the final liposome composition contained four times more liposome components (i.e., phospholipids, cholesterol, irinotecan, and sucrosophate) per milliliter than Sample 5.

[0034] [Figure 4B] FIG. 4B is a graph of the relative amount of lyso-PC (mol%) versus months of storage at 4° C. for two irinotecan liposomal compositions having a stability ratio of approximately 1047 and pH values ​​of 7.25 and 7.5 after manufacture but before storage.

[0035] [Figure 4C] FIG. 4C is a graph of the relative amount of lyso-PC (mol%) versus months of storage at 4° C. for two irinotecan liposomal compositions having a stability ratio of approximately 785 and pH values ​​of 7.25 and 7.5 after manufacture but before storage.

[0036] [Figure 5] FIG. 5 is a graph of lyso-PC concentration (mg / mL) versus months of storage at 4° C. for three irinotecan liposomal compositions having stability ratios of 1046 to 1064 and a pH of 7.3 after manufacture but before storage.

[0037] [Figure 6] FIG. 6 is a graph of the concentration of lyso-PC (mg / mL) versus months of storage at 4° C. in three irinotecan liposomal compositions having stability ratios of 1046 to 1064 and a pH of 7.3 after manufacture but before storage.

[0038] [Figure 7] FIG. 7 is a graph of the estimated rate of lyso-PC formation (mg / mL / month) during storage at 4° C. in irinotecan liposomal compositions with varying amounts of substituted ammonium (protonated TEA).

[0039] [Figure 8] 8 is a graph of the gram equivalents of irinotecan and sucrose in precipitates formed by combining irinotecan hydrochloride trihydrate and triethylammonium sucrose in aqueous solution in various ratios, i.e., gram equivalent ratios ranging from 1:9 to 9:1. The x-axis shows the relative gram equivalents of the total amount of triethylammonium sucrose (SOS) in the sample to the gram equivalents of anhydrous irinotecan free base.

[0040] [Figure 9] Figure 9 shows a graph containing a linear regression to the data obtained for each sample plotting the mean particle size of 12 different irinotecan octasulfate sucrose liposome product lot numbers stored at 4°C over periods ranging from 12 to 36 months.

[0041] [Figure 10] FIG. 10 is a graph containing a linear regression of the particle size polydispersity index (PDI) of the irinotecan octasulfate sucrose product lot numbers shown in FIG. 9 to the data obtained for each sample.

[0042] [Figure 11A] FIG. 11A is a graph containing linear regressions of the pH of 13 different irinotecan octasulfate sucrose product lot numbers stored at 4° C. over periods ranging from 12 to 36 months onto the data obtained for each sample.

[0043] [Figure 11B]FIG. 11B is a graph containing linear regressions of the pH of 16 different irinotecan octasulfate sucrose product lot numbers stored at 4° C. over a 12-month period onto the data obtained for each sample.

[0044] [Figure 12] FIG. 12 is a graph of the concentration (mg / mL) of lyso-PC in two irinotecan liposomal compositions over 36 months and the best-fit linear regression to each data point obtained from each irinotecan liposomal sample.

[0045] [Figure 13A] FIG. 13A is a representative chromatogram at full scale for Method A.

[0046] [Figure 13B] FIG. 13B is a representative chromatogram on an expanded scale for Method A. DETAILED DESCRIPTION OF THE INVENTION

[0047] Detailed Description Stabilized camptothecin compositions can include liposomes encapsulating one or more camptothecin compounds. Liposomes can be used to administer pharmaceuticals, including chemotherapeutic drugs. The present invention provides stabilized phospholipid-containing compositions of camptothecin compounds, such as liposomal irinotecan, which produce lower amounts of lyso-phospholipids, e.g., lyso-PC.

[0048] Camptothecin liposomes contain an entrapment agent (e.g., phosphorus) inside the lipid composition. Camptothecin can be encapsulated by lipid-containing vesicles. For example, FIG. 1A shows a schematic diagram illustrating an irinotecan liposome having a diameter of approximately 110 nm and a lipid membrane encapsulating irinotecan. The lipid membrane in this schematic contains the ester-containing phospholipid MPEG-2000-DSPE. The MPEG-2000-DSPE lipid is located in the inner and outer lipid layers of the bilayer membrane, such that the PEG moiety is located within the liposome or on the outer surface of the liposome, respectively. FIG. 1B shows a cross-sectional view of a particular embodiment of the liposome generally depicted in FIG. 1A, in which the unilamellar lipid bilayer membrane contains DSPC, cholesterol, and MPEG-2000-DSPE and encapsulates irinotecan sucrose octasulfate.

[0049] It has now been discovered that novel stabilized irinotecan liposome compositions comprising ester-containing phospholipids can be prepared that have low levels of lyso-PC even after prolonged storage at 2-8°C, such as 4°C, including liposomes encapsulating irinotecan sucrose octasulfate (SOS) (irinotecan-SOS liposomes), and that significantly reduce lyso-PC formation during refrigerated storage. The present invention is based, in part, on several unexpected observations. First, irinotecan-SOS liposome compositions surprisingly have substantially less lyso-PC during refrigerated storage as the amount of encapsulated irinotecan increases relative to the amount of co-encapsulated SOS scavenger. Second, irinotecan-SOS liposome compositions surprisingly have low lyso-PC during refrigerated storage when the pH of the aqueous medium containing irinotecan-SOS liposomes after manufacture but before storage is greater than 6.5. Third, irinotecan-SOS liposome compositions surprisingly have low lyso-PC when the amount of residual ammonium / substituted ammonium cations of the liposome-entrapped agent assayed in the composition is less than 100 ppm. Constituent lipids of the liposomal camptothecin composition

[0050] A variety of lipids, especially phospholipids, can be the components of liposomes, such as phosphatidylethanolamine and phosphatidylserine, are known in the art, and it is within the skill of the art to use such other phospholipids to make liposomes.In some embodiments, the liposome of the present invention is composed of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol and methoxy-terminated polyethylene glycol (MW2000)-distearoylphosphatidylethanolamine (MPEG-2000-DSPE).With regard to the lipids present in the liposome preparation disclosed herein, the following preferred embodiments are described.

[0051] The liposome components can be selected to produce a liposome bilayer membrane that forms unilamellar and / or multilamellar vesicles that encapsulate and retain the active agent until it is delivered to the tumor site. Preferably, the liposome vesicles are unilamellar. The liposome components are selected for their properties that, when combined, produce liposomes that can actively load and retain the active agent while maintaining low protein binding in vivo, thereby extending their circulation life.

[0052] DSPC is preferably the predominant lipid component in the bilayer of the liposomes that encapsulate irinotecan (e.g., accounting for 74.4% of the total weight of all lipid components). DSPC has a phase transition temperature (Tm) of 55°C.

[0053] Cholesterol can preferably account for about 24.3% of the total weight of all lipid components. Cholesterol can be incorporated in an amount effective to stabilize the liposomal phospholipid membrane so that it is not disrupted by plasma proteins, reducing the binding of plasma opsonins responsible for rapid clearance of liposomes from the circulation and reducing the permeability of solutes / drugs combined with the bilayer-forming phospholipids.

[0054] MPEG-2000-DSPE can preferably account for about 1.3% of the total weight of all lipid bilayer components. Its amount and presence on the surface of irinotecan liposomes can be selected to achieve a minimal steric barrier that prevents liposome aggregation. Liposomes coated with MPEG-2000-DSPE of the present invention have been shown to be stable in terms of size and drug encapsulation.

[0055] In some embodiments, the lipid membrane of the liposome preparation is preferably composed of the following components: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and methoxy-terminated polyethylene glycol (MW2000)-distearoylphosphatidylethanolamine (MPEG-2000-DSPE) in a ratio of approximately 1 polyethylene glycol (PEG)-modified phospholipid molecule for every 200 non-PEG-phospholipid molecules.

[0056] In a preferred embodiment, the liposomes of the present invention are made from a mixture of DSPC, cholesterol, and MPEG-2000-DSPE combined in a molar ratio of 3:2:0.015. In a preferred embodiment, the liposome preparation of the present invention comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) at a concentration of about 6.81 mg / mL, cholesterol at a concentration of about 2.22 mg / mL, and methoxy-terminated polyethylene glycol (MW2000)-distearoylphosphatidylethanolamine (MPEG-2000-DSPE) at a concentration of about 0.12 mg / mL.

[0057] In a further preferred embodiment, the liposome preparation of the present invention comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) at a concentration of 6.81 mg / mL, cholesterol at a concentration of 2.22 mg / mL, and methoxy-terminated polyethylene glycol (MW2000)-distearoylphosphatidylethanolamine (MPEG-2000-DSPE) at a concentration of 0.12 mg / mL. Camptothecin Composition Scavenger

[0058] In some embodiments, the liposomes of the present invention encapsulate one or more agents (hereinafter referred to as "trapping agents") that trap pharmaceuticals within the liposome. The trapping agent preferably comprises a polyanionic compound having multiple negatively charged groups, or a combination of two or more different such compounds. In a non-limiting example, the polyanionic trapping agent is a divalent anion, a trivalent anion, a polyvalent anion, a polymeric polyanion, a polyanionized polyol, or a polyanionized sugar. In the context of the present invention, the polyanionic trapping agent can be a polyanionized polyol or sugar, such as a polyol or sugar whose hydroxyl groups have been completely or partially modified or replaced (anionized) by anionic groups. In a non-limiting example, the polyanionized polyol or polyanionized sugar can comprise a polyol or sugar moiety with an anionic group attached thereto. Preferably, when in aqueous medium, at least one anionic group of the polyanionized sugar or polyanionized polyol sequestering agent is greater than 50% ionized in the pH range of 3 to 12, preferably 6.5 to 8, or alternatively, the anionic group(s) have a pKa of 3 or less, preferably 2 or less. In preferred embodiments, the sequestering agent contains a sulfate moiety having a pKa of 1.0 or less. In non-limiting examples, the polyanion sequestering agent can have a charge density of at least 2, 3, or 4 negatively charged groups per unit, e.g., per carbon atom or ring in a carbon chain, or per monosaccharide unit in a sugar.

[0059] In some embodiments of the present invention, the release rate of a liposome composition can be enhanced by using as a sequestering agent a mixture of a polyanionized sugar or polyanionized polyol with one or more other mono- or polyvalent anions, such as chloride, sulfate, phosphate, etc. In another non-limiting example of enhancing the release rate of a sustained release composition, a mixture of different polyanionized sugars and / or polyols with varying degrees of polyanionization is used as a sequestering agent.

[0060] In some embodiments, the degree of polyanionization inside the liposomes of the present invention will be, for example, greater than 90%, greater than 99%, or between 0.1% and 99%, 10% and 90%, or 20% and 80% of the total anion(s) inside the liposome due to the camptothecin or camptothecin derivative compound entrapped in the liposome.

[0061] In some embodiments, the sequestrant is a sulfated sugar and / or a polyol. Exemplary sulfated sugars of the present invention are sulfated sucrose, including, but not limited to, sucrose hexasulfate, sucrose heptasulfate, and sucrose octasulfate (Ochi, K. et al., 1980, Chem. Pharm. Bull., Vol. 28, pp. 638-641). Similarly, in the presence of a base catalyst, Reaction with phosphorus oxychloride or diethylchlorophosphate results in polyphosphorylated polyols or sugars. Polyphosphorylated polyols can also be isolated from natural sources. For example, inositol polyphosphates, such as inositol hexaphosphate (phytic acid), can be isolated from corn. Various sulfated, sulfonated, and phosphorylated sugars and polyols suitable for practicing the present invention are disclosed, for example, in U.S. Pat. No. 5,783,568, the entire contents of which are incorporated herein by reference. Complexation of polyols and / or sugars with more than one molecule of boric acid also results in polyanionized (polyboronated) products. Reaction of polyols and / or sugars with carbon disulfide in the presence of alkali results in polyanionized (polydithiocarbonated, polyxanthated) derivatives. The polyanionized polyol or sugar derivative can be isolated in its free acid form and neutralized with a suitable base, such as an alkali metal hydroxide, ammonium hydroxide, or preferably with a substituted amine, such as the amine corresponding to the substituted ammonium salt of the present invention, either neat or in the form of a substituted ammonium hydroxide to provide the polyanionic salt of the substituted ammonium salt of the present invention. Alternatively, the sodium, potassium, calcium, barium, or magnesium salt of the polyanionized polyol / sugar can be isolated and converted to a suitable form, such as the substituted ammonium salt, by any known method, such as by ion exchange. Non-limiting examples of sulfated sugar sequestrants are sulfated sucrose compounds, including, but not limited to, sucrose hexasulfate, sucrose heptasulfate, and sucrose octasulfate (SOS). Exemplary polyol sequestrants include inositol polyphosphates, such as inositol hexaphosphate (also known as phytic acid or IHP) or sulfated forms of other disaccharides.

[0062] In a preferred embodiment of the present invention, the sequestering agent is a sulfated polyanion, a non-limiting example of which is sucrose octasulfate (SOS). Sucrosophate is also referred to as sucrose octasulfate or sucrooctasulfate (SOS). Methods for preparing sucrosophate in various salt forms, such as ammonium, sodium, or potassium salts, are well known in the art (e.g., U.S. Pat. No. 4,990,610, the entire contents of which are incorporated herein by reference). Sucrose octasulfate (also referred to as sucrosophate) in its fully protonated form has the structure of formula (II): [ka] is a fully substituted sulfate of sucrose having the formula:

[0063] Methods for preparing sucrosophate in various salt forms, such as the ammonium, sodium, or potassium salts, are well known in the art (see, e.g., U.S. Pat. No. 4,990,610, incorporated herein by reference in its entirety). Similarly, sulfated forms of other disaccharides, such as lactose and maltose, which yield octasulfate lactose and octasulfate maltose, are also contemplated.

[0064] In some embodiments, the liposome formulations of the present invention comprise a camptothecin compound, such as irinotecan or topotecan, and an anionic sequestering agent, such as SOS. The liposomes of the present invention preferably comprise the camptothecin compound in a stoichiometric ratio with the anionic sequestering agent. For example, an irinotecan liposome formulation can encapsulate irinotecan and sucrose octasulfate at a molar ratio of about 8:1. The stabilized composition of the liposome can encapsulate an irinotecan composition of formula (I), where x is about 8: [ka] The liposomal irinotecan may comprise a composition of formula (I) encapsulated in a liposome. Preferably, the composition of formula (I) is formed (e.g., precipitated) within a liposome comprising cholesterol and one or more phospholipids (e.g., including PEG-containing phospholipids). For example, the compound of formula (I) may be formed within a liposome by reacting (1) a camptothecin compound(s) (e.g., irinotecan, topotecan, etc.) with (2) a liposome encapsulating a polysulfated anionic scavenger (e.g., sucrose octasulfate) in the step of forming a stabilized liposomal irinotecan composition. Preferably, the liposomal irinotecan composition has a pH greater than 6.5 (e.g., 7.0 to 7.5, including 7.25, 7.3, and 7.5).

[0065] A preferred stabilized camptothecin composition comprises liposomal irinotecan.

[0066] The stabilized camptothecin composition comprises a liposomal formulation of a high density camptothecin compound(s) containing irinotecan or a salt thereof at an irinotecan moiety concentration equivalent to that provided by 4.5-5.5 mg / mL of irinotecan hydrochloride trihydrate (i.e., 3.9-4.8 mg / mL of anhydrous irinotecan free base), and DSPC at a concentration of 6.13-7.49 mg / mL (preferably about 6.81 mg / mL), 2-2.4 mg The present invention comprises liposomal compositions of pharmaceutical camptothecin compound(s) that can be stored under refrigeration (i.e., 2-8°C) for at least the first six months, preferably at least the first nine months, after manufacture, without the formation of lyso-PC levels greater than 20 mol%. More preferably, the present invention provides compositions containing an amount of irinotecan moiety equivalent to that provided by between 4.7 and 5.3 mg / mL of irinotecan hydrochloride trihydrate (i.e., 4.1 to 4.6 mg of the anhydrous free base of the irinotecan moiety) (irinotecan can be present as the octasucrose sulfate salt encapsulated within liposomes), together with 6.4 to 7.2 mg / mL (DSPC), 2.09 to 2.35 mg / mL cholesterol, and about 0.113 to 0.127 mg / mL MPEG-2000-DSPE, which compositions contain no more than 20 mol% lyso-PC at 6 or 9 months when stored at 2-8°C, or no more than 2 mg / mL lyso-PC at 21 months when stored at 2-8°C. Calculation of the gram equivalent ratio (ER) of irinotecan / sulfate compounds

[0067] For each irinotecan liposomal preparation, the gram equivalent ratio (ER) of irinotecan / sulfate compound was determined by determining the molar amounts of irinotecan (I) and sulfate compound (S) co-encapsulated by the liposome per unit of liposome composition (e.g., 1 mL) using the following formula: ER = I / (SN), where N is the valency of the sulfate compound anion (e.g., for sucrosofate, N is 8, and the free sulfate SO4 2- (where N is 2). For example, a liposomal irinotecan-sucrose composition containing 7.38 mM irinotecan and 1.01 mM sucrose (N=8) would have an ER of 7.38 / (1.01 x 8) = 0.913. Preferably, the sulfate compound (S) is sucrose octasulfate, which contains 8 sulfate moieties per mole of SOS. The liposomal composition has a pH of 7.1-7.5 and one of the following ER ranges: preferably, 0.85-1.2, 0.85-1.1, or, most preferably, 0.9-1.05, such as about 1.02. Alternatively, the liposome composition has an irinotecan moiety in an amount equivalent to that provided by 500 g (±10%) of anhydrous irinotecan free base per mole of phospholipid and has one of the following ER ranges: preferably, 0.85 to 1.1, most preferably, 0.9 to 1.05, such as about 1.02. pH of stabilized camptothecin compositions

[0068] The pH of the liposome composition can be adjusted or otherwise selected to achieve the desired storage stability characteristics (e.g., reduced intraliposomal lyso-PC formation during storage at 4°C for 180 days) by, for example, preparing a composition at a pH of about 6.5 to 8.0, or any suitable pH value therebetween (e.g., including 7.0 to 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. Liposome compositions having a particular pH value and equivalent irinotecan free base concentration (mg / mL) to that provided by anhydrous irinotecan free base can be used. Irinotecan liposomes containing irinotecan moieties and varying concentrations of sucrose octasulfate were prepared as described in further detail herein. More preferably, the pH after manufacture and before storage is between 7.1 and 7.5, even more preferably between about 7.2 and 7.3, and most preferably about 7.25. The pH can be adjusted by standard means, for example, by using 1N HCl or 1N NaOH, as appropriate.

[0069] In some embodiments of the invention, the pH of the liposomal irinotecan preparation after manufacture but before storage is greater than 6.5, preferably between 7.2 and 7.3. In some embodiments of the invention, the pH is between 7.2 and 7.5. Compound Gram Equivalent Ratio ("ER") of Stabilized Camptothecin Compositions

[0070] The stabilized liposomal camptothecin composition can have a pH greater than 6.5 and includes liposomes encapsulating irinotecan and a sulfate polyanionic sequestering agent having a gram equivalent ratio ("ER") of irinotecan / sulfate compound greater than 0.9 (e.g., 0.9-1.1). For irinotecan SOS liposomal preparations, the ER is determined by the molar amount of irinotecan (I) and sulfate compound (S) co-encapsulated by the liposome per unit of liposomal composition (e.g., 1 mL) using the following formula: ER = I / (SN), where N is the valency of the sulfate compound anion (e.g., for sucrosophate, N is 8, and the free sulfate S04 is 0.04). 2- where N is 2, I is the concentration of encapsulated irinotecan in the liposomal irinotecan composition, and S is the concentration of sulfate groups of sucrose octasulfate encapsulated in the liposomal irinotecan composition. Preferably, the sulfate compound (S) is sucrose octasulfate, which contains 8 sulfate moieties per mole of SOS.

[0071] Although it is preferable to directly determine the sulfate group concentration (S·N) of the sucrose octasulfate encapsulated in the liposomal irinotecan composition, S·N can be determined from the liposomal phospholipid concentration (P, mol / L), the SOS sulfate group concentration in the liposomal interior space (SOS sulfate group concentration in the solution used to prepare the entrapment liposomes; parameter B, see the definition of the stability ratio herein), and the liposomal (entrapped) volume per unit of liposomal phospholipid, i.e., the volume sequestered within the interior space of the liposomal vesicle (Ve, L / mol of phospholipid): S N = P Ve B

[0072] As an example, for phosphatidylcholine-cholesterol liposomes obtained by extrusion through 100 nm polycarbonate filters, the entrapped volume can approach 1.7 L / mol of phospholipid (Mui et al., 1993, Biophys. J., 65:44). In this case, quantitative loading of irinotecan (molecular weight 586.7) into SOS-encapsulated liposomes was achieved at 471 g / mol of phospholipid and a SOS sulfate group concentration of 0.45 M. (471 / 586.7) / (1.7 0.45)=1.049 It becomes ER.

[0073] On the other hand, at an SOS concentration of 0.65M sulfate groups, the ER (471 / 586.7) / (1.7 0.65)=0.726 This becomes:

[0074] Similarly, quantitative loading of irinotecan (molecular weight 586.7) into SOS-encapsulated liposomes at 500 g (±10%) / mol of phospholipid and an SOS sulfate group concentration of 0.45 M resulted in an ER of approximately 1.11, whereas an SOS sulfate group concentration of 0.65 M resulted in an ER of approximately 0.77. Preparation of Stabilized Camptothecin Compositions

[0075] The stabilized camptothecin composition can comprise camptothecin liposome. Liposomes are used for the administration of pharmaceuticals, including chemotherapeutic drugs. Various techniques related to drug-encapsulated liposomes and their preparation methods are generally known in the art, and therefore will not be described in any further detail herein.See, for example, U.S. Patent No. 8,147,867, the entire contents of which are incorporated herein by reference.

[0076] In some embodiments, liposomes encapsulating one or more camptothecin compounds within the vesicles comprise at least one phospholipid. The camptothecin compounds can be loaded or otherwise entrapped within the liposomes in a multi-step process including, for example, (a) forming a sequestering agent liposome encapsulating an anionic sequestering agent and a cation within a liposomal vesicle comprising a phospholipid(s), and (b) then contacting the sequestering agent liposome with the camptothecin compound(s) under conditions effective to load the sequestering agent liposomes with the camptothecin compound(s) and retain the camptothecin compound(s) inside the sequestering agent-containing liposomes to form camptothecin liposomes.

[0077] Camptothecin compound(s) can be loaded into the capture agent liposomes using a gradient across the liposome membrane, which forces the camptothecin compound(s) into the capture agent liposomes, resulting in the formation of camptothecin liposomes. Preferably, the capture agent liposomes have a transmembrane concentration gradient of a membrane-permeable cation, such as ammonium or a substituted ammonium, effective to cause the exchange of the camptothecin compound(s) for ammonium / substituted ammonium in the capture agent liposomes when heated above the phase transition temperature of the lipid components of the liposomes. Preferably, the capture agent has a higher concentration in the capture agent liposome than in the medium surrounding the capture agent liposomes. Furthermore, the capture agent liposomes can contain one or more transmembrane gradients in addition to the gradient generated by the ammonium / substituted ammonium cations. For example, the liposomes contained in the capture agent liposome composition can additionally or alternatively contain a transmembrane pH gradient, an ion gradient, an electrochemical potential gradient, and / or a solubility gradient.

[0078] In some embodiments, the sequestering agent (e.g., SOS and / or another sulfated polyol sequestering agent (including acceptable salts thereof)) used to prepare liposomes has a sulfate group concentration of 0.3-0.8, 0.4-0.5, 0.45-0.5, 0.45-0.0475, 0.45-0.5, 0.3, 0.4, 0.45, 0.475, 0.5, 0.6, 0.7, or 0.8 M (e.g., ±10% of these specified values). In preferred embodiments, the sequestering agent used to prepare liposomes is SOS and has a sulfate group concentration of about 0.45 or about 0.475 M. In more preferred embodiments, the sequestering agent used to prepare liposomes is SOS and has a sulfate group concentration of 0.45 or 0.475 M.

[0079] Preferably, the camptothecin compound(s) are loaded into the capture agent liposomes by incubating the camptothecin compound(s) with the capture agent liposomes in an aqueous medium at a suitable temperature, e.g., above the first-order phase transition temperature of the constituent phospholipids, during loading, and then lowering the temperature to below the first-order phase transition temperature of the constituent phospholipids, preferably about room temperature, after loading. The incubation time typically depends on the constituent lipids, the nature of the camptothecin compound(s) loaded into the liposomes, and the incubation temperature. Incubation times ranging from a few minutes (e.g., 30-60 minutes) to several hours are typically sufficient.

[0080] Because high entrapment efficiencies, greater than 85%, typically greater than 90%, are achieved, it is often not necessary to remove unentrapped entities. However, if such is required, the unentrapped camptothecin compound(s) can be removed from the composition by various means, such as, for example, size exclusion chromatography, dialysis, ultrafiltration, adsorption, and precipitation.

[0081] In some embodiments, the camptothecin liposomes are irinotecan liposomes. Irinotecan liposomes can be prepared by a process comprising: (a) preparing liposomes containing triethylamine (TEA) as the triethylammonium salt of sucrosophate (TEA-SOS); and (b) subsequently contacting the TEA-SOS liposomes with irinotecan under conditions effective to allow irinotecan to enter the liposomes and a corresponding amount of TEA to exit the liposomes (thereby eliminating or reducing the TEA concentration gradient across the resulting liposomes). Extraliposomal ionic strength during drug loading of camptothecin liposomes

[0082] In some embodiments of the present invention, liposomal camptothecin loading is carried out in aqueous solution at an ionic strength of less than 50 mM NaCl equivalent, or more preferably less than 30 mM NaCl equivalent. After drug loading, a higher concentration salt solution, e.g., NaCl solution, can be added to increase the ionic strength to greater than 50 mM NaCl equivalent, or more preferably greater than 100 mM NaCl equivalent, preferably between about 140-160 mM NaCl equivalent. Scavenger cation

[0083] The cations of the present invention can be encapsulated in the sequestrant liposomes in an amount effective to achieve loading of the camptothecin compound(s) into the sequestrant liposomes when heated above the phase transition temperature of the lipid components. The cations are selected so that the cations can exit the sequestrant liposomes while the liposomes are loaded with the camptothecin compound(s). The extraliposomal cations can be removed after preparation of the liposomes loaded with the camptothecin compound(s).

[0084] In some embodiments of the present invention, the cation in the liposome combined with the sequestering agent is a substituted ammonium compound. In some embodiments of the present invention, the substituted ammonium compound has a pKa of at least about 8.0. In some embodiments of the present 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 present invention, the substituted ammonium compound has a pKa of about 8.0-12.0, about 8.5-11.5, or about 9.0-11. In preferred embodiments, the pKa is about the pKa of TEA or about the pKa of DEA.

[0085] Non-limiting examples of such substituted ammonium compounds include compounds of the formula: N(R1)(R2)(R3)(R4) + wherein R1, R2, R3, and R4 are each independently hydrogen or an organic group having a total of up to 18 carbon atoms, and at least one of R1, R2, R3, and R4 is an organic group that is a hydrocarbon group having up to 8 carbon atoms, which can be an alkyl, alkylidene, heterocyclic alkyl, cycloalkyl, aryl, alkenyl, or cycloalkenyl group, or a hydroxyl-substituted derivative thereof, and optionally contains one or more S, O, or N atoms in the hydrocarbon portion that form ether, ester, thioether, amine, or amide bonds. The substituted ammonium can be a sterically hindered ammonium compound (e.g., having at least one organic group with a secondary or tertiary carbon atom directly bonded to the ammonium nitrogen atom). Similarly, at least one of R1, R2, R3, and R4 must be hydrogen. Preferably, the substituted ammonium cation is triethylammonium (protonated TEA) or diethylammonium (protonated DEA).

[0086] A camptothecin compound is loaded into liposomes encapsulating an anionic sequestering agent under conditions effective to form camptothecin compound liposomes, thereby reducing the concentration of substituted ammonium cations within the sequestering agent liposomes. Liposomes of the present invention can include an anionic sequestering agent and ammonium or substituted ammonium cations, which are subsequently removed and / or replaced by the camptothecin compound loaded into the liposomes in a subsequent drug loading step.

[0087] In a preferred embodiment, the concentration of ammonium or substituted ammonium cations within the camptothecin compound liposomes is sufficiently low to result in low amounts of lyso-PC after extended refrigerated storage of camptothecin liposome preparations containing phospholipids. For example, as discussed in Example 3, including the data in Figure 7, reduced lyso-PC formation was observed in irinotecan SOS liposome preparations having less than about 100 ppm, preferably between 20 and 80 ppm, preferably less than about 50 ppm, even more preferably less than about 40 ppm, and even more preferably less than 30 ppm of substituted ammonium cations.

[0088] In some embodiments, irinotecan SOS liposomes (e.g., Samples 24-29; Table 10 in the Examples) contain less than 100 ppm, or about 15-100 ppm, of substituted ammonium SOS scavenger counterions. In some embodiments, irinotecan SOS liposomes (e.g., Samples 24-29; Table 10 in the Examples) contain about 15-80 ppm of substituted ammonium. In some embodiments, irinotecan SOS liposomes (e.g., Samples 24-29; Table 10 in the Examples) contain about 40-80 ppm of substituted ammonium. In some embodiments, irinotecan SOS liposomes (e.g., Samples 24-29; Table 10 in the Examples) contain about 80-100 ppm of substituted ammonium. In preferred embodiments, the substituted ammonium present at any of the above ppm concentrations is derived from TEA or DEA. Stability Ratio of Stabilized Camptothecin Compositions

[0089] When phospholipid-based camptothecin-containing liposomes are prepared by reacting (1) the camptothecin drug with (2) liposomes encapsulating a polysulfated anionic sequestering agent, the stability of the resulting drug-loaded liposomes depends on the ratio of camptothecin, anionic sequestering agent, and liposome-forming phospholipid, as defined below, by a stability ratio of at least about 950. This stability ratio depends on the initial concentration of sulfate groups in the sequester-liposome and the ratio of camptothecin to phospholipid encapsulated in the liposome. As used herein, this stability ratio ("SR") is defined as follows: SR=A / B (In the formula, a. A is the amount of irinotecan moiety entrapped in the entrapment agent liposome during the drug loading step in gram equivalents relative to anhydrous irinotecan free base per mole of phospholipid in the composition; b. B is the concentration of sulfate groups in the sucrosophate (or other sequestering agent) solution used to make the sequestering agent liposomes, expressed in moles / L (based on the concentration of sulfate groups).

[0090] For the determination of the stability ratio, the number of moles of phospholipid in the liposome preparation is determined by an assay such as that described in the Examples, and the amount of irinotecan moiety (A above) is calculated accordingly to achieve liposome loading.

[0091] For stability ratio determination, the concentration of sulfate groups in a sucrosophate (or other sequestering agent) solution, B, expressed in moles / L, is calculated as the concentration of sucrosophate (or other sequestering agent disclosed herein) (moles / L) in the solution added to the lipid (which is typically dissolved in a volume of alcohol that is 10% or less of the volume of the sequestering agent solution added to the lipid). Thus, for sucrosophate, the concentration of sulfate groups, B, is the concentration of sucrosophate multiplied by 8 (the number of sulfate groups in one sucrosophate molecule), or multiplied according to the number of sulfate groups in the particular sequestering agent used. (See Example 1.)

[0092] In some embodiments of the present invention, both the stability ratio and pH are increased to greater than 6.5. Thus, in certain preferred embodiments of the present invention, the stability ratio is 942-1130, the pH is 7.2-7.5, and the irinotecan composition and the SOS scavenger 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 the SOS scavenger are present in the liposome at a molar ratio of 8:1. The amount of lyso-PL, particularly lyso-PC, in liposomal formulations encapsulating other camptothecin compounds can be similarly controlled.

[0093] For example, the novel stabilized irinotecan liposome preparation can have 80% less lyso-PC than irinotecan SOS liposomes prepared according to other processes (e.g., 80% less lyso-PC than observed in comparative sample 12 after 9 months of refrigerated storage). Sample 12 (comparison) liposomal irinotecan was prepared at a stability ratio of approximately 724 by heating a lipid mixture of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and methoxy-terminated polyethylene glycol (MW2000)-distearoylphosphatidylethanolamine (MPEG-2000-DSPE) in a molar ratio of 3:2:0.015 in the presence of triethylamine (TEA) and sucrose octasulfate ("SOS" or "sucrose sulfate") [(TEA)8SOS] in a molar ratio of 8:1 at a sulfate group concentration of 0.65 M to generate TEA-SOS sequestered liposomes. After removing the (TEA)8SOS not encapsulated in the TEA-SOS scavenger liposomes, the TEA was removed, and the resulting preparation containing TEA-SOS scavenger liposomes was loaded with irinotecan using a solution of irinotecan under conditions such that the total amount of irinotecan loaded into the liposomes was 500 g (±10%) of anhydrous irinotecan free base per mole of phospholipid in the TEA-SOS scavenger liposome preparation. The pH of the irinotecan liposome composition was 6.5 (measured according to the subsection "pH Measurement" in the Examples section of this specification), and each mL of irinotecan liposome composition contained 4.3 mg of irinotecan moieties. These phosphatidylcholine-containing liposomal irinotecan compositions generated levels of lyso-PC exceeding 30 mol% (relative to the total amount of phosphatidylcholine in the irinotecan liposomal composition) during 3 months of refrigerated storage (2-8°C) (and generated over 35 mol% lyso-PC over 9 months). Calculation of Stability Ratio and Lyso-PC Amount in Exemplary Embodiments

[0094] A series of different irinotecan liposome preparations were made according to the methods described herein (additional experimental details regarding the preparation and characterization of each sample are included in the Examples below). The amount of lyso-PC measured in each of the irinotecan liposome preparations is summarized in Table 1A (lyso-PC measurements taken after 9 months of refrigerated storage) and Table 1B (lyso-PC measurements taken after 6 months of refrigerated storage for a subset of the samples listed in Table 1A). Each irinotecan liposome preparation contained unilamellar bilayer liposomes with a diameter of approximately 110±20 nm, preferably 110±10 nm, encapsulating irinotecan via a sucrose octasulfate sequestering agent. Liposomes were formed from a mixture of DSPC, cholesterol, and MPEG-2000-DSPE with a molar ratio of 3:2:0.015 and then loaded with irinotecan at a concentration of approximately 471 g of irinotecan moiety per mole of phospholipid (irinotecan or its salt, resulting in an amount of irinotecan moiety equivalent to 500 g (±10%) of anhydrous irinotecan HCl). Each irinotecan liposome preparation contained different amounts of SOS scavenger and was formulated at different pH values. The amount of lyso-PC was measured in each irinotecan liposome preparation at various times, including measurements of all samples after 9 months of continuous refrigerated storage (4°C). All samples in Table 1A were loaded using protonated TEA as a counterion to SOS (i.e., irinotecan was loaded into liposomes encapsulating various concentrations of TEA8SOS, as specified in Table 1A). [Table 1A] a Measured according to Method B described herein

[0095] Figure 2A shows a plot illustrating the amount of lyso-PC measured in each sample in Table 1A after 9 months of storage at 4°C. Sample 12 is marked as a comparison in Table 1A and Figure 2A. Samples having both a stability ratio greater than about 900 and a 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°C. Figure 2C is a graph of stability ratio values ​​versus the relative amount of lyso-PC (mol%) for liquid irinotecan liposomal compositions after 6 months of storage at 4°C (data from Table 6). Data points marked with open circles correspond to irinotecan samples having a pH greater than 6.5 (7.25 or 7.5) measured after manufacture but before storage. Data points marked with diamonds correspond to irinotecan samples having a pH of 6.5 measured after manufacture but before storage. The stability ratio was calculated as defined herein during the manufacture of each sample. The mol% of lyso-PC was measured after the first 6 months of storage after manufacture of each sample. [Table 1B] b Measured according to Method B described herein

[0096] Figure 2B shows a plot illustrating the amount of lyso-PC measured in each sample in Table 1B after 6 months of storage at 4° C. Samples having both a stability ratio greater than about 989 and a 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° C.

[0097] 3A-3D are plots showing the mol% of lyso-PC in irinotecan liposomal preparations selected from Tables 1A and 1B, having a pH of 6.5. Lyso-PC was determined after storage of each sample at 4°C for 0, 1, 3, 6, 9, and / or 12 months. These plots include linear regression lines for the data as estimates for the rate of increase of lyso-PC (mol%) over time in each sample. For each figure, the slope, y-intercept, and R 2A summary of the values ​​is shown in Table 1C below. [Table 1C]

[0098] In some embodiments, the stability of irinotecan liposomal preparations containing irinotecan SOS encapsulated in liposomes approximately 100 nm in diameter (e.g., 100±20 nm) is significantly improved for irinotecan liposomes with stability ratios greater than 942. The effect of stability ratio on the formation of lyso-PC in liposomal preparations was evaluated by maintaining a constant drug loading ratio of 500 g (±10%) irinotecan moieties (relative to the anhydrous free base, as described above) to total phospholipids, but varying the concentration of SOS scavenger. Table 2 provides a summary of the amount of mol% lyso-PC detected in the irinotecan liposomal preparations in Table 1 formulated at the same pH as (comparison) Sample 12 (6.5) but with various concentrations of SOS scavenger (i.e., different stability ratios). Table 2 illustrates that for irinotecan liposomes containing an SOS scavenger and irinotecan, a stability ratio greater than 942 reduces the formation of lyso-PC during refrigerated storage. Reducing the amount of SOS scavenger by up to 30% (i.e., increasing the stability ratio) compared to the control irinotecan liposome preparation resulted in a modest increase in the amount of lyso-PC by approximately 1% after 9 months of refrigerated storage. However, increasing the amount of SOS scavenger in irinotecan liposome preparations with a stability ratio greater than 942 significantly and unexpectedly reduces the amount of lyso-PC (mol%) present after 9 months of refrigerated storage at 4°C. For example, subsequent increases in the stability ratio above 942 in 5% increments (i.e., a stability ratio of 992 for Sample 2) dramatically reduced the amount of lyso-PC (mol%) present by 34% compared to Sample 3, which is equivalent to a 33% reduction in the amount of lyso-PC (mol%) compared to Sample 12 (measured at 9 months of refrigerated storage at 4°C). Overall, after 9 months of refrigerated storage at 4°C, an approximately 28-51% reduction in lyso-PC (mol%) was achieved by increasing the stability ratio of irinotecan liposomes above 942 compared to comparator Sample 12. In some embodiments, an irinotecan SOS liposome composition has a stability ratio greater than 942.In a preferred embodiment, the irinotecan SOS liposomal preparation has a stability ratio of 942-1130, or greater (eg, a stability ratio of 992-1047). [Table 2]

[0099] Table 2 illustrates the importance of having a stability ratio greater than 942 (preferably greater than 950, most preferably greater than 992) when stabilizing irinotecan liposomes containing an SOS scavenger and irinotecan at pH 6.5 to reduce intraliposomal lyso-PC formation during refrigerated storage. Overall, a reduction of intraliposomal lyso-PC of approximately 28-51% during 6 months of storage at 4°C was achieved by preparing irinotecan liposome compositions at pH 6 with stability ratios greater than 950 (e.g., 950-1050). Reducing the concentration of SOS scavenger used in preparing the scavenger liposomes by up to 30% (i.e., improving the stability ratio) compared to the corresponding concentration of SOS scavenger used to prepare the comparative irinotecan liposome preparations (compare Samples 3 and 12) resulted in a small increase in the amount of lyso-PC by approximately 1% after 9 months of refrigerated storage. However, increasing the amount of SOS scavenger used to form the scavenger liposomes prior to irinotecan loading to form irinotecan liposome preparations with stability ratios of 992 or greater significantly and unexpectedly reduced lyso-PC formation after the first 9 months of refrigerated storage of the resulting irinotecan liposomes after manufacture. For example, the data in Table 2 show that a 5% increase in the stability ratio above 942 resulted in a 34% decrease in LysoPC after 9 months of storage at 4°C (compare Sample 2 with Sample 3). Increasing the stability ratio from 992 (Sample 2) to 1047 (a 6% increase in SR) resulted in a 26% decrease in Lyso-PC produced after 9 months of storage at 4°C (compare Sample 6 with Sample 2) and an 8% increase in Lyso-PC produced after 9 months of storage at 4°C (compare Sample 1 with Sample 2). Thus, preferred irinotecan SOS liposome compositions have a stability ratio of greater than 1000, including irinotecan SOS liposome preparations having a stability ratio of 1000-1200 or greater (eg, a stability ratio of 1053-111).

[0100] In some embodiments of the present invention, the stability of irinotecan liposomal preparations containing irinotecan SOS encapsulated in liposomes approximately 100±20 nm in diameter, preferably 100±10 nm, is significantly improved by increasing the pH of the preparation above pH 6.5 after manufacture but before storage. The effect of pH on the formation of lyso-PC in liposomal preparations was evaluated by maintaining a constant drug loading ratio of 471 g or 500 g of irinotecan moieties (relative to the anhydrous free base, as described above) per mole of phospholipid, but varying the final pH of the irinotecan liposomal composition. Table 3 provides a summary of the amounts of lyso-PC in the irinotecan liposomal preparations in Table 1 formulated at different pH values. Table 3A reports data from Table 1 for an irinotecan liposome preparation formed by loading liposomes (encapsulating TEA8SOS at a sulfate concentration of 0.6 M) with a total of 471 g of irinotecan moieties per mole of phospholipid (based on the anhydrous free base, as described above) (i.e., the stability ratio is 471 / 0.6, or 785). The percent change in lyso-PC formation was calculated for both Sample 4 and Sample 9 (both of which had a pH of 6.5 after manufacture but before storage). Table 3B reports data from Table 1 for an irinotecan liposome preparation formed by loading liposomes (encapsulating TEA8SOS at a sulfate concentration of 0.45 M) with a total of 471 g of irinotecan moieties per mole of phospholipid (based on the anhydrous free base, as described above) (e.g., the stability ratio is 471 / 0.45, or 1047). The % change in lyso-PC formation was calculated for both Sample 1 and Sample 6 (both of which had a pH of 6.5 after manufacture but before storage). [Table 3A] [Table 3B]

[0101] The data in Tables 3A and 3B above show that increasing the pH from 6.5 to 7.25 or 7.5 decreased the amount of lyso-PC by approximately 15-20% for irinotecan SOS liposomes with a stability ratio of 785 (Table 3A) and by approximately 20-70% for irinotecan SOS liposomes with a stability ratio of 1047 (Table 3B). This is in line with previous reports (Grit, M et al., "Hydroxylysis of partially saturated egg phosphatidylcholine") indicating that the optimal pH for minimizing phosphatidylcholine hydrolysis is 6.5. in aqueous liposome dispersions and the effect of cholesterol incorporation on hydrolysis kinetics,” The Journal of pharmacy and pharmacology (1993), Vol. 45, No. 6, pp. 490-495) .

[0102] 4A-4C illustrate plots showing the mol% of lyso-PC in irinotecan liposomal preparations selected from Tables 1A and 1B, having a pH of 7.25 or 7.5, measured after 0, 1, 3, 6, and / or 9 months of storage of each sample at 4° C. The plots include linear regression lines for the rate of increase of lyso-PC over time in each sample. The slope, y-intercept, and R 2A summary of the values ​​is shown in Table 4 below. Lower amounts of lyso-PC were observed in irinotecan liposomal preparation samples with stability ratios greater than 942 (e.g., 1047) and a pH of 7.25 or 7.5 (e.g., compare samples 5, 7, and 13 with sample 10 at pH 7.25 in Figures 4A and 4C, or compare sample 8 in Figure 4B with sample 11 in Figure 4C at pH 7.5). Similarly, in irinotecan liposomal preparations with stability ratios less than 942, more lyso-PC was measured after 9 months (e.g., 785 in samples 10 and 11, both of which had more than 20 mol% lyso-PC after 6 months, even at a pH greater than 6.5). [Table 4] [Table 5] Additional Camptothecin Compositions

[0103] The camptothecin composition can be a sustained-release composition comprising one or more camptothecin compounds and one or more phospholipids that generates a small amount of lyso-phospholipids after a period of refrigerated storage (i.e., 2-8°C) following manufacture of the camptothecin composition (e.g., beginning when the camptothecin composition is sealed in a sterile container for pharmaceutical administration).

[0104] The stabilized sustained-release composition can include a matrix composition comprising a camptothecin compound and a phospholipid or other component(s) that can be hydrolyzed to form lyso-phospholipids. The matrix composition can be configured as a liposome that encapsulates one or more camptothecin compounds within a vesicle that includes a phospholipid(s) and other components, such as cholesterol and a lipid covalently linked to PEG.

[0105] In some embodiments of the present invention, the matrix composition is stabilized by, for example, preparing the matrix composition at a particular pH effective to reduce the amount of lyso-phospholipid formation in a medium containing an amount of an anionic sequestering agent and an amount of a camptothecin compound, and the matrix composition.

[0106] In some embodiments of the present invention, the sustained release composition is a nanoparticle comprising irinotecan in releasable association with a composition comprising triethylammonium sucrosophate (SOS) and a lipid and / or a biocompatible polymer (e.g., a biodegradable polymer such as cyclodextrin, PGA (polyglycolic acid), and / or PLGA (poly(lactic-co-glycolic acid))).

[0107] In another example, the sustained-release formulation is a matrix composition (e.g., nanoparticles or polymers that releasably entrap or retain camptothecin or camptothecin derivative compounds) containing a compound, such as topotecan, ethirinotecan, and / or irinotecan, in releasable association. The matrix composition can include a biocompatible polymer, such as polyethylene glycol (PEG) or a functionally equivalent material. In a preferred embodiment, the biocompatible polymer is polyethylene glycol (MW2000). In a more preferred embodiment, the biocompatible polymer is methoxy-terminated polyethylene glycol (MW2000).

[0108] In some embodiments, the sustained-release formulation can include a camptothecin compound conjugated to a biocompatible polymer, such as a cyclodextrin or a cyclodextrin analog (e.g., sulfated cyclodextrin). For example, the sustained-release formulation can include a cyclodextrin-containing polymer chemically bonded to a camptothecin compound (e.g., irinotecan and / or SN-38). The cyclodextrin-camptothecin conjugate compound can be administered in a pharmaceutically acceptable dose. Examples of camptothecin-cyclodextrin conjugates include cyclodextrin-containing polymer conjugates and related intermediates.

[0109] In some embodiments of the present invention, the sustained release compositions comprising lipids and / or biocompatible polymers include a lipid matrix and / or complexing agent(s), such as a cyclodextrin-containing composition, that is formulated to retain the camptothecin compound(s) during storage and then release the compound(s) within the patient's body.

[0110] In some embodiments of the present invention, the matrix composition comprises a phospholipid, such as a phosphatidylcholine derivative, that is stabilized to reduce the formation of lyso-PC during refrigerated storage.

[0111] Preferably, the sustained release composition is prepared by a multi-step process including the following steps: (a) forming a matrix composition including a trapping agent, and (b) contacting the matrix with a camptothecin compound under conditions effective to stably retain the camptothecin compound in the resulting sustained release composition, including the trapping agent and camptothecin compound associated with the matrix composition, so as to enable the desired release of the camptothecin compound within the body of the subject upon administration to the subject.

[0112] In a preferred embodiment, the sustained-release composition of the present invention contains irinotecan or a salt thereof at a concentration of irinotecan moieties equivalent to that provided by 4.3 mg / mL of anhydrous irinotecan free base per mL while containing less than about 1 mg / mL (or less than about 20 mol%) of lyso-PC upon 6 months of refrigerated storage at 4° C. In a preferred embodiment, the sustained-release composition of the present invention contains irinotecan or a salt thereof at a concentration of irinotecan moieties equivalent to that provided by 4.3 mg / mL of anhydrous irinotecan free base per mL while containing less than about 2 mg / mL (or less than about 30 mol%) of lyso-PC upon 12 months of refrigerated storage at 2-8° C., more preferably at about 4° C.

[0113] The sustained-release composition can include liposomes. Liposomes typically comprise vesicles containing one or more lipid bilayers surrounding an aqueous interior. Liposome compositions typically contain liposomes in a medium, such as an aqueous fluid outside the liposome. Liposome lipids can include amphiphilic lipid components, such as phospholipids, e.g., phosphatidylcholine, that spontaneously form bilayer membranes when in contact with an aqueous medium. Liposomes can also include membrane-hardening components, such as sterols, e.g., cholesterol. In some cases, liposomes also contain lipids conjugated to hydrophilic polymers, such as polyethylene glycol (PEG) lipid derivatives, which can reduce the tendency of liposomes to aggregate and also have other beneficial effects. One such PEG-lipid is N-(methoxy-PEG)-oxycarbonyl-distearoyl-phosphatidylethanolamine, where the PEG moiety has a molecular weight of about 2000, i.e., MPEG-2000-DSPE. Liposomes, typically having sizes in the micron or submicron range, are well recognized for their ability to transport pharmaceutical substances, including anti-cancer drugs such as irinotecan, and to alter their pharmaceutical properties in a variety of beneficial ways. Methods for preparing and characterizing pharmaceutical liposome compositions are known in the art (see, for example, Lasic D., Liposomes: From physics to applications, Elsevier, Amsterdam, 1999, which are incorporated herein by reference in their entirety for all purposes). 3 years; G. Gregoriadis (ed.), Liposome Technology, 3rd edition, vols. 1-3, CRC Press , Boca Raton, 2006; Hong et al., U.S. Pat. No. 8,147,867 sea ​​bream).

[0114] In some embodiments, liposomes are prepared as described in one or more Examples or other embodiments herein, but the concentration of the final liposome composition is increased so that the formulation contains a concentration of the irinotecan moiety equivalent to about 10, 15, 20, 25, 30, 35, 40, 45, or 50 mg / mL of irinotecan hydrochloride trihydrate. In some embodiments, the concentration of the irinotecan moiety is equivalent to between 5-10, 10-20, 20-30, 30-40, or 40-50 mg / mL of irinotecan hydrochloride trihydrate. In some embodiments, the liposome compositions specified in this section are used to treat brain tumors or any other condition in a mammal, as described in U.S. Pat. No. 8,658,203, which is incorporated herein by reference in its entirety.

[0115] The liposome formulation encapsulating irinotecan can be an injectable formulation containing liposomes (including injectable formulations that can 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 added to liposomes containing one or more entrapment agents, where the irinotecan is present at a concentration of 200 g, 300 g, 400 g, 500 g, 600 g, or 700 g of irinotecan moieties equivalent to 200-300 g, 400-550 g, 450-600 g, or 600-700 g of anhydrous irinotecan free base ... Preferably, about 500 g (±10%) of irinotecan liposomes are loaded with irinotecan per mole of liposomal phospholipid, including 471 g of irinotecan moieties per mole of liposomal phospholipid of total irinotecan. Specific examples herein include measurements of stabilized irinotecan liposomes containing 471 g of irinotecan moieties per mole of total liposomal phospholipid, and irinotecan liposomes containing 500 g of irinotecan moieties per mole of total liposomal phospholipid.

[0116] In some embodiments, the concentration of the irinotecan moiety equivalent to that provided by the anhydrous irinotecan free base in the liposome preparation 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 irinotecan moiety equivalent to that provided by the anhydrous irinotecan free base in the liposome preparation 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 a preferred embodiment, the concentration of the irinotecan moiety in the liposome preparation is about 4.3 mg / mL of anhydrous irinotecan free base per mL, and in a more preferred embodiment, 4.3 mg / mL of anhydrous irinotecan free base per mL. The liposomal preparation can be a vial containing about 43 mg of anhydrous irinotecan free base in a liposomal preparation having a volume of about 10 mL, which can then be diluted (e.g., in 500 mL of a pharmaceutically acceptable diluent) prior to intravenous administration to a patient.

[0117] Accordingly, some embodiments of the present invention provide a method for producing an irinotecan liposome preparation, comprising stabilized irinotecan liposomes encapsulating sucrose octasulfate (SOS) in unilamellar lipid bilayer vesicles composed of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and methoxy-terminated polyethylene glycol (MW2000)-distearoylphosphatidylethanolamine (MPEG-2000-DSPE), the method comprising: (a) adding an irinotecan-containing solution to a solution containing irinotecan, encapsulating sucrose octasulfate (SOS) scavenger (derived from TEA8SOS) at a sulfate concentration of 0.4-0.5 M, and triethylammonium (TEA) cations, without irinotecan; The method includes the steps of: (a) contacting the entrapment liposomes with 500 g (±10%) of an irinotecan moiety per mole of phospholipid to form irinotecan SOS liposomes; and (b) combining the irinotecan SOS liposomes with 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) to obtain an irinotecan liposome preparation having a pH of 7.25 to 7.50, and being stabilized to form less than 10 mol % (relative to the total amount of phosphatidylcholine in the irinotecan liposomes) of lyso-phosphatidylcholine (lyso-PC) during storage at 4°C for 3 months.

[0118] Storage-stable irinotecan liposomes can be prepared in multiple steps, including forming TEA-containing liposomes, followed by loading irinotecan into the liposomes as the TEA exits the liposomes. The first step can involve forming TEA-sucrose-containing liposomes by hydrating and dispersing liposomal lipids in a solution of TEA-sucrose. This can be done, for example, by dissolving lipids containing DSPC and cholesterol in heated ethanol and then heating the dissolved and heated lipid solution to the transition temperature (T) of the liposomal lipids. mThis can be accomplished by dispersing the lipid dispersion in an aqueous TEA-sucrose solution at temperatures above 100°C, e.g., 60°C or higher. This lipid dispersion can be formed into liposomes having an average size of 75-125 nm (e.g., 80-120 nm, or in some embodiments, 90-115 nm) by extrusion through a track-etched polycarbonate membrane with a defined particle size, e.g., 100 nm. The TEA-sucrose can contain at least 8 molar equivalents of TEA for each molar equivalent of sucrosophate to obtain a solution that can have a sulfate concentration of about 0.40-0.50 M and a pH selected to prevent unacceptable degradation of liposomal phospholipids during the dispersion and extrusion steps (e.g., about 6.5) (e.g., a pH selected to minimize degradation of liposomal phospholipids during these steps). Unentrapped TEA-Sucrose can then be removed from the liposomal dispersion by, for example, dialysis, gel chromatography, ion exchange, or ultrafiltration prior to encapsulation of irinotecan. These liposomes can be stabilized by loading the liposomes with enough irinotecan to reduce the amount of TEA in the resulting liposome composition to a level at which lyso-PC formation is below a given maximum level after 180 days at 4°C or, more commonly, 5±3°C, as measured in mg / mL / month, i.e., %PC conversion to lyso-PC over time (e.g., mol% lyso-PC / month). The TEA exchanged from the liposomes into the external medium during the loading process, along with any unentrapped irinotecan, is then removed from the liposomes, typically by any suitable known process(es) (e.g., by gel chromatography, dialysis, diafiltration, ion exchange, or ultrafiltration). The liposome external medium can be exchanged with an injectable isotonic fluid (e.g., isotonic sodium chloride solution) buffered at a desired pH.

[0119] In some embodiments, when the amount of TEA is less than about 25 ppm or as low as about 20 ppm, an irinotecan liposome composition containing about 3.9-4.7 mg / mL of irinotecan and less than 20% lyso-PC can be obtained after 180 days at 4° C. By increasing the pH of the irinotecan liposome composition outside the liposomes, irinotecan scotopic liposomes, which also contain more than 25 ppm of TEA, can be stored and stabilized, resulting in irinotecan liposomes with less than 20% additional lyso-PC formation after 180 days at 4° C. For example, an irinotecan liposome composition containing about 4-5 mg of irinotecan / mL and 100 ppm of TEA, with a pH of about 7-8 outside the liposomes, can also have less than 20% lyso-PC formation after 180 days at 4° C. In another example, a liposome composition containing approximately 3.9-4.7 mg / mL of irinotecan, with an external medium pH in the range of 7-8, and containing less than approximately 25 ppm (or preferably less than 20 ppm) of residual TEA, can accumulate 10 mol% or less of lyso-PC in the liposome composition over 180 days at 4°C.

[0120] Thus, the present invention provides an irinotecan liposomal composition comprising irinotecan scophore encapsulated in a phospholipid liposome having a Lyso-PC stability ratio of at least 990 (e.g., 990 to 1100 or about 1111).

[0121] The present invention also provides an irinotecan liposomal composition, comprising a 4.3 mg / mL (±10%) moiety equivalent to that provided by anhydrous irinotecan free base and a 0.4-0.5 M concentration of sulfate, encapsulated in a vesicle comprising DSPC and cholesterol in a molar ratio of 3:2, resulting in 400-600 g of irinotecan per mole of phospholipid in the vesicle.

[0122] The present invention also provides an irinotecan liposome composition containing a total irinotecan moiety of about 4.3 mg / mL, wherein at least 98% of the irinotecan is encapsulated within the liposome composition together with sucrose octasulfate (SOS) at a molar ratio of about 8:1, and the liposomes have an average size of 75 to 125 nm. The size of the stabilized high-density irinotecan liposomes is preferably about 110 nm (±20 nm), more preferably 110 nm (±10 nm) (measured after liposome drug loading). Preferably, at least about 95% of the irinotecan in the pharmaceutical composition is encapsulated within the liposomes. The liposomes preferably contain DSPC and cholesterol in a molar ratio of 3:2.

[0123] The present invention also provides a method for producing a pharmaceutical product comprising stabilized irinotecan liposomes encapsulating irinotecan sucrose octasulfate (SOS) in unilamellar lipid bilayer vesicles composed of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and methoxy-terminated polyethylene glycol (MW2000)-distearoylphosphatidylethanolamine (MPEG-2000-DSPE), the method comprising: (a) loading the irinotecan moiety onto the entrapment liposomes and allowing release of the triethylammonium (TEA) cation from the entrapment liposomes, and then sequestering the irinotecan as irinotecan-free TEA8SOS with the TEA cation and a sulfate concentration of 0.4 to 0.5 M. (b) combining the irinotecan SOS liposomes with 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) to obtain an irinotecan liposome preparation having a pH of 7.25 to 7.50, and which is stabilized to form less than 10 mol % (relative to the total amount of phosphatidylcholine in the irinotecan liposomes) of lyso-phosphatidylcholine (lyso-PC) upon storage at 4°C for 3 months; and (c) formulating the combination of irinotecan SOS liposomes and HEPES as a pharmaceutical product.

[0124] In some embodiments of these methods, the irinotecan SOS liposomes in the irinotecan liposome preparation contain less than 100 ppm total TEA. In some embodiments, the unilamellar lipid bilayer vesicles consist of 6.81 mg / mL 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 2.22 mg / mL cholesterol, and 0.12 mg / mL methoxy-terminated polyethylene glycol (MW2000)-distearoylphosphatidylethanolamine (MPEG-2000-DSPE). In some embodiments, the irinotecan liposome preparation contains a total of 500 g (±10%) of irinotecan per mole of total stabilized irinotecan liposome phospholipid, and at least 98% of the irinotecan in the irinotecan liposome preparation is encapsulated within the irinotecan liposomes. In some embodiments, the irinotecan liposome preparation further comprises 4.05 mg / mL of 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES). In some embodiments, the irinotecan liposome preparation further comprises 8.42 mg / mL of sodium chloride. In some embodiments, the irinotecan liposome preparation has an irinotecan moiety concentration equivalent to that provided by approximately 4.3 mg / mL of anhydrous irinotecan free base. In some embodiments, the stabilized irinotecan liposome encapsulates irinotecan and SOS in a compound of Formula (I) where x=8.

[0125] In some embodiments, the composition contains less than 2 mol% lyso-PC after 3 months of storage at 2-8°C. In some embodiments, the composition contains less than 5 mol% lyso-PC after 3 months of storage at 2-8°C. In some embodiments, the liposome composition contains less than 10 mol% lyso-PC after 6 months of storage at 2-8°C. In some embodiments, the composition contains less than 10 mol% lyso-PC after 9 months of storage at 2-8°C. In some embodiments, the composition contains less than 5 mol% lyso-PC after 6 months of storage at 2-8°C. In some embodiments, the composition contains less than 5 mol% lyso-PC after 9 months of storage at 2-8°C. In some embodiments, the composition contains less than 2 mol% lyso-PC after 6 months of storage at 2-8°C. In some embodiments, the composition contains less than 2 mol% lyso-PC after 9 months of storage at 2-8°C. In some embodiments, the composition contains less than 10 mol% lyso-PC after 12 months of storage at 2-8°C. In some embodiments, the composition contains less than 5 mol% lyso-PC after 12 months of storage at 2-8°C. In some embodiments, the composition contains less than 2 mol% lyso-PC after 12 months of storage at 2-8°C. In some embodiments, the composition contains less than 10 mol% lyso-PC after 24 months of storage at 2-8°C. In some embodiments, the composition contains less than 5 mol% lyso-PC after 24 months of storage at 2-8°C. In some embodiments, the composition contains less than 2 mol% lyso-PC after 24 months of storage at 2-8°C. In some embodiments, the composition contains less than 100 ppm substituted ammonium. In some embodiments, the composition contains between 20-80 ppm of a substituted ammonium compound that is protonated TEA or DEA.

[0126] In other embodiments, the stabilized camptothecin composition is provided as a kit containing vials of one or more components for preparing the camptothecin composition. For example, a kit for preparing liposomal irinotecan can include the following (stored in separate containers or separate portions of the same container): Irinotecan solution (e.g., irinotecan HCl for injection), Liposomes encapsulating a sequestrant (e.g., sequestrant liposomes formed from a sucrose octasulfate solution), and Instructions for combining the irinotecan solution and the entrapment agent liposomes to form a liposomal irinotecan composition comprising a therapeutically effective amount of irinotecan encapsulated in the liposomal irinotecan liposomes (e.g., 500 mg (±10%) irinotecan per mole of total phospholipid in the entrapment agent liposomes and 4.3 mg of total irinotecan per mL of liposomal irinotecan composition). Therapeutic Uses of Camptothecin Compositions

[0127] The camptothecin compositions of the present invention, including the irinotecan liposomes and other compositions and preparations disclosed herein, can be used in methods of therapy and treatment, and / or in the preparation of medicaments for the treatment of diseases such as cancer. In some embodiments, therapy involves the administration of a camptothecin composition to treat cancer. For example, the cancer is selected from the group consisting of basal cell carcinoma, medulloblastoma cancer, liver cancer, rhabdomyosarcoma, lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, subcutaneous or intraocular melanoma, 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 intestine cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvis cancer, neoplasms 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 adenocarcinoma, such as pancreatic cancer, optionally metastatic adenocarcinoma of the pancreas, for example, where disease progression has occurred after gemcitabine-based therapy. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is small cell lung cancer. In some embodiments, the cancer is cholangiocarcinoma.

[0128] When used as a therapy, the liposome composition may be used in a treatment regimen that includes one or more other compounds or compositions. The administration of the liposome composition with one or more other compounds or compositions can be simultaneous, separate, or sequential. The one or more other compounds or compositions may be an additional therapeutic agent, for example, an additional anti-cancer agent, or a compound designed to ameliorate a negative side effect of a therapeutic agent. In some embodiments, the liposome composition is administered together with leucovorin. In some embodiments, the liposome composition is administered together with 5-fluorouracil (5-FU). In some embodiments, the liposome composition is administered together with leucovorin and 5-fluorouracil (5-FU). This three-component regimen y regimen) can be used to treat pancreatic cancer, as discussed in the previous paragraph. 5-FU is administered at a dose of 2400 mg / m 2Leucovorin can be administered at a dose of 200 mg / m² (1-form) or 400 mg / m². 2 (l+d racemate). In some embodiments, the composition is also administered in a treatment regimen with gemcitabine.

[0129] In some embodiments in which the liposome composition is used to treat ovarian cancer, the liposome composition is administered in combination with a PARP (poly ADP ribose polymerase) inhibitor.

[0130] In some embodiments, the sustained-release matrix can be nanoparticles (e.g., silica or polymer) or polymer aggregates (e.g., PEG polymers) 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 to form a stable sustained-release formulation.

[0131] In some embodiments, the stabilized camptothecin composition is an irinotecan SOS liposomal preparation formulated for intraparenchymal administration to a patient during convection-enhanced delivery therapy. The concentration of irinotecan moieties equivalent to that provided by anhydrous irinotecan free base in the final liposomal preparation is about 17, about 20, about 25, about 30, about 35, or about 40 mg / mL. In some embodiments, the concentration of irinotecan moieties equivalent to that provided by anhydrous irinotecan free base in the final liposomal preparation is 17-20, 17-25, 17-30, 17-35, or 17-40 mg / mL. Most preferably, the sum of the total concentrations of irinotecan moieties (e.g., as irinotecan octasulfate sucrose) equivalent to that provided by anhydrous irinotecan free base in the irinotecan liposomal preparation is 17 mg / mL or 35 mg / mL. The liposome preparation can be present in a sterile container containing irinotecan octasulfate sucrose liposomes at a concentration of about 17 mg / mL or about 35 mg / mL, or equivalent to that provided by 17-35 mg / mL of anhydrous irinotecan free base, for local administration to a patient (e.g., to a location within the brain as part of a convection-enhanced delivery therapy to the brain of a patient diagnosed with glioma). The 17-35 mg / mL concentration of irinotecan liposomes can be equivalently expressed as the amount of anhydrous irinotecan free base present in 20-40 mg of irinotecan hydrochloride trihydrate per mL of irinotecan liposome preparation. For example, the liposomal irinotecan preparation can be administered to the patient's brain (e.g., via one or more catheters surgically placed at a location within the tumor) in a dose that provides a total amount of irinotecan moieties equivalent to that provided by 17 mg, 26 mg, 52 mg, or 70 mg of total anhydrous irinotecan free base. The total volume of irinotecan in the liposomal irinotecan preparation delivered to the intratumoral location within the patient's brain can be about 1-2 mL (e.g., 1.0, 1.5, or 2.0 mL) over a period of about 2-4 hours (e.g., 2-3 hours, 3-4 hours, or 2-4 hours).

[0132] Irinotecan liposomes preferably contain irinotecan sucrose encapsulated in vesicles formed from lipids containing DSPC and cholesterol in a 3:2 molar ratio. The vesicles can also contain phospholipids derivatized with polyethylene glycol (PEG), such as MPEG-2000-DSPE. The amount of MPEG-2000-DSPE can be less than 1 mol % of the liposomal lipids (e.g., about 0.3 mol % in vesicles consisting of DSPC, cholesterol, and MPEG-2000-DSPE in a 3:2:0.015 molar ratio). PEG can be distributed both inside and outside the liposomal lipid vesicles surrounding irinotecan. The encapsulated irinotecan is preferably in the form of a salt with sucrose sulfate (sucrose phosphate), such as irinotecan sucrose sucrose (CAS Registry Number 1361317-83-0). Preferably, at least 95%, and most preferably at least about 98%, of the irinotecan in the irinotecan liposome composition is encapsulated within the liposome vesicles, and the concentration of the irinotecan moiety is about 3.87 to 4.73 mg of irinotecan (anhydrous free base) per mL of irinotecan liposome composition. The pH of the irinotecan liposome composition outside the liposomes is preferably about 6.5 to 8.0, or about 6.6 to 8.0, 6.7 to 8.0, 6.8 to 8.0, 6.9 to 8.0, or 7.0 to 8.0, preferably about 7.2 to 7.6. In some embodiments, the pH is about 7.2 to 7.5. In some embodiments, the pH is about 7.25. In other embodiments, the pH is about 7.25 to 7.5. In other embodiments, the pH is about 7.4 to 7.5. Combination Embodiment

[0133] Features from numbered embodiments herein may be combined with features from other embodiments disclosed herein, including both embodiments referring to compositions and embodiments referring to preparations.

[0134] The methods described above relate to the production of compositions and preparations described elsewhere herein, and therefore share common features with the embodiments of such compositions and preparations. The features disclosed with respect to the compositions and preparations may also be combined with the methods disclosed in the previous paragraph. Thus, the features found elsewhere herein, such as in the subsections above and in the numbered embodiment sections below, may be combined with the features disclosed in the methods in this subsection.

[0135] For example, the following are examples of various combinations of the embodiments disclosed and / or illustrated herein: an irinotecan liposomal composition containing about 3.9-4.7 mg / ml of irinotecan moiety and less than 20% lyso-PC after storage at 4°C for 180 days. An irinotecan liposomal composition comprising irinotecan scosofather encapsulated in phospholipid liposomes having a lyso-PC stability ratio of at least 990 (e.g., 990-1100 or about 1111). An irinotecan liposomal composition, comprising 4.3 mg / mL (±10%) irinotecan moiety and 0.4-0.5 M sulfate, encapsulated in vesicles comprising DSPC and cholesterol in a 3:2 molar ratio, with the vesicles containing 450-550 g irinotecan per mole of total phospholipid. an irinotecan liposomal composition comprising about 4.3 mg / mL of total irinotecan moieties, wherein at least 98% of the irinotecan is encapsulated within the liposomal composition together with sucrose octasulfate (SOS) at a molar ratio of about 8:1 irinotecan:SOS, and wherein the liposomes have an average size of 75-125 nm. The composition of any of the previous embodiments, wherein the irinotecan liposomes are obtained by a process comprising contacting irinotecan with triethylammonium (TEA) sucrosophate that is encapsulated within a phospholipid liposome. The composition of the previous embodiment, wherein the concentration of TEA-SOS is about 0.40 to 0.50M. The composition of any of the preceding embodiments, wherein the liposome size is about 110 nm (±10%). The composition of any of the previous embodiments, comprising about 433 g of irinotecan moieties per mole of phospholipid. The composition of any of the previous embodiments, wherein the irinotecan liposomal composition contains less than about 100 ppm triethylamine. The composition of any of the preceding embodiments, wherein the irinotecan liposome composition is a solution of liposomes in a liquid, and the liquid outside the irinotecan liposomes has a pH of about 7.0 to 8.0, e.g., 7.25 to 7.5, such as 7.25, and optionally, the liquid outside the irinotecan liposomes is a pharmaceutically acceptable injectable fluid. The composition of any of the previous embodiments, comprising an irinotecan moiety in an amount equivalent to that provided by 4.5-5.5 mg / mL of irinotecan hydrochloride trihydrate. The composition of any of the preceding embodiments, wherein at least about 95% of the irinotecan in the irinotecan liposomal composition is encapsulated within the liposomes. The composition of any of the preceding embodiments, wherein the liposomes comprise DSPC and cholesterol in a molar ratio of 3:2, such as DSPC, cholesterol and MPEG(2000)-DSPE in a molar ratio of 3:2:0.015. The composition of any of the preceding embodiments, having a stability ratio of 990 to 1200. The composition of any of the preceding embodiments, having a gram equivalent ratio of irinotecan / sucrosofate encapsulated in the liposomes 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 of the preceding embodiments, wherein the liposomal phospholipids contain no more than 20 mol % lyso-PC after storage at about 4° C. for 180 days. 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 the irinotecan liposomes, comprising 4.3 mg / mL of irinotecan calculated as the free base, and optionally obtained by a process comprising contacting the irinotecan with triethylammonium (TEA) sucrosophate encapsulated within phospholipid liposomes, optionally having a concentration of encapsulated TEA sucrosophate of about 0.40-0.50 N. The composition of any of the previous embodiments, wherein the composition comprises about 433 g of irinotecan moieties per mole of phospholipid and about 100 ppm or less of triethylammonium encapsulated within phospholipid liposomes. The composition of any of the preceding embodiments, having an encapsulated irinotecan / sucrosofa gram equivalent ratio 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 sucrosophate is in the precipitate or gel form of a stoichiometric salt comprising 8 molecules of irinotecan per molecule of sucrosophate. The composition of any of the previous embodiments, wherein at least 98%, such as at least 99%, of the encapsulated irinotecan sucrosophate is in the form of a precipitate or gel of a stoichiometric salt comprising 8 molecules of irinotecan per molecule of sucrosophate. The irinotecan liposomal composition of any of the previous embodiments, having about 100 ppm or less triethylammonium (TEA). The irinotecan liposomal composition of any of the previous embodiments, having about 20 ppm or less triethylammonium (TEA). The irinotecan liposomal composition of any of the preceding embodiments, having a total volume of about 10 mL. The irinotecan liposomal composition of any of the previous embodiments, comprising 6.81 mg / mL 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 2.22 mg / mL cholesterol, and 0.12 mg / mL methoxy-terminated polyethylene glycol (MW2000)-distearoylphosphatidylethanolamine (MPEG-2000-DSPE). The irinotecan liposome composition of any of the previous embodiments, comprising polyethylene glycol on both the inside and outside of the irinotecan liposome. 1 m of patient's body surface area 2 1. A stabilized injectable unit dose irinotecan liposomal composition formulated for administration to a patient, the composition comprising a dose of irinotecan sufficient to deliver 70 mg of irinotecan per injectable unit dose, At least 99% of the irinotecan is encapsulated in vesicles comprising phospholipids and cholesterol, wherein up to 20 mol.% of the phospholipid is lyso-PC and the remainder is DSPC, and the vesicles are in an injectable fluid having a pH in the range of 7.0 to 8.0; or o the injectable unit dose of the liposomal composition is a unit dose of the liposomal composition of any one of the above embodiments; A stabilized injectable unit dose liposomal irinotecan composition. an injectable liposomal irinotecan unit dosage form, comprising: o irinotecan, wherein at least about 98% of the irinotecan in the unit dosage form is encapsulated in liposomes comprising phospholipids, the phospholipids containing about 20 mol.% or less of lyso-PC; and o A liposome composition according to any one of the above embodiments 1. An injectable liposomal irinotecan unit dosage form comprising: The unit dosage form as disclosed in the above embodiment, wherein irinotecan is encapsulated in vesicles surrounded by a lipid membrane consisting essentially of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and methoxy-terminated polyethylene glycol (MW2000)-distearoylphosphatidylethanolamine (MPEG-2000-DSPE). The unit-dosage form of embodiment 29 or 30, wherein said unit-dosage form comprises at least about 6.81 mg / mL of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), about 2.22 mg / mL of cholesterol, and about 0.12 mg / mL of methoxy-terminated polyethylene glycol (MW2000)-distearoylphosphatidylethanolamine (MPEG-2000-DSPE)L. The unit dosage form of any of embodiments 29-31, wherein said 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 liposome composition according to any one of embodiments 1 to 27 or a unit dose according to any one of embodiments 29 to 32 for use in therapy. A liposome composition or unit dose disclosed in an embodiment herein for use in treating cancer. The liposome composition or unit dose for the uses disclosed in the embodiments herein, wherein the cancer is selected from the group consisting of basal cell carcinoma, medulloblastoma cancer, liver cancer, rhabdomyosarcoma, lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, subcutaneous or intraocular melanoma, 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 intestine cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvis cancer, neoplasms of the central nervous system, primary central nervous system lymphoma, spinal axis tumor, brain stem glioma and pituitary adenoma, or a combination of one or more of these cancers. The liposome composition or unit dose according to any of the above embodiments, wherein the cancer is pancreatic cancer, optionally an adenocarcinoma of the pancreas, such as a metastatic adenocarcinoma of the pancreas, for example, where disease progression has occurred following gemcitabine-based therapy. The liposome composition or unit dose according to any of the above embodiments, wherein the cancer is colon cancer. The liposomal composition or unit dose is for use with leucovorin and / or 5-fluorouracil, and optionally, The liposome composition or unit dose according to any of the above embodiments, wherein the administration of the composition or unit dose, leucovorin and / or 5-fluorouracil is simultaneous, separate or sequential. Liposomes: 80mg / m 2 The liposome composition or unit dose according to any one of the above embodiments, wherein the liposome composition or unit dose is administered in a dose that results in an amount of irinotecan equivalent to 100 mg of irinotecan hydrochloride trihydrate. A method of treating metastatic adenocarcinoma of the pancreas after disease progression following gemcitabine-based therapy in a patient in need thereof, comprising administering to the patient 80 mg / m 210. A method of administering intravenously an injectable irinotecan liposomal unit dosage form of any of the embodiments herein or a unit dose according to any of the above embodiments, comprising at least about 98% irinotecan in a unit dosage form encapsulated in liposomes comprising phospholipids containing less than about 20% lyso-PC, in an amount that results in an amount of irinotecan equivalent to 100 mg of irinotecan hydrochloride trihydrate. A storage-stable liposomal irinotecan composition comprising a dispersion of irinotecan liposomes encapsulating sucrose irinotecan octasulfate in unilamellar bilayer vesicles composed of cholesterol and the phospholipids 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and methoxy-terminated polyethylene glycol (MW2000)-distearoylphosphatidylethanolamine (MPEG-2000-DSPE), at a concentration of irinotecan moieties equivalent, in grams of anhydrous irinotecan free base, to 500 mg irinotecan per mmol of total liposomal phospholipid and 4.3 mg irinotecan per mL of liposomal irinotecan composition, wherein the storage-stable liposomal irinotecan composition is stabilized to form less than 1 mg / mL Lyso-PC during 6 months of storage at 4°C. (a) forming a lipid dispersion in a solution made from DEA8SOS having a sulfate concentration of 0.4-0.5M and a pH between 5-7, wherein the lipids in the dispersion are DSPC, cholesterol, and MPEG-2000-DSPE in a molar ratio of about 3:2:0.015, respectively; (b) extruding the lipid dispersion through at least one 0.1 μm membrane at between 60 and 70° C. to form liposomes; (c) substantially removing DEA8SOS and / or ions derived from DEA8SOS present outside the liposomes; (d) contacting the liposomes with a solution made using irinotecan free base or an irinotecan salt at a temperature between 60-70°C, thereby forming a preparation of liposomes encapsulating irinotecan; (e) substantially removing material derived from the TEA8SOS and / or DEA8SOS and irinotecan components outside the liposomes; and (f) adjusting the pH of the composition to 7.0 to 7.5; The liposomal irinotecan composition of the above embodiment, made by a process comprising: The liposomal irinotecan composition of any of the above embodiments, wherein the lipid dispersion is extruded through at least two laminated 0.1 μm polycarbonate membranes. The liposomal irinotecan composition of any of the above embodiments, wherein the liposomes have an average size of 110 nm as determined by dynamic light scattering, wherein the size is determined by the cumulants method. The liposomal irinotecan composition of any of the above embodiments, having a total irinotecan moiety content equivalent to 4.3 mg / ml of anhydrous irinotecan free base. In step (a), liposomes are formed from DEA8SOS having a sulfate concentration between 0.43 and 0.47M; In step (d), the solution made using irinotecan free base or irinotecan salt has an irinotecan moiety content equivalent to 500 g (±10%) of anhydrous irinotecan free base per mole of DSPC; In step (f), the pH of the composition is adjusted to 7.2 to 7.3. The liposomal irinotecan composition of any of the above embodiments. The liposome composition of any one of the previous embodiments, containing less than 1 mol% lyso-phosphatidylcholine (lyso-PC) before storage at about 4°C, and containing 20 mol% or less (relative to total liposomal phospholipids) lyso-PC after 180 days of storage at about 4°C. The liposome composition of any of the above embodiments, containing 20 mol % or less (relative to total liposomal phospholipids) lyso-phosphatidylcholine (lyso-PC) after 6, 9 or 12 months of storage at about 4° C. The liposomal irinotecan composition of any of the above embodiments, comprising a total of 6.1-7.5 mg DSPC / ml, 2-2.4 mg cholesterol / ml, and 0.11-0.13 mg MPEG-2000-DSPE / ml, all in an aqueous isotonic buffer. The liposomal irinotecan composition of any of the above embodiments, wherein the liposomal irinotecan comprises irinotecan liposomes at a concentration of between 2 and 20 mM in an isotonic HEPES aqueous buffer. The liposomal irinotecan composition of any of the above embodiments, further comprising sodium chloride at a concentration of 130-160 mM. The liposomal irinotecan composition of any of the above embodiments, wherein the irinotecan encapsulated in the liposomes is in a gel or precipitated state as a salt of sucrose octasulfate. The liposomal irinotecan composition of any of the above embodiments, wherein the irinotecan liposomes have a diameter of 95-115 nm as measured by quasi-elastic light scattering. The liposomal irinotecan composition of any of the above embodiments, comprising a total of 6.81 mg DSPC / ml, 2.22 mg cholesterol / ml, and 0.12 mg MPEG-2000-DSPE / ml, 4.05 mg / mL HEPES aqueous buffer, and 8.42 mg sodium chloride / mL. The liposomal irinotecan composition of any of the above embodiments, wherein the irinotecan liposomes have a diameter of 110 nm as measured by quasi-elastic light scattering and have a pH of 7.25. The liposomal irinotecan composition of any of the above embodiments, which forms less than 1 mg / mL of lyso-phosphatidylcholine (lyso-PC) after 6 months of storage at about 4° C. Follow these steps: (a) forming a lipid dispersion in a solution of DEA8SOS having a sulfate concentration of about 0.45 M and a pH of about 6.5, wherein the lipids in the dispersion consist of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and methoxy-terminated polyethylene glycol (MW2000)-distearoylphosphatidylethanolamine (MPEG-2000-DSPE) in a molar ratio of 3:2:0.015, respectively; (b) extruding the lipid dispersion through at least one 0.1 μm membrane at between 60 and 70° C. to form liposomes; (c) removing ions derived from DEA8SOS present outside the liposomes; (d) contacting the liposomes with a solution made using irinotecan hydrochloride trihydrate at a temperature between 60-70°C to form a liposome preparation encapsulating about 500 g (±10%) of irinotecan per mole of total liposomal phospholipid; (e) removing materials derived from the TEA8SOS and irinotecan components outside the liposomes; and (f) adjusting the pH of the composition to about 7.3. The liposomal irinotecan composition of any of the above embodiments, made by a process comprising: The liposomal irinotecan composition of any of the above embodiments, comprising less than 100 ppm total DEA. The liposomal irinotecan composition of any of the above embodiments, comprising less than 100 ppm total DEA. The liposomal irinotecan composition of any of the above embodiments, wherein after 6 months of storage at about 4° C., at least 98% of the irinotecan is encapsulated in the irinotecan liposomes. an irinotecan liposome preparation comprising stabilized irinotecan liposomes encapsulating sucrose irinotecan octasulfate (SOS) in unilamellar lipid bilayer vesicles having a diameter of approximately 110 nm, the unilamellar lipid bilayer vesicles consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and methoxy-terminated polyethylene glycol (MW2000)-distearoylphosphatidylethanolamine (MPEG-2000-DSPE), wherein the stabilized irinotecan liposomes are (a) loading the entrapment liposomes with 500 g (±10%) of irinotecan moieties per mole of total liposomal phospholipid and contacting irinotecan with entrapment liposomes encapsulating diethylammonium (DEA) cations and a 0.4-0.5 M concentration (relative to sulfate group concentration) of SOS entrapment agent as irinotecan-free TEA8SOS under conditions effective to permit release of DEA cations from the entrapment liposomes to form irinotecan sucrose octasulfate (SOS) liposomes; (b) combining irinotecan SOS liposome with 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) to obtain an irinotecan liposome preparation having a pH of 7.25 to 7.50, and being stabilized to form less than 10 mol % (relative to the total amount of phosphatidylcholine in the irinotecan liposome) of lyso-phosphatidylcholine (lyso-PC) during storage at 4° C. for 3 months. A liposomal irinotecan preparation obtained by a process comprising: The irinotecan liposomal preparation of any of the above embodiments, wherein the irinotecan SOS liposomes in the irinotecan liposomal preparation contain less than 100 ppm total TEA. The irinotecan liposomal preparation of any of the above embodiments, wherein the unilamellar lipid bilayer vesicles consist of 6.81 mg / mL 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 2.22 mg / mL cholesterol, and 0.12 mg / mL methoxy-terminated polyethylene glycol (MW2000)-distearoylphosphatidylethanolamine (MPEG-2000-DSPE). The irinotecan liposomal preparation of any of the above embodiments, comprising a total of 500 mg of irinotecan per mole of phospholipid in all stabilized irinotecan liposomes, and wherein at least 98% of the irinotecan in the irinotecan liposomal preparation is encapsulated within the irinotecan liposomes. The irinotecan liposomal preparation of any of the above embodiments, wherein the irinotecan liposomal preparation further comprises about 4.05 mg / mL of 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) at a pH of about 7.25 to 7.50. The irinotecan liposomal preparation of any of the above embodiments, wherein said irinotecan liposomal preparation further comprises about 8.42 mg / mL sodium chloride. The irinotecan liposomal preparation of any of the above embodiments, having a total of about 4.3 mg of irinotecan per mL of the irinotecan liposomal preparation. The composition of any of the previous embodiments, wherein the irinotecan liposomes are obtained by a process comprising contacting irinotecan with ammonium that is encapsulated within a phospholipid liposome. an irinotecan liposome preparation comprising stabilized irinotecan liposomes encapsulating sucrose irinotecan octasulfate (SOS) in unilamellar lipid bilayer vesicles having a diameter of approximately 110 nm, the unilamellar lipid bilayer vesicles consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and methoxy-terminated polyethylene glycol (MW2000)-distearoylphosphatidylethanolamine (MPEG-2000-DSPE), wherein the stabilized irinotecan liposomes are (a) contacting irinotecan with a sequester liposome encapsulating ammonium cations and an SOS sequester under conditions effective to load the sequester liposome with 500 g (±10%) of irinotecan moieties per mole of total liposomal phospholipid and to permit release of ammonium cations from the sequester liposome to form irinotecan sucrose octasulfate (SOS) liposomes; (b) combining irinotecan SOS liposome with 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) to obtain an irinotecan liposome preparation having a pH of 7.25 to 7.50, and being stabilized to form less than 10 mol % (relative to the total amount of phosphatidylcholine in the irinotecan liposome) of lyso-phosphatidylcholine (lyso-PC) during storage at 4° C. for 3 months. A liposomal irinotecan preparation obtained by a process comprising: An SN38 liposome preparation comprising stabilized liposomes containing irinotecan and / or SN-38 in liposomes comprising 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and methoxy-terminated polyethylene glycol (MW2000)-distearoylphosphatidylethanolamine (MPEG-2000-DSPE), which are stabilized to form less than 10 mol % (based on the total amount of phosphatidylcholine in the liposome) of lyso-phosphatidylcholine (lyso-PC) during storage at 4°C for 3 months. The irinotecan liposomal preparation of any of the above embodiments, wherein in the compound of formula (I) where x is 8, the stabilized irinotecan liposomes encapsulate 30-100 ppm of TEA or DEA, irinotecan, and SOS. [ka]

[0136] In one embodiment, the irinotecan liposomal composition disclosed herein is a stabilized irinotecan liposomal composition comprising irinotecan scosophate encapsulated in phospholipid liposomes having a Lyso-PC stability ratio of at least 990 (e.g., 990 to 1100 or about 1111), wherein the liposomal composition comprises at least one of the following features: (i) the size of the liposomes is approximately 110 nm (±10%); (ii) the composition comprises about 433 g or at least about 433 g of irinotecan moiety per mole of phospholipid; (iii) the composition contains less than about 100 ppm triethylamine; (iv) the composition comprises a pharmaceutically acceptable injectable fluid having a pH of about 7.25 outside the irinotecan liposome; (v) the liposomes comprise DSPC and cholesterol in a molar ratio of 3:2; (vi) the composition has a gram equivalent ratio of liposomally encapsulated irinotecan to sucrosophate 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 sucrosophate is in the form of a stoichiometric salt precipitate or gel containing 8 molecules of irinotecan per molecule of sucrosophate.

[0137] In one embodiment, the irinotecan liposomal composition disclosed herein is a stabilized irinotecan liposomal composition comprising irinotecan scophosphate encapsulated in phospholipid liposomes having a Lyso-PC stability ratio of at least 990 (e.g., 990 to 1100 or about 1111): (i) the size of the liposomes is approximately 110 nm (±10%); (ii) the composition comprises about 433 g or at least about 433 g of irinotecan moieties per mole of phospholipid; (iii) the composition contains less than about 100 ppm triethylamine; (iv) the composition comprises a pharmaceutically acceptable injectable fluid having a pH of about 7.25 outside the irinotecan liposome; (v) the liposomes comprise DSPC and cholesterol in a molar ratio of 3:2; (vi) the composition has a gram equivalent ratio of liposomally encapsulated irinotecan to sucrosophate of at least 0.9, at least 0.95, at least 0.98, or substantially 1.0; (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 sucrosophate is in the form of a stoichiometric salt precipitate or gel containing 8 molecules of irinotecan per molecule of sucrosophate.

[0138] Embodiment 1: A liposomal irinotecan composition having a pH of 7.00-7.50, and at a concentration of irinotecan moieties equivalent to 500 mg (±10%) of irinotecan moieties per mmol of total liposomal phospholipid and 4.3 mg of irinotecan moieties per mL of liposomal irinotecan composition, comprising cholesterol and the phospholipid 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and methoxy-terminated polyethylene glycol (M 1. A storage-stable liposomal irinotecan composition comprising a dispersion of irinotecan liposomes encapsulating sucrose irinotecan octasulfate in vesicles composed of MPEG-2000-distearoylphosphatidylethanolamine (MPEG-2000-DSPE), wherein the storage-stable liposomal irinotecan composition is stabilized such that it forms less than 20 mol % Lyso-PC during the first six months of storage at 4°C.

[0139] Embodiment 2: A liposomal irinotecan composition comprising cholesterol and the phospholipid 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and methoxy-terminated polyethylene glycol, having a pH of 7.00 to 7.50, and at a concentration of irinotecan moieties equivalent, in grams of anhydrous irinotecan free base, to 500 mg (±10%) of irinotecan moieties per mmol of total liposomal phospholipid and 4.3 mg of irinotecan moieties per mL of liposomal irinotecan composition. A storage-stable liposomal irinotecan composition comprising a dispersion of irinotecan liposomes encapsulating sucrose irinotecan octasulfate in unilamellar bilayer vesicles composed of (MW2000)-distearoylphosphatidylethanolamine (MPEG-2000-DSPE), wherein the storage-stable liposomal irinotecan composition has a gram equivalent ratio of irinotecan / sulfate compound of 0.85 to 1.2.

[0140] Embodiment 3: A storage-stabilized liposomal irinotecan composition that is stabilized to form less than 20 mol % Lyso-PC during the first 6 months of storage at 4° C., wherein the storage-stabilized liposomal irinotecan composition is prepared by the following steps: (a) forming a lipid dispersion in a solution made from TEA8SOS and / or DEA8SOS having a sulfate concentration of 0.4-0.5M and a pH between 5-7, wherein the lipids in the dispersion are DSPC, cholesterol, and MPEG-2000-DSPE in a molar ratio of about 3:2:0.015, respectively; (b) extruding the lipid dispersion through at least one 0.1 μm membrane at a temperature between 60-70° C. to form liposomes; (c) substantially removing ions derived from TEA8SOS and / or DEA8SOS present outside the liposomes; (d) contacting the liposomes with a solution made using irinotecan free base or an irinotecan salt at a temperature between 60-70°C, thereby forming a preparation of liposomes encapsulating irinotecan; (e) substantially removing material derived from the TEA8SOS and / or DEA8SOS and irinotecan components that is outside the liposomes; and (f) adjusting the pH of the composition to 7.0 to 7.5; 1. A storage-stable liposomal irinotecan composition made by a process comprising:

[0141] Embodiment 4: The steps of: (a) forming a lipid dispersion in a solution made from TEA8SOS having a sulfate concentration of 0.4-0.5M and a pH between 5-7, wherein the lipids in the dispersion are DSPC, cholesterol, and MPEG-2000-DSPE in a molar ratio of about 3:2:0.015, respectively; (b) extruding the lipid dispersion through at least one 0.1 μm membrane at a temperature between 60-70° C. to form liposomes; (c) substantially removing ions derived from TEA8SOS present outside the liposome; (d) contacting the liposomes with a solution made using irinotecan free base or an irinotecan salt at a temperature between 60-70°C, thereby forming a preparation of liposomes encapsulating irinotecan; (e) substantially removing material from the TEA8SOS and irinotecan components outside the liposomes; and (f) adjusting the pH of the composition to 7.0 to 7.5; 4. The liposomal irinotecan composition of any one of embodiments 1 to 3, made by a process comprising:

[0142] Embodiment 5: The liposomal irinotecan composition of embodiment 4, wherein the lipid dispersion is extruded through at least two laminated 0.1 μm polycarbonate membranes.

[0143] Embodiment 6: The liposomal irinotecan composition of any one of the preceding embodiments, wherein the liposomes have an average size of 110 nm as determined by dynamic light scattering, wherein the size is determined by the cumulants method.

[0144] Embodiment 7: The liposomal irinotecan composition of any one of the previous embodiments, having a total irinotecan moiety content equivalent to 4.3 mg / ml of anhydrous irinotecan free base.

[0145] Embodiment 8: In step (a), the liposomes are formed from TEA8SOS having a sulfate concentration of between 0.43 and 0.47M; In step (d), the solution made using irinotecan free base or an irinotecan salt has an irinotecan moiety content equivalent to 500 g (±10%) of anhydrous irinotecan free base per mole of DSPC; In step (f), the pH of the composition is adjusted to 7.2 to 7.3. The liposomal irinotecan composition according to any one of embodiments 3 to 6.

[0146] Embodiment 9: The liposome composition of any one of the previous embodiments, containing less than 1 mol% lyso-phosphatidylcholine (lyso-PC) before storage at about 4°C, and containing 20 mol% or less (relative to total liposomal phospholipids) lyso-PC after 180 days of storage at about 4°C.

[0147] Embodiment 10: The liposome composition of embodiment 9, containing 20 mol % or less (relative to total liposomal phospholipids) lyso-phosphatidylcholine (lyso-PC) after 6, 9, or 12 months of storage at about 4° C.

[0148] Embodiment 11: The liposomal irinotecan composition of any one of the preceding embodiments, comprising a total of 6.1-7.5 mg DSPC / ml, 2-2.4 mg cholesterol / ml, and 0.11-0.13 mg MPEG-2000-DSPE / ml, all in an aqueous isotonic buffer.

[0149] Embodiment 12: The liposomal irinotecan composition of any one of the preceding embodiments, wherein the liposomal irinotecan comprises the irinotecan liposomes at a concentration of between 2 and 20 mM in an isotonic HEPES aqueous buffer.

[0150] Embodiment 13: The liposomal irinotecan composition according to any one of the preceding embodiments, further comprising sodium chloride at a concentration of 130-160 mM.

[0151] Embodiment 14: The liposomal irinotecan composition of any one of the preceding embodiments, wherein the irinotecan encapsulated in the liposomes is in a gel or precipitated state as a salt of sucrose octasulfate.

[0152] Embodiment 15: The liposomal irinotecan composition of any one of the preceding embodiments, wherein the irinotecan liposomes have a diameter of 95 to 115 nm as measured by quasi-elastic light scattering.

[0153] Embodiment 16: The liposomal irinotecan composition of any one of the preceding embodiments, comprising a total of 6.81 mg DSPC / ml, 2.22 mg cholesterol / ml and 0.12 mg MPEG-2000-DSPE / ml, 4.05 mg / mL HEPES aqueous buffer and 8.42 mg sodium chloride / mL.

[0154] Embodiment 17: The liposomal irinotecan composition of any one of the preceding embodiments, having a pH of 7.25 and wherein the irinotecan liposomes have a diameter of 110 nm as measured by quasi-elastic light scattering.

[0155] Embodiment 18: The liposomal irinotecan composition of any one of the preceding embodiments, wherein the composition forms less than 1 mg / mL of lyso-phosphatidylcholine (lyso-PC) after 6 months of storage at about 4° C.

[0156] Embodiment 19: The steps of: (a) forming a lipid dispersion in a solution of TEA8SOS having a sulfate concentration of about 0.45 M and a pH of about 6.5, wherein the lipids in the dispersion consist of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and methoxy-terminated polyethylene glycol (MW2000)-distearoylphosphatidylethanolamine (MPEG-2000-DSPE) in a molar ratio of 3:2:0.015, respectively; (b) extruding the lipid dispersion through at least one 0.1 μm membrane at a temperature between 60-70° C. to form liposomes; (c) removing ions derived from TEA8SOS present outside the liposome; (d) contacting the liposomes with a solution made using irinotecan hydrochloride trihydrate at a temperature between 60-70°C to form a liposome preparation encapsulating about 500 g (±10%) of irinotecan per mole of total liposomal phospholipid; (e) removing materials derived from the TEA8SOS and irinotecan components that are outside the liposomes; and (f) adjusting the pH of the composition to about 7.3. 3. The liposomal irinotecan composition of any one of the preceding embodiments, made by a process comprising:

[0157] Embodiment 20: The liposomal irinotecan composition of any of the previous embodiments, comprising less than 100 ppm total TEA.

[0158] Embodiment 21: The liposomal irinotecan composition of any one of the previous embodiments, comprising a total of 30-100 ppm TEA or DEA.

[0159] 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 liposomes after 6 months of storage at about 4°C.

[0160] Embodiment 23: The irinotecan liposomes contain a compound of Formula (I): [ka] (wherein x is 8) 3. The liposomal irinotecan composition of any one of the preceding embodiments, comprising an irinotecan composition of formula: [Example]

[0161] The synthesis and characterization of several irinotecan liposome preparations are described in the following examples. Unless otherwise indicated in the examples, these irinotecan liposomes can be obtained by the following multi-step process. Thus, the present invention also provides methods for making irinotecan liposomes within the scope of the preparation methods described in this subsection and in the examples, as well as variations and combinations thereof.

[0162] First, liposome-forming lipids were dissolved in heated ethanol. These lipids included DSPC, cholesterol, and MPEG-2000-DSPE. Unless otherwise indicated, the DSPC, cholesterol, and MPEG-2000-DSPE were present in a molar ratio of 3:2:0.015. The resulting ethanol-lipid composition was dispersed in an aqueous medium containing substituted ammonium ions and polyanions under conditions effective to form appropriately sized (e.g., 80-120 nm or 95-115 nm), substantially unilamellar liposomes containing substituted ammonium ions and polyanions. This liposome dispersion can be formed, for example, by mixing an ethanolic lipid solution with an aqueous solution containing substituted ammonium ions and a polyanion at a temperature above the lipid transition temperature, e.g., 60-70°C, and extruding the resulting lipid suspension (multilamellar liposomes) under pressure through one or more track-etched membrane filters, e.g., polycarbonate, with defined pore sizes, e.g., 50 nm, 80 nm, 100 nm, or 200 nm. Preferably, the substituted ammonium is protonated triethylamine (TEA) or diethylamine (DEA), and the polyanion is sucrose octasulfate (SOS), preferably in a stoichiometric ratio (e.g., TEA8SOS). The concentration of TEA8SOS can be selected based on the amount of irinotecan loaded into the liposomes (e.g., to substantially or completely eliminate the concentration loading gradient across the liposomes and / or to result in liposomes containing SOS and irinotecan in a molar ratio of approximately 1:8). For example, to prepare irinotecan SOS liposomes having 471 g or 500 g of irinotecan moieties per mole of phospholipid, the TEA8SOS used preferably has a concentration of about 0.4-0.5 M sulfate groups (e.g., 0.45 M or 0.475 M sulfate groups, or 0.45 M or 0.475 M SOS). All or substantially all unentrapped TEA or SOS is then removed (e.g., by gel filtration, dialysis, or ultrafiltration / diafiltration).

[0163] The resulting entrapment liposomes (e.g., encapsulating a substituted ammonium compound such as TEA8SOS or DEA8SOS) are then contacted with an irinotecan solution under conditions effective to load the entrapment liposomes with irinotecan (i.e., conditions that allow irinotecan to enter the liposomes in exchange for TEA exiting the liposomes). The irinotecan loading solution (e.g., 15 mg / ml anhydrous irinotecan-HCl, which can be prepared using a corresponding amount of irinotecan-HCl trihydrate) preferably contains an osmotic agent (e.g., 5% dextrose) and a pH of 6.5 (unless otherwise specified, pH values ​​specified herein are determined at room temperature). Drug loading is facilitated by raising the temperature of the composition above the transition temperature of the liposomal lipids (e.g., 60-70°C), accelerating the transmembrane exchange of the substituted ammonium compound (e.g., TEA) and irinotecan. In some embodiments, the irinotecan closophate within the liposome is in a gel or precipitated state.

[0164] Loading of irinotecan by exchange with a substituted ammonium compound (e.g., TEA or DEA) across the liposomes is preferably continued until all or substantially all of the substituted ammonium compound (e.g., TEA) has been removed from the liposomes, thereby eliminating all or substantially all of the concentration gradient across the liposomes. Preferably, the irinotecan liposome loading step continues 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 in the 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 step continues until at least 90%, at least 95%, at least 98%, or at least 99%, or more, of the TEA has been removed from the liposome interior. In some embodiments of the present invention, the irinotecan SOS liposome composition prepared by this method using TEA8SOS contains less than 100 ppm of TEA. In some embodiments of the present invention, the irinotecan SOS liposome composition prepared by this method using TEA8SOS contains 20-100 ppm, 20-80 ppm, 40-80 ppm, or 40-100 ppm of TEA.

[0165] The extraliposomal irinotecan and substituted ammonium compounds (e.g., TEA or DEA) are removed to obtain the final irinotecan liposomal product. This removal can be facilitated by various methods, including, but not limited to, gel (size exclusion) chromatography, dialysis, ion exchange, and ultrafiltration / diafiltration. The extraliposomal medium is replaced with an injectable, pharmacologically acceptable fluid, such as buffered (pH between 7.1 and 7.5, preferably between 7.2 and 7.3) isotonic saline. Finally, the liposomal composition is sterilized, for example, by 0.2 micron filtration, dispensed into single-dose vials, labeled, and stored in a refrigerator, for example, at 2-8°C, until use. The extraliposomal medium can be replaced with a pharmacologically acceptable fluid simultaneously with the removal of remaining extraliposomal irinotecan and ammonium / substituted ammonium ions (e.g., TEA). Scavenger quantification

[0166] For purposes of the present invention, the liposome sequestering agent and substituted ammonium compound counterion (e.g., TEA8SOS) are quantified based on the concentration used in preparing liposomes and calculated based on the number of sulfate groups in the sequestering agent. For example, each molecule of SOS has eight sulfate groups, so 0.1 M TEA8SOS is expressed herein as 0.8 M / L of sulfate. If a different sequestering agent is used, this calculation is adjusted accordingly depending on the number of anionic groups (e.g., sulfate groups) per sequestering agent molecule. Quantitation of Lyso-PC in Irinotecan Liposomal Preparations

[0167] The amount of lyso-PC in the irinotecan octasulfate sucrose liposome preparation tested to obtain the data in Figures 11B and 12 was obtained by an HPLC method ("Method A"), which is described in Example 9.

[0168] A different preparative (TLC) method (herein, "Method B") was used to obtain lyso-PC measurements from Samples 1-23 herein; lyso-phospholipids were determined by the following TLC method followed by phosphate analysis, rather than the HPLC method (Method A) discussed immediately above. To measure lyso-PC by Method B, the following steps were followed: An aliquot of liposome sample (e.g., 0.05 mL of 10 mM PL liposome solution) containing approximately 500 nmol of phospholipids (PL) was desalted using a PD-10 column (GE Healthcare) equilibrated with water. The sample was eluted from the column with water, divided into three portions containing approximately 150 nmol of PL each, and then dried under vacuum using a centrifugal concentrator (Savant Speed ​​Vac Concentrator, Model No. SVC100X). The dried lipids were dissolved in 30 μl of chloroform / methanol (5 / 1, v / v) and applied to the non-adsorbed area of ​​a normal-phase silica gel TLC plate (Uniplate by Analtech, catalog number 44921) using a glass syringe. TLC was performed using a mobile phase consisting of chloroform / methanol / 30% ammonium hydroxide / water (60 / 40 / 2.5 / 3.75, v / v / v / v), and lipids were visualized using iodine vapor. PL determination was performed by scraping the spots corresponding to phospholipids and lyso-phospholipids on the TLC and placing them into separate 12 × 75 mm borosilicate tubes for subsequent phosphate analysis.

[0169] Quantitation of the molar amounts of irinotecan and sulfate compounds co-encapsulated by liposomes is presented in this example. material

[0170] For the preparation of Samples 1-5 and 13 in 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 Number 100286-90-6)) was purchased from SinoPharm (Taipei, Taiwan). 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and methoxy-terminated polyethylene glycol (MW-2000)-distearoylphosphatidylethanolamine (MPEG-2000-DSPE) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Ultra-pure cholesterol (Chol) was obtained from Calbiochem (La Jolla, CA, USA). Sucrose octasulfate was obtained from Euticals (Lodi, Italy).

[0171] For the preparation of Samples 6 to 11 in 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)-distearoylphosphatidylethanolamine (MPEG-2000-DSPE) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Ultra-pure cholesterol (Chol) was obtained from Calbiochem (La Jolla, CA, USA). Sucrose octasulfate was obtained from Euticals (Lodi, Italy).

[0172] For the preparation of Samples 19-23 in Example 8, vinorelbine (VNB) was obtained from a pharmacy as a 10 mg / mL solution of vinorelbine tartrate (Glaxo-SmithKline), and topotecan (TPT) powder was obtained as a complimentary product from Taiwan Liposome Company (Taipei, Taiwan).

[0173] All other chemicals of analytical grade and higher purity were obtained from common suppliers.

[0174] Methods: Unless otherwise specified below, the following methods were used to prepare Samples 1-5 and 13 (Example 1), and Samples 6-11 and 19-23 (Example 2), and Samples 12 and 14-18 (Example 3). Preparation of triethylammonium sucrose octasulfate

[0175] Sucrose triethylammonium octasulfate (TEA8SOS) and sucrose diethylammonium octasulfate (DEA8SOS) were prepared from the sodium salt of sucrose octasulfate using ion exchange chromatography. Briefly, 15 g of sucrose octasulfate (sodium salt) was dissolved in water to obtain a sulfate concentration of 2.64 M. The acid form of sucrose octasulfate was prepared using Dowex 50W-8X-200 cation exchange resin. The resin was washed twice with 2 volumes of 1N NaOH, then with ddH2O (double-distilled water) to neutral pH, twice with 2 volumes of 1N HCl, and finally with ddH2O to neutrality, then repeated. The column was poured into a 450 mL volume of resin, washed with 3 volumes of 3N HCl, and then rinsed with ddH2O until the conductivity reached less than 1 μS / cm. A sucrose octasulfate (sodium salt) solution (approximately 10% of the column volume) was loaded onto the column and eluted with ddH2O. A conductivity detector was used to monitor the column eluate and detect the elution of sucrose octasulfate from the column. The acidified sucrose octasulfate was then titrated with triethylamine or diethylamine to a pH between 6 and 7, and the sulfate content was determined using a method modified from B. Sorbo et al., Methods in Enzymology, 143:3-6, 1984 (see Sulfate Determination). Finally, the solution was diluted to a sulfate concentration corresponding to 0.65 M sulfate. The pH was typically in the range of 6 to 7. Residual sodium was determined using a sodium electrode, and any solutions with more than 1 mol% residual sodium were not further utilized. Determination of sulfate groups

[0176] The sulfate content in sucrose octasulfate solutions was determined using a turbidity-based assay. The solutions consisted of: (1) 15 g PEG 6000 and 1.02 g barium acetate in 100 mL of water; (2) 142 mg sodium sulfate in 1 mL of water; (3) barium working solution: 0.1 mL of sodium sulfate solution was added dropwise to 100 mL of barium solution while stirring. This solution should be equilibrated for 1 hour before use and should not be stored for longer than 1 week; (4) 0.4 M trisodium citrate solution (118 mg trisodium citrate / 1 mL water); and (5) 10 mM sulfate standard diluted in water from 1 N sulfuric acid. Standards and solutions were prepared in borosilicate test tubes to a final volume of 100 μl. Standards were prepared ranging from 0.2 to 1 μmol sulfate (20 to 100 μl of 10 mM standard). For samples of 0.6 M sulfate solution, a 1 / 100 dilution and a volume of 100 μl (0.6 μmol) were used. 100 μl of each sample / standard was treated with 100 μl of 70% perchloric acid and heated at 110–120°C for 12 minutes. After cooling, 0.8 mL of 0.4 M trisodium citrate solution was added and then vortexed. A 0.25 mL volume from the stirred barium working solution was transferred to each tube and immediately vortexed. All samples / standards were equilibrated for 1 hour, after which they were vortexed and absorbance measured at 600 nm. A linear standard curve of SO4 concentration versus OD600 was used to determine unknown SO4 concentrations. Determination of sucrose octasulfate by HPLC

[0177] The concentration of sucrose octasulfate (mg / mL) in the sample can be calculated based on the area of ​​the sucrose octasulfate peak generated from standards of known concentration. The calculated concentration of sucrose octasulfate is then used to calculate the concentration of sulfate (mM) in the sample.

[0178] The sample to be analyzed is chromatographed by HPLC using a Phenomenex Bondclone 10μ NH2, 300 × 3.90 mm, PN 00H-3128-C0 or a Waters μBondapak NH2 10μm 125Å, (3.9 mm × 300 mm), part number WAT084040, using a mobile phase of 0.60 M ammonium sulfate (pH 3.0) eluted at 1.00 mL / min at a column temperature of 40 °C. Samples are detected by a refractive index detector, for example, using an Agilent HPLC equipped with a refractive index detector, also at 40 °C. Potassium sucrose octasulfate heptahydrate, USP (CAS 76578-81-9, catalog number 1551150), is used as the reference standard.

[0179] The SOS assay standard and assay control samples are integrated using the baseline-to-baseline integral. The TEA-SOS sample is then integrated using the baseline-to-baseline integral. This can be done manually, starting from the baseline before the void volume valley to the end of the SOS tail, and then drawing a line from the beginning of the TEA peak to the low point between the two peaks. Note: If a single baseline from the beginning before the void volume valley to the end of the SOS tail intersects the low point between the TEA and SOS peaks, two additional lines approximating the baseline-to-baseline approach can be used. The TEA-SOS sample shows a TEA peak with a relative retention time of approximately 0.45 relative to the retention time of the SOS peak. Drug Analysis

[0180] HPLC analysis of irinotecan was performed using Supelco C 18 C with guard column installed in front 18 Reversed-phase silica column (Supelco C 18The analysis was performed on a Dionex system using a column (250 mm i.d. × 4 mm, 5 μm particle size). A sample injection volume of 50 μl was used and the column was eluted isocratically with a mobile phase consisting of 0.21 M aqueous triethylammonium acetate (pH 5.5) and acetonitrile (73:27, vol:vol) at a flow rate of 1.0 mL / min. Irinotecan and SN-38 typically eluted at 5.1 and 7.7 minutes, respectively. A diode array detector was used to detect irinotecan by absorbance at 375 nm, and SN-38 by fluorescence (excitation at 370 nm and emission at 535 nm). Phosphate determination

[0181] The following phosphate determination method was used to analyze samples 1-23. Phospholipids (PL) can be measured using a modified Bartlett phosphate assay. Standards ranging from 10 to 40 nmol of phosphate were placed in 12 x 75 mm borosilicate tubes and processed precisely as samples. Sulfuric acid (100 μL of 6 M H2SO4) was added to each tube in a heating block and heated to 180 °C for 45 minutes. Hydrogen peroxide (20 μL of a 30% solution) was added to each tube, which was then heated to 150 °C for 30 minutes. Subsequently, ammonium molybdate (0.95 mL of a 2.2 g / L solution) and ascorbic acid (50 μL of a 10% aqueous solution) were added to each tube. After vortexing, the tubes were placed in boiling water for 15 minutes and then cooled to room temperature. For lysolipid analysis using thin layer chromatography (TLC), silica was pelleted by centrifugation at 1000 rpm for 5 minutes, and the blue color was measured in the supernatant by reading the absorbance at 823 nm. Samples that did not contain silica could omit the centrifugation step. Drug retention and stability

[0182] The stability (in terms of drug retention) of liposomal irinotecan was determined by separating liposomal irinotecan from extraliposomal 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 phospholipid determination) before and after separation of extraliposomal irinotecan. Irinotecan degradation was determined by observing additional peaks in the chromatogram after HPLC analysis. The irinotecan-to-phospholipid ratio and drug encapsulation efficiency were calculated using the following Equations 1 and 2, respectively.

number

[0183] The liposomal irinotecan and free (unencapsulated) irinotecan in the irinotecan closopher liposomal compositions of Examples 3 and 4 were determined using a cartridge adsorption method. An Oasis 60 mg 3 cc HLB cartridge (Waters) was conditioned by sequentially passing 2 mL of methanol, 1 mL of HEPES-buffered saline (HBS; 5 mM HEPES, 140 mM NaCl, pH 6.5), and 0.5 mL of 10% human serum albumin in normal saline, followed by 1 mL of HBS. The liposomal irinotecan closopher composition was diluted with normal saline to approximately 2.2 mg / mL of irinotecan, and a 0.5 mL aliquot was applied onto the cartridge. The eluate was collected, and the cartridge was rinsed with two portions of HBS (1.5 mL, 3 mL). The washes were combined with the eluate to form the liposome fraction. The cartridge was further rinsed with 1.5 mL of HBS and eluted with two 3 mL portions of methanolic HCl (90% by volume methanol, 10% by volume 18 mM HCl). The eluates were combined to form the free drug fraction. The liposome drug fraction was transferred to a 25 mL volumetric flask, and the free drug fraction was transferred to a 10 mL volumetric flask, filled to the mark with methanolic HCl, mixed thoroughly, and the flask containing the liposome fraction was heated at 60°C for 10 minutes to dissolve the drug. Upon cooling, the solution was filtered, and irinotecan in both fractions was quantified using reverse-phase HPLC on a Phenomenex Luna C18(2) column eluted isocratically with a mixture of 20 mM potassium phosphate (pH 3.0) and methanol (60:40 by volume) with UV detection at 254 nm. The drug peak was integrated, and the amount of irinotecan in the sample was calculated by comparison with a linear standard curve obtained under identical conditions using a US Pharmacopoeia reference standard for irinotecan hydrochloride trihydrate. The drug encapsulation ratio was calculated as the percentage of encapsulated drug relative to the sum of free and encapsulated drug in the sample. pH measurement

[0184] The pH was always measured at ambient temperature (i.e., 20-25°C) using the potentiometric standard glass electrode method. Thus, the pH of the liposome formulation was measured by inserting a glass electrode into the liposome formulation to obtain a pH reading. TEA / DEA ppm sample analysis

[0185] Capillary gas chromatography (GC) column (50 m x 0.32 mm x 5 μm Sample analysis was performed by headspace GC separation using gradient temperature elution on a Restek Rtx-5 (5% phenyl-95% dimethylpolysiloxane) followed by flame ionization detection (FID). Sample and standard preparations were analyzed to compare the area responses of the resulting peaks. The amount of residual amine (e.g., TEA or diethylamine (DEA)) was quantified using external standards. For TEA, the standards were ≥99%. Other reagents included triethylene glycol (TEG), sodium hydroxide, and deionized (DI) water.

[0186] The GC conditions were as follows: carrier gas: helium; column flow rate: 20 cm / sec (1.24 mL / min); split ratio: 10:1 (can be adjusted as long as all system suitability criteria are met); injection mode: split 10:1; liner: 2 mm straight slot (recommended but not required); injection port temperature: 140°C, detector temperature: 260°C (FID); initial column oven temperature: 40°C; column oven temperature program: Speed ​​(℃ / min) Temperature (℃) Holding time (min) n / a 400 0 2 100 0 20 240 17 Duration: 54 minutes

[0187] Headspace parameters: Platen temperature: 90 °C; Sample loop temperature: 100 °C; Transfer line temperature: 100 °C; Equilibration time: 60 minutes; Injection time: 1 minute; Vial pressure: 10 psi; Pressurization time: 0.2 minutes; Shaking: On (medium); Injection volume: 1.0 mL of headspace; GC cycle time: 60 minutes (recommended but not necessary).

[0188] If TEA is not detected, report "Nothing detected". If the result of TEA is < 30 ppm, report < QL(30 ppm). If the result of TEA is ≥ 30 ppm, report it as an integer. Determination of liposome size

[0189] Liposome particle size was measured using the cumulant method, at 23 - 25 °C in an aqueous buffer (e.g., 10 mM NaCl, pH 6.7), using dynamic light scattering (DLS) with a Malvern ZetaSizer Nano ZS (trademark) or similar instrument. The z-average particle size and polydispersity index (PDI) were recorded. Instrument performance was confirmed using a 100 nm polymer Nanosphere NIST traceable standard (Thermo Scientific 3000 Series Nanosphere Size Standard P / N 3100A, or something equivalent with an analytical certificate including hydrodynamic diameter). As used herein, "DLS" refers to the dynamic light scattering method and "BDP" refers to bulk drug products. (Example 1) Effect of SOS scavenger concentration and pH on the storage stability of liposomal irinotecan preparations

[0190] The goal of this study was, among other things, to determine any changes in the physical and chemical stability of liposomes encapsulating irinotecan and the octasulfate sucrose (SOS) scavenger when stored at about 4 °C for a certain period. In this study, the liposomal concentration of the SOS scavenger was decreased while maintaining the ratio of the irinotecan moiety at 471 g per total mole of phospholipid.

[0191] A series of irinotecan SOS liposome preparations were prepared using a multi-step process that used different concentrations of SOS scavenger and adjusted the pH of the final liposome preparation to different pH values. Each irinotecan SOS liposome preparation contained an irinotecan moiety concentration equivalent to 5 mg / mL of irinotecan hydrochloride trihydrate. Samples 1-5 and 13 of the irinotecan SOS liposome preparations were prepared by the multi-step process described in Example 1.

[0192] DSPC, cholesterol (Chol), and PEG-DSPE were weighed in amounts corresponding to a molar ratio of 3:2:0.015 (e.g., 1264 mg / 412.5 mg / 22.44 mg), respectively. The lipids were dissolved in chloroform / methanol (4 / 1, v / v), thoroughly mixed, and divided into four aliquots (A–D). Each sample was evaporated to dryness using a rotary evaporator at 60 °C. Residual chloroform was removed from the lipids by placing them under vacuum (180 μtorr) at room temperature for 12 h. The dried lipids were dissolved in ethanol at 60 °C, and the appropriate concentration of pre-warmed TEA8SOS was added to bring the final alcohol content to 10% (v / v). The lipid concentration was approximately 75 mM. This lipid dispersion was extruded 10 times at approximately 65°C through two stacked 0.1 μm polycarbonate membranes (Nuclepore™) using a Lipex thermobarrel extruder (Northern Lipids, Canada) to produce liposomes with a typical mean diameter of 95–115 nm (determined by quasi-elastic light scattering; see the "Liposome Size Determination" subsection). The pH of the extruded liposomes was adjusted as needed to correct for pH changes during extrusion. Liposomes were purified by a combination of ion exchange and size exclusion chromatography. Dowex™ IRA910 resin was first treated with 1N NaOH, then washed three times with deionized water, then three times with 3N HCl, and then washed multiple times with water. Liposomes were passed through the prepared resin, and the conductivity of the eluted fraction was measured using a flow cell conductivity meter (Pharmacia, Uppsala, Sweden). Fractions were considered acceptable for further purification if their conductivity was less than 15 μS / cm. The liposome eluate was then applied to a Sephadex G-75 (Pharmacia) column equilibrated with deionized water, and the conductivity of the collected liposome fractions (typically less than 1 μS / cm) was measured.Transmembrane isotonicity was achieved by adding 40% dextrose solution to a final concentration of 5% (wt / wt), and buffer (Hepes) from a stock solution (0.5 M, pH 6.5) was added to a final concentration of 10 mM.

[0193] Considering the water content and impurity levels obtained from each batch's certificate of analysis, a stock solution of irinotecan was prepared by dissolving irinotecan HCl trihydrate powder in deionized water to 15 mg / mL anhydrous irinotecan HCl. Drug loading was initiated by adding irinotecan at a concentration of 500 g anhydrous irinotecan HCl per mole of liposomal phospholipid (equivalent to 471 g anhydrous irinotecan free base) and heating to 60 ± 0.1 °C in a hot water bath for 30 minutes. Upon removal from the water bath, the solution was rapidly cooled by immersion in ice-cold water. Extraliposomal drug was removed by size-exclusion chromatography using a Sephadex G75 column equilibrated and eluted with Hepes-buffered saline (10 mM Hepes, 145 mM NaCl, pH 6.5). Samples were analyzed for irinotecan by HPLC and for phosphate by the method of Bartlett (see subsection "Phosphate Determination"). For storage, samples were divided into 4 mL aliquots, pH adjusted using 1 N HCl or 1 N NaOH, sterile filtered under aseptic conditions, filled into sterile clear glass vials, sealed under argon with Teflon-lined screw caps, and placed in a refrigerator thermostatically controlled at 4°C. At defined time points, aliquots were withdrawn from each sample and tested for appearance, liposome size, drug / lipid ratio, and chemical stability of the drug and lipid.

[0194] For Example 1, liposome size distribution was determined in diluted samples by dynamic light scattering using a Coulter Nano-Sizer at a 90 degree angle and expressed as the mean ± standard deviation (nm) obtained by the cumulants method.

[0195] Irinotecan liposome preparations, Samples 1-5 and 13, were further obtained as follows. Freshly extruded liposomes contained two groups, each incorporating TEA8SOS as a scavenger at concentrations of (A) 0.45 M sulfate groups (112.0 ± 16 nm), (B) 0.475 M sulfate groups (105.0 ± 16 nm), (C) 0.5 M sulfate groups (97 ± 30 nm), and (D) 0.6 M sulfate groups (113 ± 10 nm). Samples 1-5 and 13 were loaded at an initial ratio of 471 g of anhydrous irinotecan free base per mole of total liposomal phospholipid and purified as described above in the description of Example 1 (equivalent to 500 g of anhydrous irinotecan HCl). Samples 1, 5, and 13 were derived from extruded sample (A). Sample 2 was derived from extruded sample (B). Samples 3 and 4 were derived from extruded samples (C) and (D), respectively. After purification, pH adjustment was performed using 1N HCl or 1N NaOH prior to sterilization and filling into vials. Data from Samples 1-5 are shown in Table 7 (Example 1), and data from Sample 13 is shown in Table 8 (Example 2).

[0196] Irinotecan liposome preparations, Samples 6-11, were further obtained as follows. Freshly extruded liposomes contained two groups, each incorporating TEA8SOS as a scavenger at concentrations of (A) 0.45 M sulfate groups (116 ± 10 nm) and (B) 0.6 M sulfate groups (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, pH adjustment was performed as needed by adding 1N HCl or 1N NaOH, as appropriate. Sample 12 was prepared as described in Example 2 and is included in Table 7 for comparison purposes.

[0197] Irinotecan liposomes with various extraliposomal pH values, irinotecan free base concentrations (mg / mL), and sucrose octasulfate concentrations for certain irinotecan liposome compositions are listed below in Table 6 (storage at 4°C for 6 months) and Table 7, and were prepared as described herein and presented in more detail.

[0198] Figures 4A-4C are plots showing the mol% of lyso-PC in selected irinotecan liposomal preparations from Table 7 having a pH greater than 6.5 (i.e., 7.25 or 7.5, as indicated in each figure). Lyso-PC was determined using Method B (TLC) disclosed herein after storage of each sample at 4°C for the first 1, 3, 6, and / or 9 months. These plots include a linear regression line to the data for each sample as an estimate for the rate of increase of lyso-PC (mol%) over time in each sample. Surprisingly, increasing the pH of the irinotecan liposomal preparations above 6.5 (e.g., 7.25 and 7.5) reduced the amount of Lyso-PC measured during refrigerated storage at 4°C compared to irinotecan liposomes formed at comparable stability ratios. This trend was evident at various concentrations of liposomal irinotecan. For example, for liposomal irinotecan compositions prepared at a strength of about 4.3 mg / mL of irinotecan moiety, the mol% lyso-PC levels measured in Samples 5 and 7 were significantly lower at all data points (after the first 1, 6, and 9 months of storage at 4° C. after manufacture) than the mol% lyso PC levels measured for Sample 1 at pH 6.5 (data in Table 7). Similarly, for liposomal irinotecan compositions prepared at a strength of about 18.8 mg / mL of irinotecan moiety, the mol% lyso-PC levels measured in Sample 13 were significantly lower at all data points (after the first 1 and 9 months of storage at 4° C. after manufacture) than the mol% lyso PC levels measured for either Sample 12 or Sample 14 at pH 6.5 (data in Table 8). [Table 6]

[0199] Further results from the comparative stability study in Example 1 are presented in Table 7 below. The mol% of lyso-PC was determined after storing the liposome preparations at 4°C for 1, 3, 6, 9, and / or 12 months, as indicated in Table 7. For each sample, Table 7 presents the concentration of SOS used to prepare the liposomes, expressed as the molar concentration of sulfate groups (one SOS molecule contains eight sulfate groups). Unless otherwise indicated, all irinotecan liposomes in Table 7 were prepared using an irinotecan moiety (relative to the anhydrous free base, as described above) to total phospholipid ratio of 471 g of irinotecan moiety per mole of total liposomal phospholipid (equivalent to the amount of irinotecan moiety in 500 g of anhydrous irinotecan HCl salt), respectively. Table 7 also includes the stability ratio for each sample, calculated as the ratio of 471 g of irinotecan moiety (based on anhydrous free base) per mole of phospholipid divided by the concentration of sulfate groups (moles / L) used to prepare the liposomes. Each of the sample liposomes listed in Table 7 had a measured size (volume-weighted average) between approximately 89 and 112 nm and an irinotecan encapsulation efficiency of at least 87.6%. The encapsulation efficiency was determined according to the subsection "Drug Retention and Stability." [Table 7] c Measured according to Method B described herein

[0200] Results from this storage stability study demonstrated that decreasing the concentration of SOS scavenger (measured as molar sulfate) used in preparing the liposomes, while keeping the ratio of anhydrous irinotecan free base (g) to total liposomal phospholipid (mol) constant, further improved the storage stability of irinotecan SOS liposomes, as measured by the amount of lyso-PC detected in the irinotecan liposome preparation after 6 and 9 months of refrigerated storage at 4° C. For liposome preparations manufactured at a pH of 6.5 (see the "pH Measurement" method described herein), decreasing the concentration of SOS scavenger during liposome manufacturing resulted in a decrease in the amount of lyso-PC detected in the liposome preparation after storage at 4° C.

[0201] Without being bound by theory, it is believed that once the liposomes are purified from the extraliposomal sequestering agent during preparation, the interior space of the liposomes becomes acidic. This may be because, after the removal of the extraliposomal TEA8SOS, the amine components of the sequestering agent redistribute from the inside to the outside of the liposome, each time this occurs, hydrogen ions accumulate within the liposome. Added drugs, such as irinotecan, that can be protonated also distribute between the exterior and interior spaces of the liposome. Protonation of the drug distributed within the liposome and binding of the protonated drug to the sucrosophate result in the loading of the drug into the liposome interior, leading to a decrease in the concentrations of both TEA and hydrogen ions within the liposome and a decrease in the degree of acidification within the liposome. In the case of irinotecan liposomes, a drug loading of 500 g of irinotecan hydrochloride (i.e., 471 mg of irinotecan) per mole of liposomal phospholipid using an SOS with a sulfate concentration of 0.6 M is hypothesized to result in incomplete clearance of excess intraliposomal TEA. While not essential for retaining the drug in the liposome, this may result in an acidic liposomal interior, which may contribute to degradation of the drug and lipid components of the liposome, as seen in the cases of Samples 7 and 13. In contrast, Samples 8 and 5 have the same drug loading of 500 g of irinotecan hydrochloride (i.e., 471 mg of irinotecan moiety) per mole, but have lower SOS concentrations of 0.45 M sulfate and 0.475 M sulfate, respectively. In these specific examples, the measured lysolipid levels are lower. Finally, it appears that the most stable liposomal formulations combine a higher drug / entrapment ratio with a higher external pH (ie, pH 7.25).

[0202] The irinotecan liposomes of Samples 1-11 maintained good colloidal stability for up to 9 months at 4°C, as judged by the absence of precipitation and relatively narrow and reproducible particle size distributions, corresponding to an irinotecan moiety concentration of 4.71 mg / mL of anhydrous irinotecan free base. Irinotecan was effectively and stably entrapped with minimal leakage (<10%) over extended storage periods (see "Drug Retention and Stability" method described herein).

[0203] Samples 1 and 2 had the same initial loading of approximately 471 g of irinotecan moiety per mole of phospholipid (based on the anhydrous free base, as described above), but lower SOS concentrations of 0.45 M and 0.475 M sulfate groups, respectively. Similarly, samples 6, 7, and 8 had a lower SOS concentration of 0.45 M sulfate, but the same drug loading of 471 g of irinotecan moiety per mole of phospholipid (based on the anhydrous free base, as described above) resulted in significantly lower lyso-lipid content (7-17% after 9 months).

[0204] In samples with a pH of 6.5, an increase in lyso-PC levels was measured during liposome preparation, regardless of drug loading or sequestering agent concentration, with phospholipids reaching up to 35 mol% in some samples (1, 2, and 3). When the pH was adjusted to 7.25, liposomes underwent less lyso-PC formation, with levels reaching 9.72% total PC (e.g., compare lyso-PC levels in samples 1 and 13). After 9 months, samples with higher drug-to-sequestering agent concentration ratios and higher pHs formed less lyso-lipid, as seen in samples 7 and 8, which had 7–8 mol% lyso-lipid. Combining a higher drug-sequestering agent ratio with a higher pH (e.g., compare sample 12) reduced lyso-lipid formation. The most stable liposomal formulations combine a higher drug / entrapment ratio (i.e., a stability ratio greater than 942, defined in terms of the amount of irinotecan free base) with an external pH greater than 6.5 (e.g., compare samples 1 and 13).

[0205] Furthermore, over a 9-month period, the %SN38 measured in irinotecan liposomal preparations 1-11 was only approximately 0.05% SN38 (i.e., the relative amount of SN38 compared to irinotecan), while the irinotecan liposomal preparation in sample 12 had 0.20-0.50% SN38 measured over the same period (as determined by the "Drug Analysis" method described herein). In each of samples 1-5 and 13, irinotecan was stably entrapped with low leakage from the liposomes (less than 13%; as determined by the "Drug Retention and Stability" method described herein), and the conversion rate to active cytotoxic SN-38 was low, less than 0.1%, and in samples stored at a higher pH (7.25), less than 0.05%. Example 2 Increasing the concentration of irinotecan liposomes in liquid preparations

[0206] The purpose of this storage stability study was to determine any changes in the physical and chemical stability of liposomal irinotecan SOS when stored at 4° C. During this study, the concentration of sucrose octasulfate (SOS) scavenger used in the liposome preparation was determined based on the following: (1) the initial counterion of the SOS scavenger during the preparation of irinotecan liposomes (TEA8SOS or DEA8SOS was used), (2) the ratio of the amount of anhydrous irinotecan free base (grams) to the amount of phospholipid (moles) (approximately 471 g or 707 g of irinotecan moiety (relative to the anhydrous free base, as described above) per mole of phospholipid), and (3) the amount of anhydrous irinotecan in the liquid irinotecan preparation. The concentration of irinotecan free base (irinotecan encapsulated at 4.7 mg / mL or 18.8 mg / mL in the liquid irinotecan liposomal preparation (relative to the equivalent concentration of irinotecan moieties derived from irinotecan hydrochloride trihydrate)), (4) the pH to which the irinotecan liposomal preparation was adjusted (pH 6.5 or 7.25), and (5) the buffer of the irinotecan liposomal preparation (HEPES or histidine) were varied while maintaining a sulfate concentration of 0.65 M.

[0207] Formulation parameters investigated 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 (mol%) formation.

[0208] A series of irinotecan SOS liposome preparations were prepared using a multi-step process that used different concentrations of SOS scavenger compared to the encapsulated irinotecan and adjusted the pH of the final liposome preparation to different values. DSPC, cholesterol (Chol), and PEG-DSPE were weighed in amounts equivalent to a 3:2:0.015 molar ratio (730.9 mg / 238.5 mg / 13.0 mg), respectively. The lipids were dissolved in chloroform / methanol (4 / 1, v / v), mixed thoroughly, and divided into two aliquots. Each sample was evaporated to dryness using a rotary evaporator at 60 °C. Residual chloroform was removed from the lipids by placing them under vacuum (180 μtorr) at room temperature for 12 hours. Dry lipids were dissolved in ethanol at 60°C, and prewarmed TEA8SOS or DEA8SOS (at a sulfate concentration of 0.65 M) was added to a final alcohol content of 10% (vol / vol). The samples were designated A and B, respectively. The lipid concentration was approximately 75 mM. The lipid dispersion was extruded 10 times through a 0.1 μm polycarbonate membrane (Nuclepore™) to produce liposomes with a typical mean diameter of 95–115 nm. The pH of the extruded liposomes was adjusted to the pH of the selected preparation (with 1N NaOH) as needed. Liposomes were purified by a combination of ion exchange and size exclusion chromatography. Dowex™ IRA910 resin was first treated with 1N NaOH, then washed three times with deionized water, then three times with 3N HCl, and then multiple times with water. The conductivity of the eluted fractions was measured using a flow cell conductivity meter (Pharmacia, Uppsala, Sweden). Fractions were considered acceptable for further purification if their conductivity was less than 15 μS / cm. The liposome eluate was then applied to a Sephadex G-75 (Pharmacia) column equilibrated with deionized water, and the conductivity of the collected liposome fractions (typically less than 1 μS / cm) was measured.Transmembrane isotonicity was achieved by adding 40% dextrose solution to a final concentration of 5% (wt / wt), and buffer (Hepes) from a stock solution (0.5 M, pH 6.5) was added to a final concentration of 10 mM.

[0209] Considering the water content and impurity levels obtained from each batch's certificate of analysis, a stock solution of irinotecan was prepared by dissolving 326.8 mg of irinotecan HCl trihydrate powder in 20.0 mL of deionized water to a concentration of 15 mg / mL anhydrous irinotecan HCl. Drug loading was initiated by adding anhydrous irinotecan free base at 500 g / mol or 750 g / mol of phospholipid and heating to 60 ± 0.1 °C for 30 minutes in a hot water bath. Upon removal from the water bath, the solution was rapidly cooled by immersion in ice-cold water. Extraliposomal drug was removed by size-exclusion chromatography using a Sephadex G75 column equilibrated and eluted with Hepes-buffered saline (10 mM) (HBS) (pH 6.5) for Sample A and histidine-buffered saline (pH 7.25) for Sample B. Samples were analyzed for irinotecan by HPLC and for phosphate by the method of Bartlett (see phosphate determination).

[0210] For storage, samples were divided into 4 mL aliquots, pH adjusted if necessary using 1 N HCl or 1 N NaOH, sterile filtered under aseptic conditions, filled into sterile clear glass vials, sealed under argon with Teflon-lined screw caps, and placed in a refrigerator thermostatically controlled at 4° C. At defined time points, aliquots were withdrawn from each sample and tested for appearance, size, drug / lipid ratio, and chemical stability of the drug and lipid.

[0211] Liposome size was determined in diluted samples by dynamic light scattering using a Coulter Nano-Sizer at a 90 degree angle and expressed as the mean ± standard deviation (nm) obtained by the cumulants method.

[0212] Results from the comparative stability studies are presented in Table 8 (for samples prepared using the TEA8SOS scavenger starting material) and Table 9 (for samples prepared using the DEA8SOS scavenger starting material). [Table 8] d Measured according to Method B described herein

[0213] Sample 13 (Example 2, Table 8) was stored at a concentration four times higher (20 mg irinotecan / mL) than Samples 1-5 (Example 1) and still retained good colloidal stability with no observable aggregation or precipitation. [Table 9] e Measured according to Method B described herein

[0214] Freshly extruded liposomes encapsulated either (A) TEA8SOS (113.0 ± 23.8 nm) with 0.65 M sulfate or (B) DEA8SOS (103.2 ± 21.1 nm) with 0.65 M sulfate (the only exception was sample 13, which had 0.45 M sulfate). Samples 12 and 14 were derived from (A), and samples 15–18 were derived from (B). Samples 12, 14, 15, and 16 were loaded with 471 g of anhydrous irinotecan free base (equivalent to 500 g of anhydrous irinotecan HCl) per mole of total liposomal phospholipid, and samples 16–18 were loaded with 750 g of irinotecan moiety per mole of phospholipid (based on the anhydrous free base, as described above). After purification, pH adjustment was made using 1N HCl or 1N NaOH to either pH 6.5 or 7.25, as appropriate and as noted in Tables 7 and 8. Sample 12 was prepared as described in Example 1 and is included in Table 8 for comparison purposes.

[0215] These data indicated that these liposomes retained good colloidal stability for up to 1 year at 4° C., as judged by the absence of precipitation and relatively narrow and reproducible particle size distributions. Second, colloidal stability was also evident for the more concentrated samples when stored at high pH and high drug-to-phospholipid ratios, indicating that at concentrations of irinotecan moieties equivalent to 20 mg / mL and 40 mg / mL irinotecan hydrochloride trihydrate, liposomes were stable and resistant to aggregate formation.

[0216] In all cases, irinotecan was stably entrapped in the liposomes with low leakage and low conversion to the active cytotoxic SN-38 (i.e., the relative amount of SN38 compared to irinotecan), i.e., less than 0.5 mol% SN-38 in all cases, with the exception of sample 12, which had less than 0.1 mol% SN-38. Data were obtained by the "Drug Retention and Stability" and "Drug Analysis" methods described herein.

[0217] For samples 12 and 14, an increase in the level of lyso-PC was measured in samples adjusted to pH 6.5 and prepared at a ratio of 471 g of irinotecan moiety per mole of phospholipid (using an equivalent amount of 500 g of anhydrous irinotecan HCl, as explained above), reaching 36–37 mol% (of total phosphatidylcholine), whereas when the pH was adjusted to 7.25, the liposomes were less susceptible to lyso-lipid formation, and for sample 15, the lyso-PC level only approached 11 mol% (of total phosphatidylcholine) after 1 year.

[0218] Changing the pH of the liposomes from 6.5 to 7.25 had no detrimental effect on colloidal stability or drug leakage. Example 3 Storage stability of stabilized irinotecan liposomes with various amounts of TEA (SOS scavenger counterion)

[0219] Irinotecan liposomes were prepared by loading irinotecan into liposomes containing sucrose octasulfate (SOS) and substituted ammonium counterions (e.g., protonated TEA). The effect of varying the residual amount of substituted ammonium in the drug-loaded irinotecan SOS liposomes was evaluated by preparing irinotecan SOS liposomes containing various amounts of residual substituted ammonium ions, storing these irinotecan SOS liposomes under refrigeration at 4°C for 6 months, and then measuring the amount of Lyso-PC (mol%) in these irinotecan SOS liposomes.

[0220] These data demonstrated that reducing the amount of substituted ammonium ions in irinotecan SOS liposomes resulted in lower levels of lyso-PC after 6 months of refrigerated storage at 4°C. In particular, irinotecan SOS liposomes with less than 100 ppm of substituted ammonium (e.g., 20-100 ppm TEA) showed lower levels of lyso-PC formation after 6 months of refrigerated storage at 4°C.

[0221] Six lots of liposomal irinotecan closophate (samples 24-29) were prepared according to certain embodiments of the present invention, following the protocol described herein, and had stability ratios of 1046-1064, with lipid compositions of DSPC, cholesterol, and MPEG-2000-DSPE in molar ratios of 3:2:0.015, respectively.

[0222] The amount of lyso-PC in Table 10 was determined by HPLC (Method A herein). [Table 10] f Measured according to Method A described herein g Measured according to Method A described herein

[0223] Liposomes (100-115 nm) were obtained by extrusion of lipids dispersed in TEA-SOS solution (0.4-0.5 M sulfate) through a 100 nm polycarbonate membrane (Nuclepore). They were purified from extraliposomal TEA-SOS by tangential flow diafiltration buffer exchange against osmotically balanced dextrose solution. They were loaded with irinotecan by heating to 68°C and stirring for 30 min, rapidly cooled, and purified from extraliposomal TEA and any unencapsulated drug by tangential flow diafiltration buffer exchange against buffered physiological sodium chloride solution. The irinotecan-sucrose liposome composition was filter-sterilized through a 0.2 μm membrane filter, aseptically dispensed into sterile glass vials, and incubated under refrigerated conditions (5 ± 3°C). At approximately 0, 3, 6, 9, and in some cases 12 months of refrigerated storage time, duplicate vials from each lot were withdrawn and analyzed for the amount of accumulated lyso-PC using an HPLC method with an evaporative scattering detector. The liposome compositions were also characterized by particle size, irinotecan and liposomal phospholipid concentrations, liposome composition pH, irinotecan / sucrose gram equivalent ratio (Iri / SOS ratio), and residual triethylammonium (protonated TEA) (as triethylamine). The mean particle size (D) and polydispersity index (PDI) were determined by DLS method using a Malvern ZetaSizer NanoZS™. The irinotecan concentration in the liposome compositions was determined by HPLC. Total phospholipids were determined spectrophotometrically by the blue phosphorus molybdate method after digestion of the liposomes in a sulfuric acid / hydrogen peroxide mixture.

[0224] The drug / lipid (DL) ratio was calculated by dividing the amount of drug (as anhydrous free base) in the liposome preparation by the molar amount of liposomal phospholipid. Liposome-entrapped SOS was quantified after passing the liposomes through a Sephadex G-25 gel chromatography column (PD-10, GE Healthcare) eluted with normal saline. To determine the gram-equivalent irinotecan / SOS ratio, 0.1 mL aliquots of triplicate eluted liposome fractions were mixed with 0.05 mL of 70% perchloric acid, hydrolyzed at 95-100°C for 1 hour, neutralized with 0.8 mL of 1 M sodium acetate, and filtered to remove insoluble lipid products. The amount of sulfate groups derived from the sucrosophate in the filtrate was quantified turbidimetrically using a barium-PEG reagent, essentially as described in the methods. Triplicate aliquots of another set of identical liposome eluates were dissolved in 70% acidified (0.1 M HCl) aqueous isopropanol and assayed for irinotecan spectrophotometrically at 365 nm. The gram equivalent ratio of irinotecan to sucrosophate (Iri / SOS ratio) was calculated for each eluted liposome fraction by dividing the measured molar concentration of drug by the measured molar concentration of sulfate groups. pH was measured as described in the subsection "pH Measurement." TEA was quantified by headspace GC separation using gradient temperature elution on a capillary gas chromatography (GC) column followed by flame ionization detection (FID). Results are expressed as ppm (parts per million) of TEA. TEA levels were determined by external quantification against standards.

[0225] Data in Figures 5, 6, 7, 10, 11A, 11B, and 12 were obtained from liposomal irinotecan samples prepared by loading approximately 400-600 mg (e.g., approximately 500 g) of irinotecan moiety per mole of total phospholipid onto 0.4-0.5 M TEA8SOS sequestering agent liposomes (stability ratios ranging from approximately 1000-1200), with a pH of approximately 7.0-7.5 (e.g., approximately 7.25) after manufacture. The amount of lyso-PC in each of these liposomal irinotecan samples was measured at the time points indicated in Figures 5-7 using the HPLC method described in Example 9.

[0226] Lyso-PC accumulation data (mg lyso-PC / mL of liposome composition) were plotted against storage time as shown in Figure 5 (samples 24-26 / lots 1-3) or Figure 6 (samples 27-29 / lots 4-6). A linear correlation was observed, with lyso-PC accumulation varying from approximately 0.008 mg / mL / month to approximately 0.06 mg / mL / month, with greater rates characteristic of compositions with higher TEA amounts. The amount of lyso-PC accumulated by 180 days (approximately 6 months) of storage was determined from a linear fit of the multipoint data (Figure 6). 5O Call Figure 6 ), expressed as mol% of PC, taking the molecular weight of lyso-PC equal to 523.7 g / mol. All six lots (samples 24–29; see Table 10) accumulated less than 20 mol% lyso-PC by 180 days of refrigerated storage. Lots with less than 20 ppm TEA and a gram equivalent Iri / SOS ratio greater than 0.98 showed minimal lyso-PC accumulation (less than approximately 0.015 mg / mL / month of lyso-PC—3.0 mol% or less at 180 days). Lots with less than 80 ppm TEA accumulated lyso-PC at a rate of approximately 0.03 mg / mL / month or less and had less than 7 mol% lyso-PC at 180 days. The lot with 100 ppm residual TEA accumulated lyso-PC at a rate of approximately 0.06 mg / mL / month and had approximately 10 mol% lyso-PC at 180 days.

[0227] Figure 7 is a graph showing the rate of lyso-PC accumulation (mg / mL / month) stored at 5±3°C plotted against TEA content (ppm) in stabilized irinotecan scophore liposomal compositions, along with a linear regression line derived from the data. Five additional lots of liposomal irinotecan scophore were prepared as in Example 3. The preparations were stored at irinotecan moieties resulting in approximately 4.3 mg / mL of anhydrous irinotecan free base per mL (as described above for the anhydrous free base) and periodically analyzed for lyso-PC formation and TEA content as described in Example 3. The rate of lyso-PC accumulation was calculated for each lot as the slope of the linear regression line obtained by fitting the lyso-PC data over storage time and plotted against TEA content along with the average TEA readings for the BDP / DP paired lots (Figure 6). As can be seen from the graph, preparations with about 25 ppm or less TEA accumulated lyso-PC at a rate of less than 0.02 mg / mL / month (less than a 2.5 mol% increase in lyso-PC over the 180-day period), preparations with about 70 ppm or less TEA accumulated lyso-PC at a rate of less than 0.033 mg / mL / month (less than a 4.3 mol% increase in lyso-PC over the 180-day period), and all preparations with less than about 100 ppm TEA accumulated lyso-PC at a rate of less than 0.062 mg / mL / month (less than an 8.0 mol% increase in lyso-PC over the 180-day period).

[0228] Samples 24, 25, and 28 each had less than 20 ppm (e.g., approximately 10–20 ppm) of substituted ammonium ions (protonated TEA) and exhibited the lowest levels of lyso-PC (2.2–3 mol% lyso-PC) observed after 6 months of refrigerated storage at 4°C. Comparing samples 26 and 27, increasing the amount of residual substituted amine scavenger counterion (e.g., protonated TEA) in irinotecan SOS liposomes from approximately 39 ppm to 79 ppm (a 103% increase) was accompanied by an unexpected decrease in the amount of lyso-PC observed after 180 days (from 6.9 mol% to 5.4 mol%, a 22% decrease in lyso-PC). However, when the amount of residual substituted ammonium ions (e.g., protonated TEA) in irinotecan SOS liposomes was further increased from 79 ppm (sample 27) to 100 ppm (sample 29) (i.e., a 27% increase), the amount of lyso-PC observed after 6 months of refrigerated storage at 4°C was accompanied by a further 87% increase (i.e., from 5.4 mol% in sample 27 to 10.1 mol% in sample 29). Example 4 Interaction of irinotecan with sucrosophate

[0229] FIG. 8 is a graph showing the gram equivalents of irinotecan and sucrosophate in precipitates formed by combining irinotecan hydrochloride and triethylammonium sucrosophate in aqueous solution at various ratios of sucrosophate (SOS) as described in Example 4.

[0230] When a solution of irinotecan hydrochloride is combined with liposomes containing triethylammonium sucrosophate, hydrogen ions can be trapped, forming an irinotecan sucrosophate salt. To study the reaction between irinotecan and triethylammonium sucrosophate, we prepared a 25 mM (16.93 mg / mL) aqueous solution of irinotecan hydrochloride trihydrate (USP) and a 250 meq / L (31.25 mM) triethylammonium sucrosophate (TEA-SOS) solution (essentially as described in the "Methods" section). Aliquots of the irinotecan hydrochloride solution were diluted with water, heated to 65°C, and combined with aliquots of the TEA-SOS solution to produce a series of irinotecan-SOS solutions with gram equivalent ratios ranging from 9:1 to 1:9, with a combined total gram equivalent concentration of both compounds equal to 25 meq / L. The samples were rapidly mixed by vortexing, incubated at 65°C for 30 minutes, chilled in ice water, and equilibrated at 4-6°C overnight. Precipitation was observed in all samples. The following day, samples were centrifuged at 10,000 x g for 5 minutes and 14,000 x g for an additional 5 minutes, and the clear supernatant (above a large, loose white to slightly brown precipitate) was isolated and analyzed for the amount of unprecipitated irinotecan and SOS to determine the amount and composition of the precipitate, essentially as described in the Examples. Results were plotted against the gram-equivalent percent of SOS in the sample (Figure 8). In the range of 20-80 equivalent % SOS, the graphs for both components consist of two linear branches that meet at the value of 50 equivalent %, indicating that irinotecan and sucrosophate formed an insoluble salt with a stoichiometry of one irinotecan molecule per sulfate ester group of sucrosophate (i.e., eight molecules of irinotecan (IRI) per one molecule of sucrosophate (SOS)): 8 IRI.HCl+TEA8SOS→(IRI.H)8SOS↓+TEACl

[0231] Despite the significant differences in molecular size and shape between the protonated irinotecan molecule and the sucrosophate anion, the salts remarkably maintained close stoichiometry (eight molecules of protonated irinotecan to one sucrosophate molecule), even when either component was in large excess (Figure 8). Thus, irinotecan sucrosophate can exist in liposomes in a poorly soluble, precipitated, or gel form. The fact that the precipitated salt maintains its strict stoichiometry allows the process to proceed until almost all or substantially all of the sulfate groups on the sucrosophate are attached to the drug molecule. Consistent with the determination of the gram equivalent ratio of irinotecan-sucrosofate in Example 6, in some embodiments, the process of loading irinotecan to obtain stable liposomes of the present invention can involve stoichiometric liposomal precipitation of the drug salt until at least 90%, at least 95%, or at least 98%, and in some cases, substantially all of the free liposomal sucrosofate is lost from the liposomal aqueous phase due to precipitation and / or gelation of the irinotecan salt. Example 5 Preparation and solubility determination of irinotecan scosofer.

[0232] A quantity of 1.64 g of irinotecan hydrochloride trihydrate was added to 160 mL of water acidified with 0.008 mL of 1 N HCl and heated in a 65°C water bath with stirring until the drug dissolved. With vigorous stirring, 5 mL of 0.46 M (based on sulfate concentration) triethylammonium sucrosophate was added and stirred for an additional 5 minutes. After overnight storage at 4-6°C, the yellow oily precipitate solidified into a brittle mass. This mass was pulverized with a glass rod to give a fluffy, off-white precipitate, which was then incubated under refrigeration for 25 days. The precipitate was separated by centrifugation, and the supernatant solution was discarded. The pellet was resuspended in 5 volumes of deionized water and centrifuged. This washing step was repeated two more times until the pH of the suspension reached approximately 5.8. Finally, the pellet was resuspended in an equal volume of deionized water to yield approximately 26 mL of product with an irinotecan content of 46.0 mg / mL (free base) (84% yield of theory). Aliquots of the product were dissolved in 1 N HCl and analyzed for irinotecan (spectrophotometry at 365 nm in 70% aqueous isopropanol-0.1 N HCl) and for sulfate after hydrolysis in dilute (1:4) perchloric acid using a barium sulfate turbidimetric assay. The molar ratio of irinotecan to SO4 was found to be 1.020 ± 0.011. Aliquots of the irinotecan scosofate suspension were added to deionized water to yield final drug salt concentrations of 0.93, 1.85, and 3.71 mg / mL. The samples were incubated at 4-6°C for 22 hours with agitation, the solid material was removed by centrifugation at 14000g for 10 minutes, and the supernatant was analyzed spectrophotometrically for irinotecan. The concentrations of irinotecan in the solutions were found to be 58.9±0.90 μg / mL, 63.2±0.6 μg / mL, and 63.4±1.3 μg / mL, respectively, which corresponds to an average molar solubility of irinotecan sucrose of 1.32 × 10 -5 This is equivalent to being M. Example 6 Various irinotecan liposomes

[0233] All experiments in this example were performed using a 25 mm extruder, hollow fiber, or tangential flow filtration (TFF) setup for the initial diafiltration step, drug loading at the microscale, and TFF setup for final diafiltration, followed by EAV filtration. Due to volume limitations of the drug-loaded material, the final filtration after dilution was performed using a 20 cm filter in a biosafety cabinet instead of two EBA filters. 2 The EAV filter was used. [Table 11] h Measured according to Method A described herein

[0234] A series of different irinotecan liposomes with different amounts of lyso-PC were prepared, see Table 11. Unless otherwise indicated, irinotecan liposomes encapsulated irinotecan sucrose octasulfate in vesicles composed of DSPC, cholesterol, and MPEG2000DSPE at a molar ratio of 3:2:0.015.

[0235] Sample 30 (Lot 1) was prepared by preparing liposomes as described in Example 1 (except as indicated in this Example), extruding the liposomes, and then holding the extruded liposomes at 72°C for 8 hours. At the end of the 8-hour period, the pH was adjusted to 6.2-6.9, resulting in a composition with approximately 45 mol% lyso-PC (i.e., approximately 1.7 mg / mL). The time of MLV preparation was considered time 0. This experiment was performed using an aliquot from Baseline Experiment 1. The composition of Sample 30 (Lot 1) was prepared using liposomes with a lower DSPC:cholesterol molar ratio (approximately 2:1 instead of 3:1 in the other samples). The resulting irinotecan liposome composition had a high level of lyso-PC (i.e., greater than 1 mg / mL and greater than 40 mol% lyso-PC).

[0236] Samples 31a and 31b (Lots 2a and 2b) were prepared using the process of Example 1, with modifications to test the effect of increasing the concentration of TEA-SOS solution in the liposomes prior to irinotecan drug loading and the effect of decreasing the irinotecan drug loading ratio by 15% on the characteristics of the resulting irinotecan liposome composition. Sample 31a (2a) material was obtained by forming liposomes with vesicles comprising DSPC and cholesterol (in the ratios presented in Table 11) encapsulating a solution of TEA-SOS at a sulfate group concentration of 0.5 M to form multilamellar vesicles (MLVs), and contacting these liposomes with an irinotecan hydrochloride solution in an amount of 510 g of anhydrous irinotecan free base / mol (PL) to load the drug onto the liposomes. The material for Sample 31b (2b) was obtained by maintaining the liposomal composition of Sample 31a (2a) for 1 week at 40° C. and then re-analyzing this sample. The resulting irinotecan liposomal compositions of Samples 31a and 31b (2a and 2b) both contained very low levels of lyso-PC (i.e., less than about 0.06 mg / mL or 4 mol% in Sample 31a (2a) and about 0.175 mg / mL in Sample 31b (2b)).

[0237] Samples 32a and 32b (Lots 3a and 3b, respectively) were prepared using the process of Example 1 with modifications selected to explore the combined effects of formulation buffer pH and reduced irinotecan drug loading. The material for Sample 32a (3a) was obtained by forming liposomes with vesicles comprising DSPC and cholesterol (in the ratios presented in Table 10) encapsulating a solution of TEA-SOS to form MLVs, and contacting these liposomes with irinotecan in a buffer selected to provide a pH of approximately 6.50 (instead of the approximately 7.25 pH of Sample 30(1)) to load the drug onto the liposomes and form irinotecan octasulfate sucrose within the liposomes at the irinotecan drug loading ratios shown in Table 11 (less irinotecan drug loading ratios than Samples 33(4) and 34(5)). The material for Sample 32b (3b) was obtained by maintaining the composition of Sample 3a for 1 week at 40° C. and then re-analyzing this sample. The resulting irinotecan liposomal compositions 32a (3a) and 32b (3b) both contained low levels of lyso-PC, 0.076 mg / mL and 0.573 mg / mL, respectively.

[0238] Samples 33(4) and 34(5) were prepared according to the method described in Example 1. The materials for Samples 33(4) and 34(5) were obtained by forming liposomes with vesicles containing DSPC and cholesterol (in the ratios presented in Table 11) encapsulating a solution of TEA-SOS to form MLVs, and then contacting these liposomes with irinotecan in a buffer selected to provide a pH of approximately 7.25 (instead of the approximately 6.5 pH of Samples 3a and 3b) to load the drug onto the liposomes and form irinotecan octasulfate sucrose in the liposomes at 500 g of irinotecan moiety (based on the anhydrous free base) per mole of phospholipid. Both the resulting irinotecan liposome compositions 3a and 3b contained low levels of lyso-PC, 0.24 mg / mL and 0.79 mg / mL, respectively.

[0239] Figure 12 is a graph showing the amount of lyso-PC measured in Sample 33(4) (circles, lower line) and Sample 34(5) ("+" data points, upper line). The rate of lyso-PC formation was greater in Sample 34(5) than in Sample 33(4). A linear fit to the data points in Figure 12 was as follows: Sample 33(4): lyso-PC, mg / mL = 0.0513596 + 0.0084714 × accumulation period (Age) Sample 34(5): lyso-PC, mg / mL = 0.1766736 + 0.0279783 × accumulation period The total lyso-PC concentrations of the irinotecan liposomal preparation in samples 33 and 34 were 0.24 mg / mL and 0.79 mg / mL, respectively, at 22 months. Example 7 Irinotecan liposome injection (ONIVYDE®)

[0240] One preferred example of a storage-stable liposomal irinotecan preparation is the product marketed as ONIVYDE® (irinotecan liposome injection) (Merrimack Pharmaceuticals, Inc., Cambridge, MA). The ONIVYDE® product is a topoisomerase inhibitor in which irinotecan hydrochloride trihydrate is formulated in a liposomal dispersion for intravenous use. The ONIVYDE® product is indicated in combination with fluorouracil and leucovorin for the treatment of patients with metastatic adenocarcinoma of the pancreas after disease progression following gemcitabine-based therapy.

[0241] The recommended dose of ONIVYDE® product is 70 mg / m administered by intravenous infusion over 90 minutes once every two weeks. 2 The ONIVYDE® product is administered in combination with leucovorin and fluorouracil to treat certain forms of pancreatic cancer. *In those pancreatic cancer patients known to be homozygous for the 28 allele, the recommended starting dose of ONIVYDE® products is 50 mg / m administered by intravenous infusion over 90 minutes. 2 In subsequent cycles, the ONIVYDE® product dose is increased to 70 mg / m as tolerated. 2 For patients with serum bilirubin above the upper limit of normal, there is no recommended dose of ONIVYDE® products.

[0242] ONIVYDE® product is administered to patients as follows: First, the calculated volume of ONIVYDE® product is subtracted from the vial. Next, this amount of ONIVYDE® product is diluted into 500 mL of 5% Dextrose Injection, USP or 0.9% Sodium Chloride Injection, USP and mixed by gentle inversion. This dilution should be protected from light. This dilution should then be refrigerated within 4 hours of preparation if stored at room temperature, or under refrigerated conditions [2°C to 8°C (36°F)]. o F~46 o If stored under 5°C (F), administer within 24 hours of preparation. This diluted solution is allowed to warm to room temperature before administration and should not be frozen. This diluted solution is then infused over 90 minutes without the use of an in-line filter, and any unused portion is discarded.

[0243] The ONIVYDE® product is a liposomal dispersion formulation of the topoisomerase inhibitor irinotecan hydrochloride trihydrate for intravenous use. The chemical name for irinotecan hydrochloride trihydrate is (S)-4,11-diethyl-3,4,12,14-tetrahydro-4-hydroxy-3,14-dioxo1H-pyrano[3',4':6,7]-indolizino[1,2-b]quinolin-9-yl-[1,4'bipiperidine]-1'-carboxylate monohydrochloride trihydrate. The empirical formula is C 33 H 38 It is N4O6·HCl·3H2O and has a molecular weight of 677.19 g / mol. Its molecular structure is: [ka]

[0244] The ONIVYDE® product is supplied as a white to slightly yellow, opaque, isotonic, sterile liposomal dispersion. Each 10 mL single-dose vial contains the equivalent of 43 mg of irinotecan free base per mL (i.e., 4.3 mg / mL irinotecan moiety), at a concentration of 4.3 mg / mL anhydrous irinotecan free base. The liposomes are unilamellar lipid bilayer vesicles approximately 110 nm in diameter that enclose an aqueous space containing irinotecan in a gel or precipitated state as the salt of sucrose octasulfate. The vesicles are composed of 6.81 mg / mL 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 2.22 mg / mL cholesterol, and 0.12 mg / mL methoxy-terminated polyethylene glycol (MW2000)-distearoylphosphatidylethanolamine (MPEG-2000-DSPE). Each mL also contains 4.05 mg / mL of 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) as a buffer, and 8.42 mg / mL of sodium chloride as an isotonicity agent.

[0245] Irinotecan liposomal 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, reversibly bind to the topoisomerase 1-DNA complex and prevent religation of single-strand breaks, resulting in exposure-dependent double-strand DNA damage and cell death. In mice bearing human tumor xenografts, irinotecan liposomes administered at a 5-fold lower irinotecan HCl equivalent dose achieved similar intratumoral exposure to SN-38.

[0246] The plasma pharmacokinetics of total irinotecan and total SN-38 were evaluated in cancer patients receiving ONIVYDE® products at doses between 50 and 155 mg / m2 as single agents or as part of combination chemotherapy using population pharmacokinetic analysis and in 353 cancer patients.

[0247] The pharmacokinetic parameters of total irinotecan and total SN-38 following administration of the ONIVYDE® product at 70 mg / m2 as a single agent or as part of combination chemotherapy are shown below. [Table 12]

[0248] Over the dose range of 50 to 155 mg / m, the Cmax and AUC of total irinotecan increased with dose. Furthermore, the Cmax of total SN-38 increased proportionally with dose. However, the AUC of total SN-38 increased less than proportionally with dose.

[0249] Direct measurements of irinotecan liposomes showed that 95% of irinotecan remained encapsulated in liposomes, and the ratio between the total form and the encapsulated form did not change with time from 0 to 169.5 hours after dosing.

[0250] ONIVYDE® products should be stored at room temperature between 2°C and 8°C (36°F). o F~46 o F), protected from light, and should not be frozen.

[0251] Several ONIVYDE® product formulations were studied for long-term stability and analyzed over 12 to 36 months of storage at 2 to 8°C (refrigerated conditions). The results are plotted in the graphs in Figures 9, 10, 11A, and 11B, as described below. In one study, particle size (Figure 9) and particle size distribution (Figure 10) were measured for 12 different ONIVYDE® product formulations over 12 to 36 months. For all samples, the PDI remained well below 0.1 and below about 0.05. In another study, pH (Figure 11A) was measured for 13 different ONIVYDE® product formulations over 12 to 36 months. For all samples, the pH remained above 6.8 throughout the study. In another study, the amount of lyso-PC (Figure 11B) was measured for 16 different ONIVYDE® product formulations over 12 months of refrigerated storage. The amount of lyso-PC remained below 1 mg / mL for all samples.

[0252] To determine the irinotecan free base concentration in embodiments of the ONIVYDE® product at different time points during storage, irinotecan free base is quantified as provided in the "Examples" section. To determine the lipid composition of embodiments of the ONIVYDE® product at different time points during storage, lipids are quantified using standard HPLC methods standard in the art.

[0253] To determine the mean particle size (D) and polydispersity index (PDI) of liposomes of embodiments of the ONIVYDE® product at different time points during storage, a Malvern The DLS method was used in combination with a ZetaSizer Nano ZS™.

[0254] For purposes of determining the presence of lyso-PC in embodiments of the ONIVYDE® product at different time points during storage, lyso-PC is quantified as described in the "Examples" section. Additionally, within the context of the present invention, it is also contemplated that lyso-PC can be quantified by HPLC as described herein. Example 8 Topotecan and vinorelbine liposomes

[0255] The purpose of this storage stability study was to determine any changes in the physical and chemical stability of topotecan (TPT) and vinorelbine (VNB) liposomes prepared with a sucrose octasulfate sequester when stored at 4°C. Specifically, the study examined whether reducing the sucrose octasulfate (SOS) sequester concentration from 0.6 M to 0.45 M sulfate groups during liposome preparation, while maintaining the topotecan or vinorelbine to phospholipid ratio per mole as shown below, affected 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 whether such an increase in pH reduced the presence of lyso-PC in the liposome composition. TPT and VNB were encapsulated with an SOS sequester in liposomes containing DSPC, cholesterol (Chol), and PEG-DSPE in a molar ratio of 3:2:0.015. The formulation parameters investigated included: solution pH (6.5-7.5), sucrose octasulfate sequestering agent concentration during liposome preparation (0.45-0.6 M sulfate), encapsulated drug (TPT or VNB), and drug-to-lipid ratio (500 g TPT HCl per mol of phospholipid during liposome loading; in the case of VNB, 350-450 g VNB moieties per mol of phospholipid during liposome loading). 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.

[0256] DSPC, cholesterol (Chol), and PEG-DSPE were weighed in amounts corresponding to a molar ratio of 3:2:0.015 (790.15 mg / 257.8 mg / 14.0 mg), respectively. The lipids were dissolved in chloroform / methanol (4 / 1, v / v), thoroughly mixed, and divided into two aliquots (A and B). Each sample was evaporated to dryness using a rotary evaporator at 60 °C. Residual chloroform was removed from the lipids by placing them under vacuum (180 μtorr) at room temperature for 12 h. The dried lipids were dissolved in ethanol at 60 °C, and the appropriate concentration of pre-warmed TEA8SOS was added to bring the final alcohol content to 10% (v / v). The total phospholipid concentration was approximately 75 mM. The lipid solution was extruded 10 times through a 0.1 μm polycarbonate membrane (Nuclepore™) to produce liposomes with a typical mean diameter of 95–115 nm. The pH of the extruded liposomes was adjusted to pH 6.5 (with 1N NaOH) as needed. Liposomes were purified by a combination of ion exchange and size exclusion chromatography. Dowex™ IRA910 resin was first treated with 1N NaOH, then washed three times with deionized water, then three times with 3N HCl, and then multiple times with water. The conductivity of the eluted fractions was measured using a flow cell conductivity meter (Pharmacia, Uppsala, Sweden). Fractions were considered acceptable for further purification if their conductivity was less than 15 μS / cm. 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 (typically less than 1 μS / cm) was measured. A 40% dextrose solution was added to achieve a final concentration of 5% (w / w), and buffer (Hepes) from a stock solution (0.5 M, pH 6.5) was added to a final concentration of 10 mM.

[0257] A stock solution of topotecan hydrochloride was prepared by dissolving 50 mg in 10 mL of deionized water. The drug was added to the liposome solution at the drug / lipid ratio indicated for each formulation in the results in Table 13. For TPT loading, the pH was adjusted to pH 6.0 before loading. Vinorelbine was added directly from the commercially available USP injection solution from a pharmacy, and the pH of the resulting mixture was adjusted to 6.5 with 1 N NaOH before heating. Drug loading was initiated by heating the liposome / drug mixture to 60°C for 30 minutes. Upon removal from the water bath, the solution was rapidly cooled by immersion in ice-cold water. Extraliposomal drug was removed by size-exclusion chromatography using a Sephadex G75 column equilibrated and eluted with Hepes (10 mL)-buffered saline (HBS) (pH 6.5). Samples were analyzed for irinotecan by HPLC and for phosphate by the method of Bartlett (see phosphate determination).

[0258] For storage, samples were divided into 4 mL aliquots, pH adjusted, if necessary, using 1 N HCl or 1 N NaOH, sterile filtered under aseptic conditions, filled into sterile clear glass vials, sealed under argon with Teflon-lined screw caps, and placed in a refrigerator thermostatically controlled at 4°C. At defined time points, aliquots were withdrawn from each sample and tested for appearance, size, drug / lipid ratio, and chemical stability of the drug and lipid. Liposome size was determined in diluted samples by dynamic light scattering using a Coulter Nano-Sizer at a 90-degree angle and expressed as the mean ± standard deviation (nm) obtained by the cumulants method.

[0259] Results from the comparative stability study are presented in Table 13. [Table 13] i Measured according to Method B described herein j500g of topotecan HCl per 1 mol of total phospholipids

[0260] No effect of storage medium pH on lyso-lipid formation in topotecan-loaded liposomes was observed for Samples 19 and 20. Even when Sample 19 was stored at pH 6.5 and Sample 20 at pH 7.25, both formulations in Samples 19 and 20 exhibited close to 30 mol% lyso-lipid after 9 months.

[0261] In contrast to both liposomal camptothecins, liposomal vinorelbine was more resistant to lipid hydrolysis, in that the highest amount of lyso-lipid measured was in sample 21, which had 9.5 mol% lyso-lipid after 9 months. We also detected a less pronounced dependence on the stability ratio and pH of the storage medium. A higher stability ratio reduced 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")

[0262] The amount of lyso-PC in the irinotecan octasulfate sucrose liposome preparation tested to obtain the data in Figures 11B and 12 was obtained using HPLC with evaporative light scattering detection. The preferred HPLC method (referred to herein as "Method A") is a quantitative method used to measure the amounts of stearic acid, lyso-PC, cholesterol, and DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) in the drug product. Liposomes are dissociated into their individual lipid components using a methanol-tetrahydrofuran solution. These lipid components are quantified using reverse-phase high-pressure liquid chromatography equipped with an evaporative light scattering detector. Sample and standard preparation Preparation of standards: LysoPC

[0263] A five-point standard curve is prepared by diluting the appropriate amount of LysoPC with 85:15 methanol-tetrahydrofuran to target final concentrations of 4, 8, 20, 32, and 40 μg / mL. stearic acid

[0264] A five-point standard curve is prepared by diluting the appropriate amount of stearic acid with 85:15 methanol-tetrahydrofuran to target final concentrations of 2, 4, 10, 16, and 20.4 μg / mL. cholesterol

[0265] A five-point standard curve is prepared by diluting the appropriate amount of cholesterol with 85:15 methanol-tetrahydrofuran to target final concentrations of 90, 144, 183.7, 224.9, and 266.6 μg / mL. DSPC

[0266] A five-point standard curve is prepared by diluting the appropriate amount of DSPC with 85:15 methanol-tetrahydrofuran to target final concentrations of 220, 352, 449, 549.8 and 651.7 μg / mL. Assay Controls

[0267] Assay controls are prepared by diluting stearic acid in diluent (85:15 methanol-tetrahydrofuran) to target final concentrations of 9.0 μg / mL and 18.0 μg / mL. Sample preparation:

[0268] Samples are prepared by diluting each sample in an 85:15 methanol-tetrahydrofuran solution to a target final DSPC concentration of 475 μg / mL. solution stability

[0269] Test sample standards and assay controls demonstrated acceptable stability in solution for up to 48 hours when stored at ambient temperature. Instruments and instrument parameters

[0270] A suitable high pressure chromatography system was equipped with an evaporative light scattering detector with the ability to change gain and filter settings throughout the run as needed to ensure proper peak detection. Instrument operating parameters are listed in Table 14. [Table 14] [Table 15]

[0271] The lipid concentrations of each lipid were determined by analyzing the sample peak area against the standard curve. A quadratic polynomial trend line was used to calculate the lipid concentrations of lyso-PC and stearic acid. A linear trend line was used to calculate the lipid concentrations of DSPC and cholesterol.

[0272] Representative chromatograms are shown in Figures 13A and 13B.

[0273] All references cited herein are incorporated by reference in their entirety. In particular embodiments, for example, the following items are provided: (Item 1) The liposomal irinotecan composition has a pH of 7.00 to 7.50 and is composed of cholesterol and phospholipids 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and methoxy-terminated polyethylene glycol (MW 20 2500) at a concentration of irinotecan moiety equivalent to 500 mg (±10%) of irinotecan moiety per mmol of total liposomal phospholipid and 4.3 mg of irinotecan moiety per mL of liposomal irinotecan composition, based on a gram amount of anhydrous irinotecan free base. 1. A storage-stable liposomal irinotecan composition comprising a dispersion of irinotecan liposomes encapsulating sucrose irinotecan octasulfate in vesicles composed of MPEG-2000-DSPE (MPEG-2000-DSPE), wherein the storage-stable liposomal irinotecan composition is stabilized such that it forms less than 20 mol % Lyso-PC during the first six months of storage at 4°C. (Item 2) and a pH of 7.00 to 7.50, and a concentration of irinotecan moieties equivalent to 500 mg (±10%) of irinotecan moieties per mmol of total liposomal phospholipid and 4.3 mg of irinotecan moieties per mL of liposomal irinotecan composition, based on a gram amount of anhydrous irinotecan free base, comprising cholesterol and phospholipids 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and methoxy-terminated polyethylene glycol (MW 1. A storage-stable liposomal irinotecan composition comprising a dispersion of irinotecan liposomes encapsulating sucrose irinotecan octasulfate in unilamellar bilayer vesicles composed of MPEG-2000)-distearoylphosphatidylethanolamine (MPEG-2000-DSPE), wherein the storage-stable liposomal irinotecan composition has a gram equivalent ratio of irinotecan to sulfate compound of 0.85 to 1.2. (Item 3) 1. A storage-stabilized liposomal irinotecan composition that is stabilized to form less than 20 mol % Lyso-PC during an initial six-month storage period at 4° C., said storage-stabilized liposomal irinotecan composition being prepared by the following steps: (a) forming a lipid dispersion in a solution made from TEA8SOS and / or DEA8SOS having a sulfate concentration of 0.4-0.5M and a pH between 5-7, wherein the lipids in the dispersion are DSPC, cholesterol, and MPEG-2000-DSPE in a molar ratio of about 3:2:0.015, respectively; (b) extruding the lipid dispersion through at least one 0.1 μm membrane at a temperature between 60-70° C. to form liposomes; (c) substantially removing ions derived from TEA8SOS and / or DEA8SOS present outside the liposomes; (d) contacting the liposomes with a solution made using irinotecan free base or an irinotecan salt at a temperature between 60-70°C, thereby forming a preparation of liposomes encapsulating irinotecan; (e) substantially removing material derived from the TEA8SOS and / or DEA8SOS and irinotecan components that is outside the liposomes; and (f) adjusting the pH of the composition to 7.0 to 7.5; 1. A storage-stable liposomal irinotecan composition made by a process comprising: (Item 4) Follow these steps: (a) forming a lipid dispersion in a solution made from TEA8SOS having a sulfate concentration of 0.4-0.5M and a pH between 5-7, wherein the lipids in the dispersion are DSPC, cholesterol, and MPEG-2000-DSPE in a molar ratio of about 3:2:0.015, respectively; (b) extruding the lipid dispersion through at least one 0.1 μm membrane at a temperature between 60-70° C. to form liposomes; (c) substantially removing ions derived from TEA8SOS present outside the liposome; (d) contacting the liposomes with a solution made using irinotecan free base or an irinotecan salt at a temperature between 60-70°C, thereby forming a preparation of liposomes encapsulating irinotecan; (e) substantially removing material from the TEA8SOS and irinotecan components outside the liposomes; and (f) adjusting the pH of the composition to 7.0 to 7.5; 4. The liposomal irinotecan composition according to any one of items 1 to 3, wherein the liposomal irinotecan composition is produced by a process comprising: (Item 5) 5. The liposomal irinotecan composition according to item 4, wherein the lipid dispersion is extruded through at least two laminated 0.1 μm polycarbonate membranes. (Item 6) 6. The liposomal irinotecan composition of any one of items 1 to 5, wherein the liposomes have an average size of 110 nm as determined by dynamic light scattering, the size being determined by the cumulant method. (Item 7) 7. The liposomal irinotecan composition according to any one of items 1 to 6, having a total irinotecan moiety content equivalent to 4.3 mg / ml of anhydrous irinotecan free base. (Item 8) In step (a), the liposomes are formed from TEA8SOS having a sulfate concentration of between 0.43 and 0.47M; In step (d), the solution made using irinotecan free base or an irinotecan salt has an irinotecan moiety content equivalent to 500 g (±10%) of anhydrous irinotecan free base per mole of DSPC; In step (f), the pH of the composition is adjusted to 7.2 to 7.3. 7. The liposomal irinotecan composition according to any one of items 3 to 6. (Item 9) 9. The liposome composition of any one of items 1 to 8, containing less than 1 mol% lyso-phosphatidylcholine (lyso-PC) before storage at about 4° C. and containing 20 mol% or less (relative to total liposomal phospholipids) lyso-PC after 180 days of storage at about 4° C. (Item 10) 10. The liposome composition according to item 9, containing 20 mol % or less (relative to total liposomal phospholipids) lyso-phosphatidylcholine (lyso-PC) after storage at about 4° C. for 6, 9, or 12 months. (Item 11) 11. The liposomal irinotecan composition according to any one of items 1 to 10, comprising a total of 6.1 to 7.5 mg DSPC / ml, 2 to 2.4 mg cholesterol / ml, and 0.11 to 0.13 mg MPEG-2000-DSPE / ml, all in an aqueous isotonic buffer solution. (Item 12) 12. The liposomal irinotecan composition according to any one of items 1 to 11, wherein the liposomal irinotecan comprises the irinotecan liposomes at a concentration of between 2 and 20 mM in an isotonic HEPES aqueous buffer. (Item 13) 13. The liposomal irinotecan composition according to any one of items 1 to 12, further comprising sodium chloride at a concentration of 130 to 160 mM. (Item 14) 14. The liposomal irinotecan composition according to any one of items 1 to 13, wherein the irinotecan encapsulated in the liposomes is in a gel state or a precipitated state as a salt of sucrose octasulfate. (Item 15) 15. The liposomal irinotecan composition according to any one of items 1 to 14, wherein the irinotecan liposomes have a diameter of 95 to 115 nm as measured by quasi-elastic light scattering. (Item 16) 16. The liposomal irinotecan composition according to any one of items 1 to 15, comprising in total 6.81 mg DSPC / ml, 2.22 mg cholesterol / ml and 0.12 mg MPEG-2000-DSPE / ml, 4.05 mg / mL HEPES aqueous buffer and 8.42 mg sodium chloride / mL. (Item 17) 17. The liposomal irinotecan composition of any one of items 1 to 16, having a pH of 7.25 and wherein the irinotecan liposomes have a diameter of 110 nm as measured by quasi-elastic light scattering. (Item 18) 18. The liposomal irinotecan composition of any one of items 1 to 17, which forms less than 1 mg / mL of lyso-phosphatidylcholine (lyso-PC) after 6 months of storage at about 4° C. (Item 19) Follow these steps: (a) forming a lipid dispersion in a solution of TEA8SOS having a sulfate concentration of about 0.45 M and a pH of about 6.5, wherein the lipids in the dispersion consist of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and methoxy-terminated polyethylene glycol (MW2000)-distearoylphosphatidylethanolamine (MPEG-2000-DSPE) in a molar ratio of 3:2:0.015, respectively; (b) extruding the lipid dispersion through at least one 0.1 μm membrane at a temperature between 60-70° C. to form liposomes; (c) removing ions derived from TEA8SOS present outside the liposome; (d) contacting the liposomes with a solution made using irinotecan hydrochloride trihydrate at a temperature between 60-70°C to form a liposome preparation encapsulating about 500 g (±10%) of irinotecan per mole of total liposomal phospholipid; (e) removing materials derived from the TEA8SOS and irinotecan components that are outside the liposomes; and (f) adjusting the pH of the composition to about 7.3. 19. The liposomal irinotecan composition according to any one of items 1 to 18, wherein the liposomal irinotecan composition is produced by a process comprising: (Item 20) 20. The liposomal irinotecan composition according to any of items 1 to 19, comprising less than 100 ppm total TEA. (Item 21) 21. The liposomal irinotecan composition according to any one of items 1 to 20, comprising a total of 30 to 100 ppm of TEA or DEA. (Item 22) 22. The liposomal irinotecan composition of any one of items 1 to 21, wherein after 6 months of storage at about 4° C., at least 98% of the irinotecan is encapsulated in the irinotecan liposomes. (Item 23) The irinotecan liposome contains a compound of formula (I): [ka] wherein x is 8. 23. The liposomal irinotecan composition according to any one of items 1 to 22, comprising an irinotecan composition of formula:

Claims

Claim 1: A storage-stabilized liposomal irinotecan composition comprising cholesterol, one or more phospholipids, and irinotecan sucrose octasulfate (SOS) encapsulated in unilamellar liposomes, comprising: (i) an irinotecan:total phospholipid ratio corresponding to a total mass of 500 g±10% of irinotecan moieties per mole of total phospholipid; (ii) a gram equivalent ratio of 0.85 to 1.2, as determined by the molar amounts of irinotecan and sucrose octasulfate co-encapsulated according to the formula: gram equivalent ratio = I / (SN), where I is the molar concentration of irinotecan, S is the molar concentration of sucrose octasulfate, and N=8; (iii) a pre-storage pH of about 7.25 to about 7.5 at room temperature; and less than 20 mol% lysophosphatidylcholine (lyso-PC) relative to the total phospholipids after the first six months of storage of the composition at a storage temperature of 2°C to 8°C. A composition having:

2. The composition described in claim 1, wherein the one or more phospholipids include phosphatidylcholine and PEG-phosphatidylethanolamine.

3. The composition described in claim 2, wherein the phosphatidylcholine is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and the PEG-phosphatidylethanolamine is N-(methoxy-poly(ethylene glycol)-oxycarbonyl)-distearoylphosphatidylethanolamine.

4. The composition of claim 3, wherein the N-(methoxy-poly(ethylene glycol)-oxycarbonyl)-distearoylphosphatidylethanolamine comprises N-methoxy-terminated polyethylene glycol (MW 2000)-distearoylphosphatidylethanolamine.

5. The composition described in claim 4, wherein before storage, the molar ratio of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) to cholesterol is approximately 3:

2.

6. The composition described in claim 5, wherein, prior to storage, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) is present at a concentration of 6.1 to 7.5 mg / mL and cholesterol is present at a concentration of 2 to 2.4 mg / mL.

7. The composition described in claim 6, wherein the composition contains less than 20 mol% lyso-PC relative to the total phospholipids when quantified by HPLC after the first 6 months of storage of the composition at a storage temperature of 2°C to 8°C.

8. The composition described in claim 7, wherein the composition contains less than 20 mol% lyso-PC when quantified by HPLC after the first 9 months of the composition at a storage temperature of 2°C to 8°C.

9. The composition described in claim 7, wherein the composition contains less than 10 mol% lyso-PC when quantified by HPLC after the first 6 months of the composition at a storage temperature of 2°C to 8°C.

10. The composition described in claim 7, wherein the composition contains less than 10 mol% lyso-PC when quantified by HPLC after the first 9 months of the composition at a storage temperature of 2°C to 8°C.

11. The composition described in claim 7, wherein the composition contains less than 10 mol% lyso-PC when quantified by HPLC after the first 12 months of the composition at a storage temperature of 2°C to 8°C.

12. The composition described in claim 7, wherein the composition contains less than 10 mol% lyso-PC when quantified by HPLC after the first 24 months of the composition at a storage temperature of 2°C to 8°C.

13. The composition described in claim 7, wherein the composition contains less than 5 mol% lyso-PC when quantified by HPLC after the first 6 months of the composition at a storage temperature of 2°C to 8°C.

14. The composition described in claim 7, wherein the composition contains less than 5 mol% lyso-PC when quantified by HPLC after the first 9 months of the composition at a storage temperature of 2°C to 8°C.

15. The composition described in claim 7, wherein the composition contains less than 5 mol% lyso-PC when quantified by HPLC after the first 12 months of the composition at a storage temperature of 2°C to 8°C.

16. The composition described in claim 7, wherein the composition contains less than 5 mol% lyso-PC when quantified by HPLC after the first 24 months of the composition at a storage temperature of 2°C to 8°C.

17. The composition of claim 6, wherein the composition further comprises triethylammonium or diethylammonium in a total amount of less than about 100 ppm.

18. The composition of claim 6, wherein the composition further comprises triethylammonium or diethylammonium in a total amount of less than 100 ppm.

19. The composition of claim 18, wherein the composition further comprises triethylammonium in a total amount of less than 79 ppm.

20. The composition of claim 17, wherein the composition further comprises triethylammonium or diethylammonium in a total amount of about 10 ppm to about 100 ppm.

21. The composition of claim 18, wherein the composition further comprises triethylammonium or diethylammonium in a total amount of 10 ppm to 100 ppm.

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

23. The composition of claim 6, further comprising a total amount of 4.05 mg / mL of 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid and a total amount of 8.42 mg / mL of sodium chloride.

24. The composition of claim 6, wherein the composition further comprises a histidine buffer.

25. The composition of claim 6, wherein the gram equivalent ratio is 0.85 to 1.

1.

26. The composition of claim 6, wherein the gram equivalent ratio is 0.9 to 1.

1.

27. The composition of claim 6, wherein the gram equivalent ratio is 0.9 to 1.

05.

28. The composition of claim 6, wherein the gram equivalent ratio is 0.95 to 1.

0.

29. The composition of claim 6, wherein the gram equivalent ratio is 0.98 to 1.

0.

30. The composition of claim 6, wherein the gram equivalent ratio is 0.99 to 1.

0.

31. The composition of claim 6, wherein the gram equivalent ratio is determined by the molar amounts of irinotecan and sucrose octasulfate co-encapsulated in the composition according to the formula: gram equivalent ratio = I / (SN) (where I is the molar concentration of irinotecan, S is the molar concentration of sucrose octasulfate, and N = 8), and when the molar amounts of irinotecan and sucrose octasulfate co-encapsulated in the composition are determined by subjecting the liposomes to size exclusion chromatography in normal saline, quantifying the amount of sulfate groups from the sucrose octasulfate in the chromatographed liposomes, and quantifying the amount of irinotecan in the chromatographed liposomes, the gram equivalent ratio is 0.85 to 1.

2.

32. The composition of claim 25, wherein the gram equivalent ratio is determined by the molar amounts of irinotecan and sucrose octasulfate co-encapsulated in the composition according to the formula: gram equivalent ratio = I / (SN) (where I is the molar concentration of irinotecan, S is the molar concentration of sucrose octasulfate, and N = 8), and when the molar amounts of irinotecan and sucrose octasulfate co-encapsulated in the composition are determined by subjecting the liposomes to size exclusion chromatography in normal saline, quantifying the amount of sulfate groups from the sucrose octasulfate in the chromatographed liposomes, and quantifying the amount of irinotecan in the chromatographed liposomes, the gram equivalent ratio is 0.85 to 1.

1.

33. The composition of claim 26, wherein the gram equivalent ratio is determined by the molar amounts of irinotecan and sucrose octasulfate co-encapsulated in the composition according to the formula: gram equivalent ratio = I / (SN) (where I is the molar concentration of irinotecan, S is the molar concentration of sucrose octasulfate, and N = 8), and when the molar amounts of irinotecan and sucrose octasulfate co-encapsulated in the composition are determined by subjecting the liposomes to size exclusion chromatography in normal saline, quantifying the amount of sulfate groups from the sucrose octasulfate in the chromatographed liposomes, and quantifying the amount of irinotecan in the chromatographed liposomes, the gram equivalent ratio is 0.9 to 1.

1.

34. The composition of claim 27, wherein the gram equivalent ratio is determined by the molar amounts of irinotecan and sucrose octasulfate co-encapsulated in the composition according to the formula: gram equivalent ratio = I / (SN) (where I is the molar concentration of irinotecan, S is the molar concentration of sucrose octasulfate, and N = 8), and when the molar amounts of irinotecan and sucrose octasulfate co-encapsulated in the composition are determined by subjecting the liposomes to size exclusion chromatography in normal saline, quantifying the amount of sulfate groups from the sucrose octasulfate in the chromatographed liposomes, and quantifying the amount of irinotecan in the chromatographed liposomes, the gram equivalent ratio is 0.9 to 1.

05.

35. The composition of claim 28, wherein the gram equivalent ratio is determined by the molar amounts of irinotecan and sucrose octasulfate co-encapsulated in the composition according to the formula: gram equivalent ratio = I / (SN) (where I is the molar concentration of irinotecan, S is the molar concentration of sucrose octasulfate, and N = 8), and when the molar amounts of irinotecan and sucrose octasulfate co-encapsulated in the composition are determined by subjecting the liposomes to size exclusion chromatography in normal saline, quantifying the amount of sulfate groups from the sucrose octasulfate in the chromatographed liposomes, and quantifying the amount of irinotecan in the chromatographed liposomes, the gram equivalent ratio is 0.95 to 1.

0.

36. The composition of claim 29, wherein the gram equivalent ratio is determined by the molar amounts of irinotecan and sucrose octasulfate co-encapsulated in the composition according to the formula: gram equivalent ratio = I / (SN) (where I is the molar concentration of irinotecan, S is the molar concentration of sucrose octasulfate, and N = 8), and when the molar amounts of irinotecan and sucrose octasulfate co-encapsulated in the composition are determined by subjecting the liposomes to size exclusion chromatography in normal saline, quantifying the amount of sulfate groups from the sucrose octasulfate in the chromatographed liposomes, and quantifying the amount of irinotecan in the chromatographed liposomes, the gram equivalent ratio is 0.98 to 1.

0.

37. The composition of claim 30, wherein the gram equivalent ratio is determined by the molar amounts of irinotecan and sucrose octasulfate co-encapsulated in the composition according to the formula: gram equivalent ratio = I / (SN) (where I is the molar concentration of irinotecan, S is the molar concentration of sucrose octasulfate, and N = 8), and when the molar amounts of irinotecan and sucrose octasulfate co-encapsulated in the composition are determined by subjecting the liposomes to size exclusion chromatography in normal saline, quantifying the amount of sulfate groups from the sucrose octasulfate in the chromatographed liposomes, and quantifying the amount of irinotecan in the chromatographed liposomes, the gram equivalent ratio is 0.99 to 1.

0.

38. The composition of claim 17, wherein the composition further comprises triethylammonium in a total amount of less than about 20 ppm.

39. The composition of claim 18, wherein the composition further comprises triethylammonium in a total amount of less than 20 ppm.

40. The composition described in claim 33, wherein the composition contains less than 10 mol% lyso-PC when quantified by HPLC after the first 6 months of storage of the composition at a storage temperature of 2°C to 8°C.

41. The composition described in claim 33, wherein the composition contains less than 5 mol% lyso-PC when quantified by HPLC after the first 6 months of storage of the composition at a storage temperature of 2°C to 8°C.

42. The composition described in claim 33, wherein the composition contains less than 10 mol% lyso-PC when quantified by HPLC after the first 12 months of the composition at a storage temperature of 2°C to 8°C.

43. The composition described in claim 33, wherein the composition contains less than 5 mol% lyso-PC when quantified by HPLC after the first 12 months of the composition at a storage temperature of 2°C to 8°C.

44. The composition described in claim 33, wherein the composition contains less than 10 mol% lyso-PC when quantified by HPLC after the first 24 months of the composition at a storage temperature of 2°C to 8°C.

45. The composition described in claim 33, wherein the composition contains less than 5 mol% lyso-PC when quantified by HPLC after the first 24 months of the composition at a storage temperature of 2°C to 8°C.

46. The composition described in claim 33, wherein after the first 6 months of the composition at a storage temperature of 4°C, the composition contains less than 1 mg / mL of lyso-PC.

47. The composition described in claim 33, wherein after the first 9 months of the composition at a storage temperature of 2°C to 8°C, the composition contains less than 1 mg / mL of lyso-PC.

48. The composition described in claim 33, wherein after the first 21 months of the composition at a storage temperature of 2°C to 8°C, the composition contains less than 2 mg / mL of lyso-PC when stored at 2°C to 8°C.

49. The composition described in claim 33, wherein after the first 12 months of the composition at a storage temperature of 2°C to 8°C, the composition contains less than 2 mg / mL of lyso-PC when stored at 2°C to 8°C.

50. The composition of claim 6, wherein the composition further comprises less than 20 ppm of substituted ammonium ions.

51. The composition of claim 6, wherein the composition further comprises 10 to 20 ppm of substituted ammonium ions.

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