Method for preparing liposomal formulations

Abstract

Provided herein is a method for purifying a liposomal formulation by washing with an acidic aqueous solution.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 222,887, filed July 16, 2021, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Provided herein are methods for preparing liposomal formulations containing encapsulated hydrophobic drugs, which may be useful formulations for treating diseases such as hyperproliferative disorders. [Background technology]

[0003] Liposomes are closed vesicles with at least one lipid bilayer surrounding an aqueous core. The intraliposomal space and lipid layer(s) can entrap a wide variety of substances, including drugs, cosmetics, diagnostic reagents, genetic materials, and bioactive compounds. Because non-toxic lipids serve as the basis of liposomes, liposomes generally exhibit low toxicity. Low toxicity, combined with the ability of liposomes to extend the plasma circulation life of drugs, makes liposomes a particularly useful vehicle for delivering pharma-ceutically active drugs. In many cases, drugs delivered by liposomes provide excellent clinical efficacy with reduced toxicity.

[0004] Passive loading of lipophilic and, to a lesser extent, amphiphilic functional compounds is somewhat more efficient than hydrophilic functional compounds, since they are distributed both in the lipid bilayer and in the intraliposomal (internal) aqueous medium. However, the final functional compound to lipid ratio and encapsulation efficiency are generally lower when passive loading is used. The concentration of drug in the liposome is equal to that of the surrounding liquid, and drug that is not trapped in the internal aqueous medium is washed out after encapsulation. Furthermore, drug loaded in the bilayer is released from the liposome very quickly when the liposome is injected into a subject. For sustained release of drug in a patient, it is preferable that the drug is encapsulated inside the liposome.

[0005] Certain hydrophilic or amphiphilic compounds can be loaded into preformed liposomes using a transmembrane pH gradient or ion gradient (D. Zucker et al., Journal of Controlled Release (2009) 139:73-80). This technique is called active loading or remote loading. Compounds suitable for active loading must be able to change from an uncharged form that can diffuse through the liposomal membrane to a charged form that cannot. Typically, a functional compound is loaded by adding it to a suspension of liposomes that have been prepared to have a low inside / high outside pH gradient or ion gradient. Through active loading, a high functional compound-to-lipid mass ratio and a high loading efficiency (up to 100%) can be achieved. An example is the active loading of the anticancer drugs doxorubicin, daunorubicin, and vincristine (PR Cullis et al., Biochimica et Biophysica Acta, (1997) 1331:187-211, and references therein).

[0006] Hydrophobic drugs are believed to be primarily capable of being loaded into liposomes through membrane intercalation via a passive loading / assembly mechanism. In describing the use of micelles to transfer poorly soluble drugs into liposome bilayers, Wasan et al. state that "drugs with hydrophobic attributes can intercalate into the lipid bilayer, which can be accomplished by adding the drug to preformed liposomes" (US2009 / 0028931). However, such loading relies on the hydrophobic drug associating with or being entrapped in the lipid bilayer, and the drug may easily leak out of the liposome, resulting in poor retention and less than desirable pharmacokinetics in vivo. In particular, if bound or entrapped in the lipid bilayer, rather than the drug being encapsulated in the aqueous core of the liposome, rapid release into the bloodstream may occur upon administration of the liposome to a patient. This may be particularly problematic for certain drugs where dose-limiting toxicity and / or low therapeutic index are clinical concerns, as is the case for many antineoplastic agents. For these reasons, particularly in the field of liposomal drug delivery, there is a need for improved methods of preparing liposome-encapsulated drugs that do not contain drug associated with the lipid bilayer. Such improved liposomal formulations may provide advantageous pharmacokinetic properties and greater clinical value. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] US2009 / 0028931 [Non-patent literature]

[0008] [Non-Patent Document 1] D. Zucker et al., Journal of Controlled Release (2009) 139:73-80 [Non-Patent Document 2] PRCullis et al., Biochimica et Biophysica Acta, (1997) 1331:187-211 Summary of the Invention

[0009] The present disclosure relates to methods for preparing purified liposomal compositions.

[0010] In one aspect, a method for preparing a purified liposome composition comprising liposomes is provided, comprising: The liposome is (a) lipid bilayer, (b) the internal medium, and (c) comprising a therapeutic agent encapsulated in the internal medium of the liposome; The therapeutic agent has low water solubility and can be protonated to a protonated form; The method comprises: (i) providing a crude liposome composition; (ii) purifying the crude liposome composition using an acidified aqueous solution.

[0011] In some embodiments, the lipid bilayer comprises a first lipid and a first sterol, and the internal medium comprises a first loading aid.

[0012] In some embodiments, the first lipid is a polymer-conjugated lipid.For example, in some embodiments, the first lipid is selected from the group consisting of 1,2-distearoyl-rac-glycero-3-methoxypoly(ethylene glycol) (such as DSG-PEG2000), 1,2-dimyristoyl-rac-glycero-3-methoxypoly(ethylene glycol) (such as DMG-PEG2000), 1,2-dipalmitoyl-rac-glycero-3-methoxypoly(ethylene glycol) (such as DPG-PEG2000), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) (such as DSPE-PEG2000).In some embodiments, the first lipid is DSG-PEG2000.

[0013] In some embodiments, the lipid bilayer further comprises a second lipid. In some embodiments, the second lipid is a phospholipid. In some embodiments, the second lipid is distearoylphosphatidylcholine (DSPC). In some embodiments, the second lipid is hydrogenated sphingomyelin.

[0014] In some embodiments, the internal medium is an aqueous internal medium. In some embodiments, the aqueous internal medium is an acidic aqueous internal medium. In some embodiments, the internal medium further comprises an additional solvent. In some embodiments, the additional solvent is an organic solvent. In some embodiments, the additional solvent is dimethylsulfoxide (DMSO).

[0015] In some embodiments, the first loading aid is an ionic loading aid. In some embodiments, the first loading aid is selected from the group consisting of ammonium sulfate (AS), sucrose ammonium octasulfate (NH4SOS), potassium sucrose octasulfate (KSOS), sucrose triethanolammonium octasulfate (TEA(OH)SOS), sucrose triethylammonium octasulfate (TEASOS), and sodium citrate. In some embodiments, the first loading aid is ammonium sulfate (AS). In some embodiments, the first loading aid is sucrose triethylammonium octasulfate (TEASOS).

[0016] In some embodiments, the internal medium further comprises a second loading aid. In some embodiments, the second loading aid is an ionic loading aid. In some embodiments, the second loading aid is selected from the group consisting of ammonium sulfate (AS), ammonium sucrose octasulfate (NH4SOS), potassium sucrose octasulfate (KSOS), triethylammonium sucrose octasulfate (TEASOS), and sodium citrate.

[0017] In some embodiments, the first loading aid is potassium sucrose octasulfate (KSOS) and the second loading aid is sodium citrate.

[0018] In some embodiments, the first sterol is cholesterol or β-sitosterol.

[0019] In some embodiments, the therapeutic agent has a cLogP of greater than about 2. In some embodiments, the protonated form of the therapeutic agent has a pKa of greater than about 2.

[0020] In some embodiments, the therapeutic agent is an antiangiogenic agent, an antimetabolite, an apoptosis inducer, a cell cycle inhibitor, a cell cycle control inhibitor, a checkpoint inhibitor, a cyclin-dependent kinase inhibitor, a cytotoxic agent, a DNA damaging agent, a DNA repair inhibitor, a mitochondrial poison, a telomerase inhibitor, a signal transduction inhibitor, a transcription inhibitor, a Bcl inhibitor, a PARP inhibitor, an HSP90 inhibitor, a JAK inhibitor, an ATR inhibitor, a tyrosine kinase inhibitor, a receptor tyrosine kinase inhibitor, a BTK inhibitor, an alkylating agent, an SMO inhibitor, an antitubulin agent, a MEK inhibitor, a topoisomerase inhibitor, a RAF inhibitor, a BRAF inhibitor, or a proteasome inhibitor. In some embodiments, the therapeutic agent is a Bcl-2 inhibitor, a Bcl-X ... L Inhibitors and Bcl-2 / Bcl-X L The Bcl inhibitor is selected from the group consisting of dual inhibitors.

[0021] In some embodiments, the liposome further comprises an additional therapeutic agent. In some embodiments, the additional therapeutic agent is encapsulated in the liposome. In some embodiments, the additional therapeutic agent is an antiangiogenic agent, an antimetabolite, an apoptosis inducer, a cell cycle inhibitor, a cell cycle control inhibitor, a checkpoint inhibitor, a cyclin-dependent kinase inhibitor, a cytotoxic agent, a DNA damaging agent, a DNA repair inhibitor, a mitochondrial poison, a telomerase inhibitor, a signal transduction inhibitor, a transcription inhibitor, a Bcl inhibitor, a PARP inhibitor, an HSP90 inhibitor, a JAK inhibitor, an ATR inhibitor, a tyrosine kinase inhibitor, a receptor tyrosine kinase inhibitor, a BTK inhibitor, an alkylating agent, an SMO inhibitor, an antitubulin agent, a MEK inhibitor, a topoisomerase inhibitor, a RAF inhibitor, a BRAF inhibitor, or a proteasome inhibitor.

[0022] In some embodiments, the liposomes have an average diameter between about 50 nm and about 250 nm.

[0023] In some embodiments, the acidified aqueous solution comprises a sugar. In some embodiments, the acidified aqueous solution comprises dextrose. In some embodiments, the acidified aqueous solution comprises sucrose. In some embodiments, the concentration of the sugar in the acidified aqueous solution is about 5% to 20% by weight. In some aspects, the concentration of the acid in the acidified aqueous solution is about 1 mM to 100 mM. In some embodiments, the acidified aqueous solution comprises methanesulfonic acid. [Brief description of the drawings]

[0024] [Figure 1A] 1 shows the normalized plasma concentration over time of Compound 2 using various liposomal formulations compared to the free drug. [Figure 1B] The change over time in normalized plasma concentrations of Compound 2 using an exemplary liposomal formulation of the present disclosure is shown compared to using a liposomal formulation prepared according to a previously published method, and compared to using the free drug. [Figure 2A]1 shows the normalized plasma concentration of Compound 9 over time using various liposomal formulations. [Figure 2B] The change over time in normalized plasma concentrations of compound 9 using an exemplary liposomal formulation of the present disclosure is shown compared to using a liposomal formulation prepared according to a previously published method, and compared to using the free drug. [Diagram 3] 1 shows the permeate concentration of compound 9 as a function of wash volume during purification of an exemplary liposomal formulation of the present disclosure. [Figure 4] 1 shows the change over time in normalized plasma concentrations of compound 7 using an exemplary liposomal formulation of the present disclosure compared to using a liposomal formulation prepared according to a previously published method. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Unless otherwise defined, all technical terms, notations, and other scientific terms or predicates used herein shall have the same meaning as commonly understood by those of ordinary skill in the art to which this disclosure belongs. In some cases, terms having commonly understood meanings are defined herein for clarity and / or ready reference, and the inclusion of such definitions herein should not necessarily be interpreted as representing a substantial difference from that commonly understood in the art. Many of the techniques and procedures described or referenced herein are well understood using conventional methodology by those of ordinary skill in the art, but are commonly used. Procedures involving the use of commercially available kits and reagents, as appropriate, are generally performed according to protocols and / or parameters defined by the manufacturer, unless otherwise noted. All patents, applications, published applications, and other publications referred to herein are incorporated by reference in their entirety. If a definition set forth in this section is contrary to or otherwise inconsistent with a definition set forth in a patent, application, published application, and other publication incorporated herein by reference, the definition set forth in this section shall take precedence over the definition incorporated herein by reference.

[0026] definition As used herein, the use of the terms "a," "an," and the like, refers to one or more, unless otherwise specified.

[0027] Reference herein to "about" a value or parameter includes (and describes) embodiments that are directed to the value or parameter itself. For example, a description that refers to "about X" includes a description of "X."

[0028] The terms "therapeutic agent" or "drug," as used herein, refer to chemical moieties used in a variety of therapeutic applications, including pharmaceutical applications.

[0029] The term "pharmaceutically acceptable salt" refers to any salt of the compounds herein that is known to be non-toxic and is commonly used in the pharmaceutical literature. In some embodiments, the pharmaceutically acceptable salt of the compound retains the biological effectiveness of the compounds described herein and is not biologically or otherwise undesirable. Examples of pharmaceutically acceptable salts can be found in Berge et al., Pharmaceutical Salts, J. Pharmaceutical Sciences, January 1977, 66(1), 1-19. Pharmaceutically acceptable acid addition salts can be formed with inorganic and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid. Organic acids from which acids can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, lactic acid, oxalic acid, malic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethylsulfonic acid, p-toluenesulfonic acid, stearic acid, and salicylic acid. Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines; substituted amines, including naturally occurring substituted amines; cyclic amines; and basic ion exchange resins. Examples of organic bases include isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. In some embodiments, the pharma- ceutically acceptable base addition salt is chosen from ammonium, potassium, sodium, calcium, and magnesium salts.

[0030] As used herein, the term "subject" refers to an animal, such as a mammal, bird, or fish. In some embodiments, the subject is a mammal. Mammals include, for example, mice, rats, dogs, cats, pigs, sheep, horses, cows, and humans. In some embodiments, the subject is a human, e.g., a human who has been or will be the object of treatment, observation, or experiment.

[0031] The term "therapeutically effective amount" or "effective amount" refers to an amount of a compound disclosed and / or described herein that is sufficient to affect such treatment when administered to a subject in need of treatment as defined herein. The therapeutically effective amount will vary depending on, for example, the subject and disease state being treated, the subject's weight and age, the severity of the disease state, the specific compound, the dosing regimen to be followed, the timing of administration, and the mode of administration, all of which can be readily determined by one of ordinary skill in the art. The therapeutically effective amount can be ascertained experimentally, for example, by assaying the blood concentration of the chemical entity, or theoretically, by calculating bioavailability.

[0032] "Treatment" (and related terms, e.g., "treat," "treated," "treating") includes one or more of preventing a disease or disorder (i.e., preventing the onset of clinical symptoms of a disease or disorder); inhibiting a disease or disorder; slowing or arresting the onset of clinical symptoms of a disease or disorder; and / or alleviating a disease or disorder (i.e., causing clinical symptoms to be alleviated or regressed). The term encompasses situations in which a disease or disorder is already experienced by a subject, as well as situations in which a disease or disorder is not currently experienced but is expected to occur. The term encompasses both complete and partial alleviation or prevention of a pathology or disorder, as well as complete or partial alleviation of clinical symptoms of a disease or disorder. Thus, the compounds described and / or disclosed herein may prevent the worsening of an existing disease or disorder, aid in the management of a disease or disorder, or alleviate or eliminate a disease or disorder. When used in a prophylactic manner, the compounds disclosed and / or described herein may prevent the onset of a disease or disorder or reduce the extent of a disease or disorder that is likely to develop.

[0033] Without being bound by theory, it is speculated that the encapsulation of therapeutic agents in liposomes causes a portion of the therapeutic agent to become associated with or entrapped within the lipid bilayer, especially when the therapeutic agent is encapsulated with low water solubility or high hydrophobicity. By purifying the liposome composition with an acidified aqueous solution, the therapeutic agent associated with or entrapped within the lipid bilayer can be removed, resulting in a liposome formulation containing the therapeutic agent entrapped only in the internal medium of the liposome. For example, as shown in FIG. 3, when an exemplary liposome formulation containing compound 9 is washed with an acidified aqueous sugar solution, compound 9 is detected in the permeate. As the washing volume increases, i.e., as the liposome composition is washed with a larger amount of acidified aqueous sugar solution, the amount of compound 9 detected in the permeate peak also increases and then decreases. It is believed that the peak is due to the rapid release of compound 9 associated with or entrapped within the lipid bilayer into the acidified aqueous sugar solution. FIG. 3 further shows that after washing with about 300-400 mL of the acidified sugar aqueous solution, almost no compound 9 is detected in the permeate, meaning that by this time, most or all of compound 9 has been removed from the lipid bilayer. In other words, purifying the liposomal composition with an acidified aqueous solution may prevent any therapeutic agent that is merely associated with or entrapped within the lipid bilayer from being released into the acidified aqueous solution and thus undergoing rapid release in the patient's bloodstream upon administration. As a result, liposomal compositions prepared according to various embodiments described herein may be expected to exhibit superior pharmacokinetic properties and enhanced clinical value.

[0034] In a first aspect, a method for preparing a purified liposome composition comprising liposomes is provided, comprising: The liposome is (a) lipid bilayer, (b) the internal medium, and (c) comprising a therapeutic agent encapsulated in the internal medium of the liposome; The therapeutic agent has low water solubility and can be protonated to a protonated form; The method comprises: (i) providing a crude liposome composition; (ii) purifying the crude liposome composition using an acidified aqueous solution.

[0035] In another embodiment, a liposomal composition is provided comprising one or more liposomes prepared as described herein, each of the one or more liposomes comprising: (a) a lipid bilayer comprising a first lipid and a first sterol; (b) an inner medium comprising a first loading aid and a first solvent; and (c) comprising a therapeutic agent encapsulated within the interior medium of said liposome.

[0036] In another aspect, there is provided a pharmaceutical composition comprising one or more liposome compositions comprising one or more liposomes prepared as described herein, wherein each of the one or more liposomes comprises: (a) a lipid bilayer comprising a first lipid and a first sterol; (b) an inner medium comprising a first loading aid and a first solvent; and (c) comprising a therapeutic agent encapsulated in the internal medium of said liposome; and The therapeutic agent is located outside one or more liposomes.

[0037] In another aspect, there is provided a method of delivering a therapeutically effective amount of a therapeutic agent to a subject, comprising administering to the subject a liposomal composition prepared as described herein, the liposome composition comprising one or more liposomes, each of the one or more liposomes comprising: (a) a lipid bilayer comprising a first lipid and a first sterol; (b) an inner medium comprising a first loading aid and a first solvent; and (c) comprising a therapeutic agent encapsulated within the interior medium of said liposome.

[0038] In another aspect, there is provided a method of treating a hyperproliferative disorder in a subject in need of treatment, comprising administering to the subject a liposomal composition prepared as described herein, the liposome composition comprising one or more liposomes, each of the one or more liposomes comprising: (a) a lipid bilayer comprising a first lipid and a first sterol; (b) an inner medium comprising a first loading aid and a first solvent; and (c) comprising a therapeutic agent encapsulated within the interior medium of said liposome.

[0039] In some embodiments of all the above aspects, the first lipid is a polymer-conjugated lipid. In some embodiments, the polymer-conjugated lipid is selected from the group consisting of 1,2-distearoyl-rac-glycero-3-methoxypoly(ethylene glycol) (such as DSG-PEG2000), 1,2-dimyristoyl-rac-glycero-3-methoxypoly(ethylene glycol) (such as DMG-PEG2000), 1,2-dipalmitoyl-rac-glycero-3-methoxypoly(ethylene glycol) (such as DPG-PEG2000), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) (such as DSPE-PEG2000). In some embodiments, the polymer-conjugated lipid is 1,2-distearoyl-rac-glycero-3-methoxypoly(ethylene glycol) or 1,2-dimyristoyl-rac-glycero-3-methoxypoly(ethylene glycol). In some embodiments, the polymer-conjugated lipid is 1,2-distearoyl-rac-glycero-3-methoxypoly(ethylene glycol). In some embodiments, the polymer-conjugated lipid is DSG-PEG2000. In some embodiments, the lipid bilayer further comprises a second lipid. In some embodiments, the second lipid is a phospholipid. In some embodiments, the phospholipid is selected from the group consisting of phosphatidylcholine, sphingolipid, and hydrogenated sphingolipid. In some embodiments, the second lipid is distearoylphosphatidylcholine (DSPC). In some embodiments, the second lipid is sphingomyelin. In some embodiments, the second lipid is hydrogenated sphingomyelin (dihydrosphingomyelin). In some embodiments, the second lipid is egg sphingomyelin. In some embodiments, the second lipid is hydrogenated sphingomyelin (dihydrosphingomyelin) or egg sphingomyelin.

[0040] In some embodiments of all the foregoing aspects, the first lipid is a first phospholipid. In some embodiments, the phospholipid is selected from the group consisting of phosphatidylcholine, sphingolipid, and hydrogenated sphingolipid. In some embodiments, the first lipid is distearoylphosphatidylcholine (DSPC). In some embodiments, the first lipid is sphingomyelin. In some embodiments, the first lipid is hydrogenated sphingomyelin (dihydrosphingomyelin). In some embodiments, the lipid bilayer further comprises a second lipid. In some embodiments, the second lipid is a second phospholipid. In some embodiments, the second phospholipid is selected from the group consisting of phosphatidylcholine, sphingolipid, and hydrogenated sphingolipid (dihydrosphingomyelin). In some embodiments, the second lipid is distearoylphosphatidylglycerol (DSPG). In some embodiments, the second lipid is sphingomyelin. In some embodiments, the second lipid is hydrogenated sphingomyelin (dihydrosphingomyelin).

[0041] In some embodiments of all the above aspects, the first solvent is an aqueous solvent. In some embodiments, the aqueous solvent is an acidic aqueous solvent.

[0042] In some embodiments of all the above aspects, the internal medium further comprises a second solvent. In some embodiments, the second solvent is an organic solvent. In some embodiments, the organic solvent is an aprotic organic solvent. In some embodiments, the second solvent is dimethylsulfoxide (DMSO).

[0043] In some embodiments of all the above aspects, the first loading aid is an ionic loading aid. In some embodiments, the first loading aid forms an ion gradient across the lipid bilayer. In some embodiments, the ion gradient is a pH gradient, a sulfate gradient, a phosphate gradient, a citrate gradient, an acetate gradient, an EDTA ion gradient, an ammonium gradient, an alkylammonium gradient, an amylammonium gradient, a Ca gradient, a Cu gradient, a Fe gradient, a Mg gradient, a Mn gradient, a Zn gradient, a Na gradient, or a K gradient. In some embodiments, the first loading aid is a sulfate, a sucrose octasulfate, or a citrate. In some embodiments, the first loading aid is selected from the group consisting of ammonium sulfate (AS), ammonium sucrose octasulfate (NH4SOS), potassium sucrose octasulfate (KSOS), sucrose triethylammonium octasulfate (TEASOS), sucrose triethanolammonium octasulfate (TEA(OH)SOS), ammonium citrate, and sodium citrate. In some embodiments, the first loading aid is selected from the group consisting of ammonium sulfate (AS), ammonium sucrose octasulfate (NHSOS), potassium sucrose octasulfate (KSOS), triethylammonium sucrose octasulfate (TEASOS), and sodium citrate. In some embodiments, the first loading aid is ammonium sulfate (AS). In some embodiments, the first loading aid is ammonium sucrose octasulfate (NHSOS). In some embodiments, the first loading aid is potassium sucrose octasulfate (KSOS). In some embodiments, the first loading aid is triethylammonium sucrose octasulfate (TEASOS). In some embodiments, the first loading aid is sodium citrate. In some embodiments, the internal medium further comprises a second loading aid. In some embodiments, the second loading aid is an ionic loading aid. In some embodiments, the second loading aid is sulfate, sucrose octasulfate, or citrate.In some embodiments, the second loading aid is selected from the group consisting of ammonium sulfate (AS), ammonium sucrose octasulfate (NHSOS), potassium sucrose octasulfate (KSOS), triethylammonium sucrose octasulfate (TEASOS), triethanolammonium sucrose octasulfate (TEA(OH)SOS), ammonium citrate, and sodium citrate. In some embodiments, the second loading aid is ammonium sulfate (AS). In some embodiments, the second loading aid is ammonium sucrose octasulfate (NHSOS). In some embodiments, the second loading aid is potassium sucrose octasulfate (KSOS). In some embodiments, the second loading aid is triethylammonium sucrose octasulfate (TEASOS). In some embodiments, the second loading aid is sodium citrate. In some embodiments, the first loading aid is potassium sucrose octasulfate (KSOS) and the second loading aid is sodium citrate.

[0044] In some embodiments of all the above aspects, the first sterol is cholesterol or a plant sterol (such as β-sitosterol). In some embodiments, the first sterol is cholesterol. In some embodiments, the first sterol is β-sitosterol.

[0045] In some embodiments, the liposomes and liposomal compositions provided thereto comprise a liposomally encapsulated therapeutic agent, In some embodiments, the liposomes and liposomal compositions provided thereto comprise a liposomally encapsulated hydrophobic therapeutic agent.

[0046] In some embodiments, the therapeutic agent is an anti-angiogenic agent, antimetabolite, an apoptosis inducer, a cell cycle inhibitor, a cell cycle control inhibitor, a checkpoint inhibitor, a cyclin dependent kinase inhibitor, a cytotoxic agent, a DNA damaging agent, a DNA repair inhibitor, a mitochondrial poison, a telomerase inhibitor, a signal transduction inhibitor, a transcription inhibitor, a Bcl inhibitor, a PARP inhibitor, a PI3K inhibitor, an HSP90 inhibitor, a JAK inhibitor, an ATR inhibitor, an HDAC inhibitor, a tyrosine kinase inhibitor, a receptor tyrosine kinase inhibitor, a BTK inhibitor, an alkylating agent, an SMO inhibitor, an anti-tubulin agent, a MEK inhibitor, a topoisomerase inhibitor, a RAF inhibitor, a BRAF inhibitor, or a proteasome inhibitor.

[0047] In some embodiments, the therapeutic agent is an HSP90 inhibitor. In some embodiments, the HSP90 inhibitor is luminespib.

[0048] In some embodiments, the therapeutic agent is an alkylating agent selected from the group consisting of bendamustine and chlorambucil.

[0049] In some embodiments, the therapeutic agent is an antitubulin agent, hi some embodiments, the therapeutic agent is an antitubulin agent selected from the group consisting of vincristine, vinorelbine, and docetaxel.

[0050] In some embodiments, the therapeutic agent is an ATR inhibitor.

[0051] In some embodiments, the therapeutic agent is a RAF inhibitor. In some embodiments, the RAF inhibitor is dabrafenib. In some embodiments, the therapeutic agent is a BRAF inhibitor. In some embodiments, the BRAF inhibitor is vemurafenib.

[0052] In some embodiments, the therapeutic agent is a BTK inhibitor. In some embodiments, the BTK inhibitor is ibrutinib.

[0053] In some embodiments, the therapeutic agent is an HDAC inhibitor. In some embodiments, the HDAC inhibitor is panobinostat.

[0054] In some embodiments, the therapeutic agent is a JAK inhibitor. In some embodiments, the JAK inhibitor is ruxolitinib.

[0055] In some embodiments, the therapeutic agent is a MEK inhibitor. In some embodiments, the therapeutic agent is a MEK inhibitor selected from the group consisting of selumetinib and cobimetinib.

[0056] In some embodiments, the therapeutic agent is a PARP inhibitor. In some embodiments, the therapeutic agent is a PARP inhibitor selected from the group consisting of talazoparib, niraparib, and rucaparib.

[0057] In some embodiments, the therapeutic agent is a PI3K inhibitor. In some embodiments, the PI3K inhibitor is idelalisib.

[0058] In some embodiments, the therapeutic agent is a proteasome inhibitor. In some embodiments, the proteasome inhibitor is carfilzomib.

[0059] In some embodiments, the therapeutic agent is an SMO inhibitor. In some embodiments, the therapeutic agent is an SMO inhibitor selected from the group consisting of sonidegib and vismodegib.

[0060] In some embodiments, the therapeutic agent is a tyrosine kinase inhibitor. In some embodiments, the therapeutic agent is a tyrosine kinase inhibitor selected from the group consisting of brigatinib, lenvatinib, afatinib, axitinib, cabozantinib, ponatinib, sorafenib, osimertinib, regorafenib, bosutinib, crizotinib, vandetanib, nilotinib, alectinib, ceritinib, dasatinib, pazopanib, sunitinib, erlotinib, imatinib, gefitinib, and lapatinib.

[0061] In some embodiments, the therapeutic agent is a topoisomerase inhibitor. In some embodiments, the therapeutic agent is a topoisomerase I inhibitor. In some embodiments, the topoisomerase inhibitor is irinotecan.

[0062] In some embodiments, the therapeutic agent is a Bcl inhibitor. In some embodiments, the Bcl inhibitor is a Bcl-2 inhibitor. In some embodiments, the Bcl inhibitor is a Bcl-X L In some embodiments, the Bcl inhibitor is a Bcl-2 / Bcl-X L In some embodiments, the Bcl inhibitor is a compound of formula (I): [ka] or a stereoisomer, tautomer, or pharma- ceutically acceptable salt thereof, wherein: V is [ka] and W is H or [ka] and X is [ka] and Y is -NO2 or -SO2CF3; Z is [ka] In some embodiments, V is selected from the group consisting of: [ka] In some embodiments, V is [ka] In some embodiments, W is H. In some embodiments, W is [ka] In some embodiments, X is [ka] In some embodiments, X is [ka] In some embodiments, Y is -NO2. In some embodiments, Y is -SO2CF3. In some embodiments, Z is [ka] In some embodiments, Z is [ka] In some embodiments, Z is [ka] In some embodiments, Z is [ka] In some embodiments, Z is [ka] In some embodiments, Z is [ka] In some embodiments, Z is [ka] In some embodiments, Z is [ka] It is. In some embodiments, the Bcl inhibitor is [ka] [ka] [ka] [ka] In some embodiments, the Bcl inhibitor is selected from the group consisting of venetoclax (ABT-199), navitoclax (ABT-263), ABT-737, obatoclax mesylate (GX15-070), sabutoclax, TW-37, (R)-(-)-gossypol acetate, HA14-1, a BH3 mimetic, and oblimersen. [ka] In some embodiments, the Bcl inhibitor is selected from the group consisting of venetoclax (ABT-199), navitoclax (ABT-263), obatoclax mesylate (GX15-070), sabutoclax, TW-37, (R)-(-)-gossypol acetate, HA14-1, a BH3 mimetic, and oblimersen. [ka] In some embodiments, the Bcl inhibitor is selected from the group consisting of venetoclax (ABT-199), navitoclax (ABT-263), obatoclax mesylate (GX15-070), sabutoclax, TW-37, (R)-(-)-gossypol acetate, HA14-1, a BH3 mimetic, and oblimersen. [ka] In some embodiments, the Bcl inhibitor is selected from the group consisting of: venetoclax (ABT-199), and navitoclax (ABT-263). [ka] In some embodiments, the Bcl inhibitor is selected from the group consisting of: venetoclax (ABT-199), and navitoclax (ABT-263). [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is venetoclax. In some embodiments, the Bcl inhibitor is navitoclax.

[0063] In some embodiments, the liposome composition provided therein includes a therapeutic agent. In some embodiments, the therapeutic agent is a hydrophobic therapeutic agent. In some embodiments, the therapeutic agent has a cLogP of greater than about 2. In some embodiments, the therapeutic agent has a cLogP of about 2 to about 12. In some embodiments, the therapeutic agent has a cLogP of about 3 to about 12. In some embodiments, the therapeutic agent has a cLogP of about 2 to about 4. In some embodiments, the therapeutic agent has a cLogP of about 2 to about 8. In some embodiments, the therapeutic agent has a cLogP of about 4 to about 12. In some embodiments, the therapeutic agent has a cLogP of about 4 to about 8. In some embodiments, the therapeutic agent has a cLogP of about 8 to about 12. In some embodiments, the therapeutic agent has a cLogP of about 10 to about 12.

[0064] In some embodiments, the therapeutic agent can be protonated and the protonated form has a pK of greater than about 2. a In some embodiments, the therapeutic agent can be protonated and has a pK of about 3 to about 11. a In some embodiments, the therapeutic agent can be protonated and the protonated form has a pK a In some embodiments, the therapeutic agent can be protonated and the protonated form has a pK of about 8 to about 11. a In some embodiments, the therapeutic agent can be protonated and the protonated form has a pK a In some embodiments, the therapeutic agent can be protonated and has a pK of about 6 to about 8. a In some embodiments, the therapeutic agent can be protonated and has a pK of about 3 to about 8. a In some embodiments, the therapeutic agent can be protonated and has a pK of about 2 to about 12 in its protonated form. a In some embodiments, the therapeutic agent can be protonated and has a pK of about 2 to about 8. a In some embodiments, the therapeutic agent can be protonated and the protonated form has a pK a In some embodiments, the therapeutic agent can be protonated and the protonated form has a pK of about 4 to about 12. a In some embodiments, the therapeutic agent can be protonated and has a pK of about 4 to about 8. a In some embodiments, the therapeutic agent can be protonated and has a pK of about 8 to about 12 in its protonated form. a In some embodiments, the therapeutic agent can be protonated and the protonated form has a pK of about 10 to about 12. a has.

[0065] In some embodiments, the liposome composition provided therein comprises an additional therapeutic agent. In some embodiments, the additional therapeutic agent is a hydrophobic therapeutic agent. In some embodiments, the additional therapeutic agent is a hydrophilic therapeutic agent. In some embodiments, the additional therapeutic agent is encapsulated in the liposome. In some embodiments, the additional therapeutic agent is external to the liposome. In some embodiments of all the foregoing aspects, the liposome further comprises an additional therapeutic agent. In some embodiments, the additional therapeutic agent is encapsulated in the liposome.

[0066] In some embodiments, the additional therapeutic agent is an anti-angiogenic agent, antimetabolite, an apoptosis inducer, a cell cycle inhibitor, a cell cycle control inhibitor, a checkpoint inhibitor, a cyclin dependent kinase inhibitor, a cytotoxic agent, a DNA damaging agent, a DNA repair inhibitor, a mitochondrial poison, a telomerase inhibitor, a signal transduction inhibitor, a transcription inhibitor, a Bcl inhibitor, a PARP inhibitor, a PI3K inhibitor, an HSP90 inhibitor, a JAK inhibitor, an ATR inhibitor, an HDAC inhibitor, a tyrosine kinase inhibitor, a receptor tyrosine kinase inhibitor, a BTK inhibitor, an alkylating agent, an SMO inhibitor, an anti-tubulin agent, a MEK inhibitor, a topoisomerase inhibitor, a RAF inhibitor, a BRAF inhibitor, or a proteasome inhibitor.

[0067] In some embodiments, the additional therapeutic agent is an HSP90 inhibitor. In some embodiments, the HSP90 inhibitor is luminespib.

[0068] In some embodiments, the additional therapeutic agent is an alkylating agent selected from the group consisting of bendamustine and chlorambucil.

[0069] In some embodiments, the additional therapeutic agent is an antitubulin agent, hi some embodiments, the additional therapeutic agent is an antitubulin agent selected from the group consisting of vincristine, vinorelbine, and docetaxel.

[0070] In some embodiments, the additional therapeutic agent is an ATR inhibitor.

[0071] In some embodiments, the additional therapeutic agent is a RAF inhibitor. In some embodiments, the RAF inhibitor is dabrafenib. In some embodiments, the additional therapeutic agent is a BRAF inhibitor. In some embodiments, the BRAF inhibitor is vemurafenib.

[0072] In some embodiments, the additional therapeutic agent is a BTK inhibitor. In some embodiments, the BTK inhibitor is ibrutinib.

[0073] In some embodiments, the additional therapeutic agent is an HDAC inhibitor, hi some embodiments, the HDAC inhibitor is panobinostat.

[0074] In some embodiments, the additional therapeutic agent is a JAK inhibitor. In some embodiments, the JAK inhibitor is ruxolitinib.

[0075] In some embodiments, the additional therapeutic agent is a MEK inhibitor. In some embodiments, the additional therapeutic agent is a MEK inhibitor selected from the group consisting of selumetinib and cobimetinib.

[0076] In some embodiments, the additional therapeutic agent is a PARP inhibitor. In some embodiments, the additional therapeutic agent is a PARP inhibitor selected from the group consisting of talazoparib, niraparib, and rucaparib.

[0077] In some embodiments, the additional therapeutic agent is a PI3K inhibitor. In some embodiments, the PI3K inhibitor is idelalisib.

[0078] In some embodiments, the additional therapeutic agent is a proteasome inhibitor. In some embodiments, the proteasome inhibitor is carfilzomib.

[0079] In some embodiments, the additional therapeutic agent is an SMO inhibitor. In some embodiments, the additional therapeutic agent is an SMO inhibitor selected from the group consisting of sonidegib and vismodegib.

[0080] In some embodiments, the additional therapeutic agent is a tyrosine kinase inhibitor. In some embodiments, the additional therapeutic agent is a tyrosine kinase inhibitor selected from the group consisting of brigatinib, lenvatinib, afatinib, axitinib, cabozantinib, ponatinib, sorafenib, osimertinib, regorafenib, bosutinib, crizotinib, vandetanib, nilotinib, alectinib, ceritinib, dasatinib, pazopanib, sunitinib, erlotinib, imatinib, gefitinib, and lapatinib.

[0081] In some embodiments, the additional therapeutic agent is a topoisomerase inhibitor. In some embodiments, the additional therapeutic agent is a topoisomerase I inhibitor. In some embodiments, the topoisomerase inhibitor is irinotecan.

[0082] In some embodiments, the additional therapeutic agent is a Bcl inhibitor, such as those described above. Liposomes and liposomal compositions

[0083] Liposomes contain one or more lipid bilayers that encapsulate an internal compartment. These liposomes can be multilamellar, bilamellar, or unilamellar vesicles. Unilamellar liposomes (also known as unilamellar vesicles or "ULVs") encapsulate a single internal aqueous compartment and are classified as either small unilamellar vesicles (SUVs) or large unilamellar vesicles (LUVs). The sizes of LUVs and SUVs range from about 50-500 nm and 20-50 nm, respectively. Bilamellar liposomes have two lipid membranes, an inner membrane that surrounds a single internal aqueous compartment and a second, larger, outer membrane that surrounds the inner membrane, thus creating a second internal aqueous compartment.

[0084] In some embodiments, the liposomes have an average diameter between about 20 nm and about 500 nm. In some embodiments, the liposomes have an average diameter between about 50 nm and about 250 nm. In some embodiments, the liposomes have an average diameter between about 80 nm and about 250 nm. In some embodiments, the liposomes have an average diameter between about 50 nm and about 150 nm. In some embodiments, the liposomes have an average diameter between about 80 nm and about 150 nm. In some embodiments, the liposomes have an average diameter between about 50 nm and about 120 nm. In some embodiments, the liposomes have an average diameter between about 80 nm and about 120 nm. In some embodiments, the liposomes have an average diameter of about 50 nm, about 80 nm, about 100 nm, about 120 nm, about 150 nm, or about 250 nm. In some embodiments, the liposomes have an average diameter of about 80 nm.

[0085] The maintenance of the size distribution of liposomes in the liposome composition may be experimentally evaluated by obtaining a size profile. The size distribution determined by quasi-elastic light scattering is typically presented as a histogram showing the average diameter of the liposomes. The most commonly used significant size distribution measurements in the art are D10, D90, D99, or standard deviation or polydispersity index (PDI). The "D99" value indicates that 99% of the liposomes are below or above the stated size. This is particularly useful, for example, when it is important to exclude either the upper or lower size limits. For example, in certain embodiments, it is desirable to ensure that there are no liposomes with an average diameter of more than 200 nm.

[0086] In some embodiments, the particle size distribution of the liposomes in the liposome composition is quantified using a polydispersity index (PDI). In some embodiments, the liposome composition has a polydispersity index (PDI) of about 0.001 to about 0.5. In some embodiments, the liposome composition has a polydispersity index (PDI) of about 0.001 to about 0.4. In some embodiments, the liposome composition has a polydispersity index (PDI) of about 0.001 to about 0.3. In some embodiments, the liposome composition has a polydispersity index (PDI) of about 0.005 to about 0.5. In some embodiments, the liposome composition has a polydispersity index (PDI) of about 0.005 to about 0.4. In some embodiments, the liposome composition has a polydispersity index (PDI) of about 0.005 to about 0.3. In some embodiments, the liposome composition has a polydispersity index (PDI) of about 0.001 to about 0.2. In some embodiments, the liposome composition has a polydispersity index (PDI) of about 0.001 to about 0.1. In some embodiments, the liposome composition has a polydispersity index (PDI) of about 0.005 to about 0.2. In some embodiments, the liposome composition has a polydispersity index (PDI) of about 0.005 to about 0.1. In some embodiments, the liposome composition has a polydispersity index (PDI) of about 0.01 to about 0.5. In some embodiments, the liposome composition has a polydispersity index (PDI) of about 0.01 to about 0.4. In some embodiments, the liposome composition has a polydispersity index (PDI) of about 0.01 to about 0.2. In some embodiments, the liposome composition has a polydispersity index (PDI) of about 0.01 to about 0.1. In some embodiments, the liposome composition has a polydispersity index (PDI) of about 0.1 to about 0.5. In some embodiments, the liposome composition has a polydispersity index (PDI) of about 0.1 to about 0.3. In some embodiments, the liposome composition has a polydispersity index (PDI) of about 0.3 to about 0.5.

[0087] In some embodiments, the liposome composition may further comprise a dispersion medium. In some embodiments, one or more liposomes are suspended in the dispersion medium. In some embodiments, the dispersion medium is a pharma- ceutically acceptable solution. In some embodiments, the dispersion medium is an aqueous dextrose solution. In some embodiments, the dispersion medium is an aqueous sucrose solution. In some embodiments, the dispersion medium is saline. In some embodiments, the dispersion medium further comprises a buffer. In some embodiments, the buffer is a HEPES buffer. In some embodiments, the buffer is a PBS buffer. In some embodiments, the buffer is a Tris buffer. In some embodiments, the buffer is a MES buffer. Therapeutic Agents

[0088] In some embodiments, the liposomes and liposomal compositions provided thereto comprise a liposomally encapsulated therapeutic agent, In some embodiments, the liposomes and liposomal compositions provided thereto comprise a liposomally encapsulated hydrophobic therapeutic agent.

[0089] In some embodiments, the therapeutic agent is an anti-angiogenic agent, antimetabolite, an apoptosis inducer, a cell cycle inhibitor, a cell cycle control inhibitor, a checkpoint inhibitor, a cyclin dependent kinase inhibitor, a cytotoxic agent, a DNA damaging agent, a DNA repair inhibitor, a mitochondrial poison, a telomerase inhibitor, a signal transduction inhibitor, a transcription inhibitor, a Bcl inhibitor, a PARP inhibitor, a PI3K inhibitor, an HSP90 inhibitor, a JAK inhibitor, an ATR inhibitor, an HDAC inhibitor, a tyrosine kinase inhibitor, a receptor tyrosine kinase inhibitor, a BTK inhibitor, an alkylating agent, an SMO inhibitor, an anti-tubulin agent, a MEK inhibitor, a topoisomerase inhibitor, a RAF inhibitor, a BRAF inhibitor, or a proteasome inhibitor.

[0090] In some embodiments, the therapeutic agent is an HSP90 inhibitor. In some embodiments, the HSP90 inhibitor is luminespib.

[0091] In some embodiments, the therapeutic agent is an alkylating agent selected from the group consisting of bendamustine and chlorambucil.

[0092] In some embodiments, the therapeutic agent is an antitubulin agent, hi some embodiments, the therapeutic agent is an antitubulin agent selected from the group consisting of vincristine, vinorelbine, and docetaxel.

[0093] In some embodiments, the therapeutic agent is an ATR inhibitor.

[0094] In some embodiments, the therapeutic agent is a RAF inhibitor. In some embodiments, the RAF inhibitor is dabrafenib. In some embodiments, the therapeutic agent is a BRAF inhibitor. In some embodiments, the BRAF inhibitor is vemurafenib.

[0095] In some embodiments, the therapeutic agent is a BTK inhibitor. In some embodiments, the BTK inhibitor is ibrutinib.

[0096] In some embodiments, the therapeutic agent is an HDAC inhibitor. In some embodiments, the HDAC inhibitor is panobinostat.

[0097] In some embodiments, the therapeutic agent is a JAK inhibitor. In some embodiments, the JAK inhibitor is ruxolitinib.

[0098] In some embodiments, the therapeutic agent is a MEK inhibitor. In some embodiments, the therapeutic agent is a MEK inhibitor selected from the group consisting of selumetinib and cobimetinib.

[0099] In some embodiments, the therapeutic agent is a PARP inhibitor. In some embodiments, the therapeutic agent is a PARP inhibitor selected from the group consisting of talazoparib, niraparib, and rucaparib.

[0100] In some embodiments, the therapeutic agent is a PI3K inhibitor. In some embodiments, the PI3K inhibitor is idelalisib.

[0101] In some embodiments, the therapeutic agent is a proteasome inhibitor. In some embodiments, the proteasome inhibitor is carfilzomib.

[0102] In some embodiments, the therapeutic agent is an SMO inhibitor. In some embodiments, the therapeutic agent is an SMO inhibitor selected from the group consisting of sonidegib and vismodegib.

[0103] In some embodiments, the therapeutic agent is a tyrosine kinase inhibitor. In some embodiments, the therapeutic agent is a tyrosine kinase inhibitor selected from the group consisting of brigatinib, lenvatinib, afatinib, axitinib, cabozantinib, ponatinib, sorafenib, osimertinib, regorafenib, bosutinib, crizotinib, vandetanib, nilotinib, alectinib, ceritinib, dasatinib, pazopanib, sunitinib, erlotinib, imatinib, gefitinib, and lapatinib.

[0104] In some embodiments, the therapeutic agent is a topoisomerase inhibitor. In some embodiments, the therapeutic agent is a topoisomerase I inhibitor. In some embodiments, the topoisomerase inhibitor is irinotecan.

[0105] In some embodiments, the therapeutic agent is a Bcl inhibitor. In some embodiments, the Bcl inhibitor is a Bcl-2 inhibitor. In some embodiments, the Bcl inhibitor is a Bcl-X L In some embodiments, the Bcl inhibitor is a Bcl-2 / Bcl-X L In some embodiments, the Bcl inhibitor is a compound of formula (I): [ka] or a stereoisomer, tautomer, or pharma- ceutically acceptable salt thereof, wherein: V is [ka] and W is H or [ka] and X is [ka] and Y is -NO2 or -SO2CF3; Z is [ka] In some embodiments, V is selected from the group consisting of [ka] In some embodiments, V is [ka] In some embodiments, W is H. In some embodiments, W is [ka] In some embodiments, X is [ka] In some embodiments, X is [ka] In some embodiments, Y is -NO2. In some embodiments, Y is -SO2CF3. In some embodiments, Z is [ka] In some embodiments, Z is [ka] In some embodiments, Z is [ka] In some embodiments, Z is [ka] In some embodiments, Z is [ka] In some embodiments, Z is [ka] In some embodiments, Z is [ka] In some embodiments, Z is [ka] It is. In some embodiments, the Bcl inhibitor is [ka] [ka] [ka] [ka] In some embodiments, the Bcl inhibitor is selected from the group consisting of venetoclax (ABT-199), navitoclax (ABT-263), ABT-737, obatoclax mesylate (GX15-070), sabutoclax, TW-37, (R)-(-)-gossypol acetate, HA14-1, a BH3 mimetic, and oblimersen. [ka] In some embodiments, the Bcl inhibitor is selected from the group consisting of venetoclax (ABT-199), navitoclax (ABT-263), obatoclax mesylate (GX15-070), sabutoclax, TW-37, (R)-(-)-gossypol acetate, HA14-1, a BH3 mimetic, and oblimersen. [ka] In some embodiments, the Bcl inhibitor is selected from the group consisting of venetoclax (ABT-199), navitoclax (ABT-263), obatoclax mesylate (GX15-070), sabutoclax, TW-37, (R)-(-)-gossypol acetate, HA14-1, a BH3 mimetic, and oblimersen. [ka] In some embodiments, the Bcl inhibitor is selected from the group consisting of: venetoclax (ABT-199), and navitoclax (ABT-263). [ka] In some embodiments, the Bcl inhibitor is selected from the group consisting of: venetoclax (ABT-199), and navitoclax (ABT-263). [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is: [ka] In some embodiments, the Bcl inhibitor is venetoclax. In some embodiments, the Bcl inhibitor is navitoclax.

[0106] In some embodiments, the liposome composition provided thereto includes a therapeutic agent. In some embodiments, the therapeutic agent is a hydrophobic therapeutic agent. In some embodiments, the therapeutic agent is encapsulated in the liposome. In some embodiments, the therapeutic agent is present outside the liposome. In some embodiments of all the foregoing aspects, the liposome further includes a therapeutic agent. In some embodiments, the therapeutic agent is encapsulated in the liposome. In some embodiments, the liposome composition provided thereto includes a therapeutic agent. In some embodiments, the therapeutic agent is a hydrophobic therapeutic agent. In some embodiments, the therapeutic agent has a cLogP greater than about 2. In some embodiments, the therapeutic agent has a cLogP of about 2 to about 12. In some embodiments, the therapeutic agent has a cLogP of about 3 to about 12. In some embodiments, the therapeutic agent has a cLogP of about 2 to about 4. In some embodiments, the therapeutic agent has a cLogP of about 2 to about 8. In some embodiments, the therapeutic agent has a cLogP of about 4 to about 12. In some embodiments, the therapeutic agent has a cLogP of about 4 to about 8. In some embodiments, the Therapeutic Agent has a cLogP of about 8 to about 12. In some embodiments, the Therapeutic Agent has a cLogP of about 10 to about 12.

[0107] In some embodiments, the therapeutic agent can be protonated and the protonated form has a pK of greater than about 2. a In some embodiments, the therapeutic agent can be protonated and has a pK of about 3 to about 11. a In some embodiments, the therapeutic agent can be protonated and the protonated form has a pK a In some embodiments, the therapeutic agent can be protonated and the protonated form has a pK of about 8 to about 11. a In some embodiments, the therapeutic agent can be protonated and the protonated form has a pK aIn some embodiments, the therapeutic agent can be protonated and has a pK of about 6 to about 8. a In some embodiments, the therapeutic agent can be protonated and has a pK of about 3 to about 8. a In some embodiments, the therapeutic agent can be protonated and has a pK of about 2 to about 12 in its protonated form. a In some embodiments, the therapeutic agent can be protonated and has a pK of about 2 to about 8. a In some embodiments, the therapeutic agent can be protonated and the protonated form has a pK a In some embodiments, the therapeutic agent can be protonated and the protonated form has a pK of about 4 to about 12. a In some embodiments, the therapeutic agent can be protonated and has a pK of about 4 to about 8. a In some embodiments, the therapeutic agent can be protonated and has a pK of about 8 to about 12 in its protonated form. a In some embodiments, the therapeutic agent can be protonated and the protonated form has a pK of about 10 to about 12. a has.

[0108] In some embodiments, the liposome composition provided therein comprises an additional therapeutic agent. In some embodiments, the additional therapeutic agent is a hydrophobic therapeutic agent. In some embodiments, the additional therapeutic agent is a hydrophilic therapeutic agent. In some embodiments, the additional therapeutic agent is encapsulated in the liposome. In some embodiments, the additional therapeutic agent is external to the liposome. In some embodiments of all the foregoing aspects, the liposome further comprises an additional therapeutic agent. In some embodiments, the additional therapeutic agent is encapsulated in the liposome.

[0109] In some embodiments, the additional therapeutic agent is an anti-angiogenic agent, antimetabolite, an apoptosis inducer, a cell cycle inhibitor, a cell cycle control inhibitor, a checkpoint inhibitor, a cyclin dependent kinase inhibitor, a cytotoxic agent, a DNA damaging agent, a DNA repair inhibitor, a mitochondrial poison, a telomerase inhibitor, a signal transduction inhibitor, a transcription inhibitor, a Bcl inhibitor, a PARP inhibitor, a PI3K inhibitor, an HSP90 inhibitor, a JAK inhibitor, an ATR inhibitor, an HDAC inhibitor, a tyrosine kinase inhibitor, a receptor tyrosine kinase inhibitor, a BTK inhibitor, an alkylating agent, an SMO inhibitor, an anti-tubulin agent, a MEK inhibitor, a topoisomerase inhibitor, a RAF inhibitor, a BRAF inhibitor, or a proteasome inhibitor.

[0110] In some embodiments, the additional therapeutic agent is an HSP90 inhibitor. In some embodiments, the HSP90 inhibitor is luminespib.

[0111] In some embodiments, the additional therapeutic agent is an alkylating agent selected from the group consisting of bendamustine and chlorambucil.

[0112] In some embodiments, the additional therapeutic agent is an antitubulin agent, hi some embodiments, the additional therapeutic agent is an antitubulin agent selected from the group consisting of vincristine, vinorelbine, and docetaxel.

[0113] In some embodiments, the additional therapeutic agent is an ATR inhibitor.

[0114] In some embodiments, the additional therapeutic agent is a RAF inhibitor. In some embodiments, the RAF inhibitor is dabrafenib. In some embodiments, the additional therapeutic agent is a BRAF inhibitor. In some embodiments, the BRAF inhibitor is vemurafenib.

[0115] In some embodiments, the additional therapeutic agent is a BTK inhibitor. In some embodiments, the BTK inhibitor is ibrutinib.

[0116] In some embodiments, the additional therapeutic agent is an HDAC inhibitor, hi some embodiments, the HDAC inhibitor is panobinostat.

[0117] In some embodiments, the additional therapeutic agent is a JAK inhibitor. In some embodiments, the JAK inhibitor is ruxolitinib.

[0118] In some embodiments, the additional therapeutic agent is a MEK inhibitor. In some embodiments, the additional therapeutic agent is a MEK inhibitor selected from the group consisting of selumetinib and cobimetinib.

[0119] In some embodiments, the additional therapeutic agent is a PARP inhibitor. In some embodiments, the additional therapeutic agent is a PARP inhibitor selected from the group consisting of talazoparib, niraparib, and rucaparib.

[0120] In some embodiments, the additional therapeutic agent is a PI3K inhibitor. In some embodiments, the PI3K inhibitor is idelalisib.

[0121] In some embodiments, the additional therapeutic agent is a proteasome inhibitor. In some embodiments, the proteasome inhibitor is carfilzomib.

[0122] In some embodiments, the additional therapeutic agent is an SMO inhibitor. In some embodiments, the additional therapeutic agent is an SMO inhibitor selected from the group consisting of sonidegib and vismodegib.

[0123] In some embodiments, the additional therapeutic agent is a tyrosine kinase inhibitor. In some embodiments, the additional therapeutic agent is a tyrosine kinase inhibitor selected from the group consisting of brigatinib, lenvatinib, afatinib, axitinib, cabozantinib, ponatinib, sorafenib, osimertinib, regorafenib, bosutinib, crizotinib, vandetanib, nilotinib, alectinib, ceritinib, dasatinib, pazopanib, sunitinib, erlotinib, imatinib, gefitinib, and lapatinib.

[0124] In some embodiments, the additional therapeutic agent is a topoisomerase inhibitor. In some embodiments, the additional therapeutic agent is a topoisomerase I inhibitor. In some embodiments, the topoisomerase inhibitor is irinotecan.

[0125] In some embodiments, the additional therapeutic agent is a Bcl inhibitor, such as those described above. In some embodiments, the additional therapeutic agent is an anti-neoplastic agent. Suitable anti-neoplastic agents include, by way of non-limiting example, the following: "signal transduction inhibitors", which disrupt or prevent the signals that cause cancer cells to grow or divide; "Cytotoxic Agents"; "cell cycle inhibitors" or "cell cycle control inhibitors", which interfere with the progression of a cell through the normal cell cycle (the lifespan of a cell from mitosis, which results in the origin of the cell, to the post-mitotic events that cause it to divide into daughter cells); "Checkpoint inhibitors", which interfere with the normal function of cell cycle checkpoints, e.g., the S / G2 checkpoint, the G2 / M checkpoint, and the G1 / S checkpoint, e.g., S / G2 or G2 / M checkpoint inhibitors, e.g., bleomycin, docetaxel, doxorubicin, etoposide, paclitaxel, vinblastine, vincristine, vindesine, and vinorelbine; G1 / early S checkpoint inhibitors; and G2 / M checkpoint inhibitors; "topoisomerase inhibitors", which interfere with the activity of topoisomerase I or II, enzymes necessary for DNA replication and transcription, such as camptothecin, irinotecan, and topotecan; "receptor tyrosine kinase inhibitors", which interfere with the activity of growth factor receptors with tyrosine kinase activity, e.g., genistein, trastuzumab, ZD1839; "apoptosis inducers", which promote programmed cell death; "Antimetabolites", such as cytidine analogs, e.g., cytarabine, 5-azacytidine, and gemcitabine (2',2'-difluorodeoxycytidine), or hydroxyurea, which closely resemble essential metabolites and thus interfere with the physiological reactions involved therein; "telomerase inhibitors", which interfere with the activity of telomerase, the enzyme that increases telomere length, thereby extending the lifespan of cells and their replicative potential; "cyclin-dependent kinase inhibitors", which interfere with cyclin-dependent kinases that control key steps during different phases of the cell cycle through the phosphorylation of cellular proteins such as histones, cytoskeletal proteins, transcription factors, and tumor suppressor genes; "DNA damaging agents" such as carboplatin, cisplatin, cyclophosphamide, doxorubicin, daunorubicin, epirubicin, mitomycin C, mitoxantrone; "DNA repair inhibitors", which include 5-fluorouracil (5-FU) or FUDR, gemcitabine, and methotrexate; "immunomodulators", which stimulate or suppress the immune system and can help the body fight cancer, infection, or other diseases; for example, specific immunomodulators such as monoclonal antibodies, cytokines, and vaccines affect specific parts of the immune system, while non-specific immunomodulators such as BCG and levamisole affect the immune system in a general manner; "Anti-angiogenic agents", which interfere with the generation of new blood vessels or the growth of existing blood vessels that occurs during tumor growth; and "Mitochondrial poisons", which directly or indirectly disrupt mitochondrial respiratory chain function.

[0126] The mechanism of action of one or more of the agents may be unknown or imprecisely specified.

[0127] Other anticancer drugs include paclitaxel, etoposide compounds, camptothecin compounds, idarubicin, carboplatin, oxaliplatin, adriamycin, mitomycin, ansamitocin, bleomycin, cytosine arabinoside, arabinosyl adenine, mercaptopolylysine, vincristine, busulfan, chlorambucil, melphalan, mercaptopurine, mitotane, procarbazine hydrochloride, dactinomycin, mitomycin, plicamycin, aminoglutethimide, and phosphate. estramustine sodium, flutamide, leuprolide acetate, megestrol acetate, tamoxifen citrate, testolactone, trilostane, amsacrine, asparaginase, interferon, teniposide, vinblastine sulfate, vincristine sulfate, bleomycin, methotrexate, valrubicin, carzelesin, paclitaxel, taxotan, camptothecin, doxorubicin, daunomycin, cisplatin, 5-fluorouracil, methotrexate; anti-inflammatory agents, e.g. indomethacin, ibuprofen, ketoprofen, flubiprofen, diclofenac, piroxicam, tenoxicam, naproxen, aspirin, and acetaminophen; sex hormones such as testosterone, estrogen, progesterone, estradiol; antihypertensives such as captopril, ramipril, terazosin, minoxidil, and parazosin; antiemetics such as ointment, serotonin, and serotonin; antifungals such as itraconazole, ketoconazole, and amphotericin; steroids such as triamcinolone acetonide, hydrocortisone, dexamethasone, prednisolone, and betamethasone; cyclosporine, as well as functionally equivalent analogs, derivatives, or combinations thereof.

[0128] In some embodiments, the liposome composition provided therein includes an additional therapeutic agent. In some embodiments, the additional therapeutic agent is a hydrophobic therapeutic agent. In some embodiments, the additional therapeutic agent is encapsulated in the liposome. In some embodiments, the additional therapeutic agent is external to the liposome. In some embodiments of all the foregoing aspects, the liposome further includes an additional therapeutic agent. In some embodiments, the additional therapeutic agent is encapsulated in the liposome. In some embodiments, the additional therapeutic agent has a cLogP greater than about 2. In some embodiments, the additional therapeutic agent has a cLogP of about -6 to about 12. In some embodiments, the additional therapeutic agent has a cLogP of about -6 to about 0. In some embodiments, the additional therapeutic agent has a cLogP of about -3 to about 0. In some embodiments, the additional therapeutic agent has a cLogP of about 0 to about 2. In some embodiments, the additional therapeutic agent has a cLogP of about -1 to about 12. In some embodiments, the additional therapeutic agent has a cLogP of about 3 to about 12. In some embodiments, the additional therapeutic agent has a cLogP of about -6 to about -1. In some embodiments, the additional therapeutic agent has a cLogP of about -1 to about 3. In some embodiments, the additional therapeutic agent has a cLogP of about 2 to about 12. In some embodiments, the additional therapeutic agent has a cLogP of about 2 to about 4. In some embodiments, the additional therapeutic agent has a cLogP of about 2 to about 8. In some embodiments, the additional therapeutic agent has a cLogP of about 4 to about 12. In some embodiments, the additional therapeutic agent has a cLogP of about 4 to about 8. In some embodiments, the additional therapeutic agent has a cLogP of about 8 to about 12. In some embodiments, the additional therapeutic agent has a cLogP of about 10 to about 12.

[0129] In some embodiments, the additional therapeutic agent can be protonated and has a pK of greater than about 2 in its protonated form. a In some embodiments, the additional therapeutic agent can be protonated and has a pK of between about -6 and about 12 in its protonated form. aIn some embodiments, the additional therapeutic agent can be protonated and has a pK of about -6 to about 11 in its protonated form. a In some embodiments, the additional therapeutic agent can be protonated and has a pK of about 3 to about 11 in its protonated form. a In some embodiments, the additional therapeutic agent can be protonated and has a pK of about 6 to about 11 in its protonated form. a In some embodiments, the additional therapeutic agent can be protonated and has a pK of about 8 to about 11 in its protonated form. a In some embodiments, the additional therapeutic agent can be protonated and has a pK of about -6 to about 3 in its protonated form. a In some embodiments, the additional therapeutic agent can be protonated and has a pK of about 3 to about 6 in its protonated form. a In some embodiments, the additional therapeutic agent can be protonated and has a pK of about 6 to about 8 in its protonated form. a In some embodiments, the additional therapeutic agent can be protonated and has a pK of about -6 to about 8 in its protonated form. a In some embodiments, the additional therapeutic agent can be protonated and has a pK of about 3 to about 8 in its protonated form. a In some embodiments, the additional therapeutic agent can be protonated and has a pK of about -6 to about 6 in its protonated form. a In some embodiments, the additional therapeutic agent can be protonated and has a pK of about 2 to about 12 in its protonated form. a In some embodiments, the additional therapeutic agent can be protonated and has a pK of about 2 to about 8 in its protonated form. a In some embodiments, the additional therapeutic agent can be protonated and has a pK of about 2 to about 4 in its protonated form. a In some embodiments, the additional therapeutic agent can be protonated and has a pK of about 4 to about 12 in its protonated form.a In some embodiments, the additional therapeutic agent can be protonated and has a pK of about 4 to about 8 in its protonated form. a In some embodiments, the additional therapeutic agent can be protonated and has a pK of about 8 to about 12 in its protonated form. a In some embodiments, the additional therapeutic agent can be protonated and has a pK of about 10 to about 12 in its protonated form. a has.

[0130] Process for making liposomes and liposomal compositions Liposomes can be prepared as described in "Liposomes: Rational Design" (AS Janoff, ed., Marcel Dekker, Inc., New York, NY), or by additional techniques familiar to those of skill in the art. Suitable liposomes include large unilamellar vesicles (LUV), multilamellar vesicles (MLV), small unilamellar vesicles (SUV), and interdigitating fusion liposomes.

[0131] Lipids Liposomes may contain therapeutic lipids, examples of which include ether lipids, phosphatidic acid, phosphonates, ceramides and ceramide analogs, sphingosine and sphingosine analogs, and serine-containing lipids. Liposomes may also contain phospholipids, such as dialiphatic chain lipids, for example, phosphatidic acid lipids, phosphatidylglycerol, phosphatidylinositol, phosphatidylcholine lipids, phosphatidylethanolamine lipids, phosphatidylserine lipids, and phosphatidylglycerol lipids. Liposomes may also contain diglycerides, dialiphatic glycolipids, or single lipids. Liposomes may be prepared to contain phosphatidylcholine lipids, such as distearoylphosphatidylcholine (DSPC). Phospholipids may be selected from the group consisting of phosphatidylcholine, sphingolipids, and hydrogenated sphingolipids. For example, the phospholipid may be egg phosphatidylcholine (egg PC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), egg sphingomyelin, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phospho-L-serine (DSPS), 1,2-distearoyl-sn-glycero-3-phospho-L-serine sodium salt (DSPS-Na), 2-oleoyl-1-palmitoyl-sn-glycero-3-phospho-L-serine (POPS), and 2-oleoyl-1-palmitoyl-sn-glycero-3-phospho-L-serine sodium salt (POPS-Na).

[0132] The liposomes may be prepared using a surface-stabilizing hydrophilic polymer-lipid conjugate. The hydrophilic polymer-lipid conjugate comprises a polymer portion and a lipid portion. The polymer portion may be a PEG portion. The lipid portion may be based on a phospholipid. The phospholipid may be selected from the group consisting of phosphatidylcholine, sphingolipid, and hydrogenated sphingolipid. For example, the phospholipid may be egg phosphatidylcholine (egg PC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), egg sphingomyelin, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phospho-L-serine (DSPS), 1,2-distearoyl-sn-glycero-3-phospho-L-serine sodium salt (DSPS-Na), 2-oleoyl-1-palmitoyl-sn-glycero-3-phospho-L-serine (POPS), and 2-oleoyl-1-palmitoyl-sn-glycero-3-phospho-L-serine sodium salt (POPS-Na). Exemplary polymer lipid conjugates include 1,2-distearoyl-rac-glycero-3-methoxypoly(ethylene glycol) (such as DSG-PEG2000 or DSG-PEG1000), 1,2-dimyristoyl-rac-glycero-3-methoxypoly(ethylene glycol) (such as DMG-PEG2000 or DMG-PEG1000), 1,2-dipalmitoyl-rac-glycero-3-methoxypoly(ethylene glycol) (such as DPG-PEG2000 or DPG-PEG1000), 1,2-dimethy ... PEG1000), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) (such as DSPE-PEG2000 or DSPE-PEG1000), N-palmitoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)]} (such as C16PEG2000 ceramide), PEG-derivatized cholesterol (such as mPEG cholesterol), sphingomyelin, or dihydrosphingomyelin. The average molecular weight of the polymer portion of the polymer lipid conjugate may vary from about 1000 g / mol to about 5000 g / mol.The average molecular weight of the polymer portion of the polymer-lipid conjugate may vary from about 1000 g / mol to about 2000 g / mol. The average molecular weight of the polymer portion of the polymer-lipid conjugate may be about 1000 g / mol. The average molecular weight of the polymer portion of the polymer-lipid conjugate may be about 2000 g / mol. The average molecular weight of the polymer portion of the polymer-lipid conjugate may be about 5000 g / mol.

[0133] The incorporation of negatively charged lipids such as phosphatidylglycerol (PG) and phosphatidylinositol (PI) can also be added to liposomal formulations to increase the circulation life of the carriers. These lipids can be used in place of or in combination with hydrophilic polymer-lipid conjugates as surface stabilizers.

[0134] Sterols The liposomes may also contain a sterol, such as cholesterol, a cholesterol derivative, or a plant sterol, such as β-sitosterol.

[0135] Liposome loading process A variety of methods can be utilized to encapsulate active agents in liposomes. "Encapsulation" includes covalent or non-covalent binding of agents to liposomes. For example, this can be by interaction of agents with the outer layer(s) of the liposome, or by entrapment of agents in the liposome, an equilibrium state achieved between different parts of the liposome. Thus, encapsulation of agents can be by association of agents with the bilayer of the liposome through covalent or non-covalent interactions with lipid components, or by entrapment in the aqueous interior of the liposome, or in equilibrium between the internal aqueous phase and the bilayer. "Loading" refers to the act of encapsulating one or more agents in a delivery vehicle.

[0136] When combinations of therapeutic agents are sought, it will be clear to those skilled in the art that the desired combination can be encapsulated either by encapsulation in separate delivery vehicles or by encapsulation in the same delivery vehicle. If encapsulation in separate liposomes is desired, the lipid composition of each liposome may be quite different to allow coordinated pharmacokinetics. By varying the composition of the vehicle, the release rate of the encapsulated drugs can be adapted to allow the desired ratio of drugs to be delivered to the tumor site. Means of altering the release rate include increasing the acyl chain length of the vesicle-forming lipids to improve drug retention, controlling the exchange of surface-grafted hydrophilic polymers such as PEG out of the liposome membrane, and incorporating membrane-rigidifying agents such as sterols into the membrane. It should be apparent to one skilled in the art that if it is desired to administer a first and second drug at a particular drug ratio, and the second drug is poorly retained in the liposomal composition of the first drug, improved pharmacokinetics can be achieved by encapsulating the second drug in a second liposomal composition. Alternatively, two or more agents may be encapsulated in the same liposome.

[0137] The technique for encapsulation varies depending on the nature of the delivery vehicle and the nature of the therapeutic agent to be encapsulated. For example, the therapeutic agent may be loaded into liposomes using both passive and active loading methods. Passive methods of encapsulating active agents into liposomes include encapsulating the agent during the preparation of liposomes. This includes the passive entrapment method described by Bangham, et al. (J. Mol. Biol. (1965) 12:238). This technique results in the formation of multilamellar vesicles (MLVs) that can be converted into large unilamellar vesicles (LUVs) or small unilamellar vesicles (SUVs) upon extrusion. Another suitable method of passive encapsulation includes the ether injection technique described by Deamer and Bangham (Biochim. Biophys. Acta (1976) 443:629) and the reverse phase evaporation technique described by Szoka and Paphadjopolos (PNAS (1978) 75:4194). In addition, another suitable method of passive encapsulation includes passive equilibration after the formation of liposomes. This process involves incubating preformed liposomes under altered or non-ambient (based on temperature, pressure, etc.) conditions and adding a therapeutic agent to the exterior of the liposomes. The therapeutic agent then equilibrates to the interior of the liposomes through the liposome membrane. The liposomes are then returned to ambient conditions and unencapsulated therapeutic agent, if present, is removed via dialysis or another suitable method.

[0138] Active methods of encapsulation include the pH gradient loading technique described in U.S. Patent Nos. 5,616,341, 5,736,155, and 5,785,987, as well as active metal loading. One method of pH gradient loading is the citrate-based loading method, which utilizes citrate as an internal buffer with a pH of 4.0 and a neutral external buffer. Other methods used to establish and maintain a pH gradient through liposomes include the use of ionophores that can insert into the liposome membrane and transport ions through the membrane in exchange for protons (see U.S. Patent No. 5,837,282). Another technique may also be used that utilizes transition metals to drive drug uptake into liposomes through complex formation in the absence of ionophores. This technique relies on the formation of drug-metal complexes rather than the establishment of a pH gradient to drive drug uptake.

[0139] Preferred methods for encapsulation of poorly water-soluble compounds are known to those skilled in the art and are described, for example, in U.S. Patent Nos. 9,737,485, 10,507,182, and 10,722,467. In such methods, the drug to be encapsulated is dissolved in a loading solvent and the resulting solution is added to a suspension of liposomes containing a loading aid to obtain a mixture containing the drug to be loaded as a non-crystalline solid, or the drug to be loaded is prepared in a non-crystalline solid form and added to a suspension of liposomes containing a loading aid. Other preferred methods for encapsulation of poorly water-soluble compounds are described in Li et al.,Pharmaceutics(2019),11,465.

[0140] Passive and active entrapment methods may be combined to prepare liposomal formulations containing multiple encapsulated drugs.

[0141] Liposome purification In one aspect, a method for preparing a purified liposome composition comprising liposomes is provided, comprising: The liposome is (a) lipid bilayer, (b) the internal medium, and (c) comprising a therapeutic agent encapsulated in the internal medium of the liposome; The therapeutic agent has low water solubility and can be protonated to a protonated form; The method comprises: (i) providing a crude liposome composition; (ii) purifying the crude liposome composition using an acidified aqueous solution.

[0142] In some embodiments, the acidified aqueous solution comprises a sugar. In some embodiments, the acidified aqueous solution comprises dextrose or sucrose. In some embodiments, the acidified aqueous solution comprises dextrose. In some embodiments, the acidified aqueous solution comprises about 5% to about 20% dextrose by weight. In some embodiments, the acidified aqueous solution comprises 5% dextrose by weight. In some embodiments, the acidified aqueous solution comprises 9% dextrose by weight. In some embodiments, the acidified aqueous solution comprises sucrose. In some embodiments, the acidified aqueous solution comprises about 5% to about 20% sucrose by weight. In some embodiments, the acidified aqueous solution comprises 5% sucrose by weight. In some embodiments, the acidified aqueous solution comprises 9% sucrose by weight.

[0143] In some embodiments, the acidified aqueous solution comprises an inorganic acid, such as a mineral acid. In some embodiments, the acidified aqueous solution comprises hydrochloric acid. In some embodiments, the acidified aqueous solution comprises about 1 mM to 100 mM hydrochloric acid. In some embodiments, the acidified aqueous solution comprises an organic acid, such as a sulfonic acid or a carboxylic acid. In some embodiments, the acidified aqueous solution comprises methanesulfonic acid. In some embodiments, the acidified aqueous solution comprises about 1 mM to 100 mM organic acid. In some embodiments, the acidified aqueous solution comprises about 1 mM to 100 mM methanesulfonic acid. In some embodiments, the acidified aqueous solution comprises about 1 mM to 100 mM methanesulfonic acid. In some embodiments, the acidified aqueous solution comprises about 5 mM methanesulfonic acid. In some embodiments, the acidified aqueous solution comprises about 10 mM methanesulfonic acid. In some embodiments, the acidified aqueous solution comprises acetic acid. In some embodiments, the acidified aqueous solution comprises about 1 mM to 100 mM acetic acid. In some embodiments, the acidified aqueous solution comprises about 25 mM acetic acid. In some embodiments, the acidified aqueous solution comprises sodium acetate. In some embodiments, the acidified aqueous solution comprises about 1 mM to 100 mM sodium acetate. In some embodiments, the acidified aqueous solution comprises about 25 mM sodium acetate. In some embodiments, the acidified aqueous solution comprises acetate. In some embodiments, the acidified aqueous solution comprises about 1 mM to 100 mM acetate. In some embodiments, the acidified aqueous solution comprises about 25 mM acetate.

[0144] In some embodiments, the acidified aqueous solution comprises a salt. In some embodiments, the acidified aqueous solution comprises a salt, the salt comprising an alkali metal or an alkaline earth metal. In some embodiments, the acidified aqueous solution comprises a sodium salt. In some embodiments, the acidified aqueous solution comprises NaF, NaCl, NaBr, or NaI. In some embodiments, the acidified aqueous solution comprises NaCl. In some embodiments, the acidified aqueous solution comprises a potassium salt. In some embodiments, the acidified aqueous solution comprises a lithium salt. In some embodiments, the acidified aqueous solution comprises a magnesium salt. In some embodiments, the acidified aqueous solution comprises a calcium salt.

[0145] In some embodiments, the pH of the acidified aqueous solution is about 1 to about 6. In some embodiments, the pH of the acidified aqueous solution is about 2 to about 5. In some embodiments, the pH of the acidified aqueous solution is about 2.3. In some embodiments, the pH of the acidified aqueous solution is about 3. In some embodiments, the pH of the acidified aqueous solution is about 4.

[0146] In some embodiments, the crude liposome composition is purified multiple times using an acidified aqueous solution. In some embodiments, the crude liposome composition is purified 8 to 24 times using an acidified aqueous solution. In some embodiments, the crude liposome composition is purified 8 to 12 times using an acidified aqueous solution. In some embodiments, the crude liposome composition is purified 12 to 24 times using an acidified aqueous solution. In some embodiments, the crude liposome composition is purified at least 8 times using an acidified aqueous solution. In some embodiments, the crude liposome composition is purified 8 times using an acidified aqueous solution. In some embodiments, the crude liposome composition is purified at least 12 times using an acidified aqueous solution. In some embodiments, the crude liposome composition is purified 12 times using an acidified aqueous solution.

[0147] How to use Also provided is a method for delivering a therapeutically effective amount of a therapeutic agent, comprising administering to a subject a liposomal composition comprising one or more liposomes prepared as described herein, each of the one or more liposomes comprising: (a) a lipid bilayer comprising a first lipid and a first sterol; (b) an inner medium comprising a first loading aid and a first solvent; and (c) comprising a therapeutic agent encapsulated within the interior medium of said liposome.

[0148] The compositions of the present invention may be administered to warm-blooded animals, including humans, as well as domestic and / or avian species. In addition to pharmaceutical compositions, formulations suitable for veterinary use may be prepared and administered in a manner suitable for the subject. Preferred veterinary subjects include mammalian species, such as non-human primates, dogs, cats, cows, horses, sheep, and poultry. Subjects may also include laboratory animals, such as rats, rabbits, mice, and guinea pigs, among others. For the treatment of human diseases, a competent physician will use established protocols to determine how the compositions of the present invention should be utilized in terms of dose, schedule, and route of administration. Such applications may also utilize dose escalation, provided that the drug encapsulated in the delivery vehicle composition of the present invention exhibits reduced toxicity to healthy tissues of the subject.

[0149] Preferably, the pharmaceutical composition of the present invention is administered parenterally, i.e., intraarterially, intravenously, intraperitoneally, subcutaneously, or intramuscularly.More preferably, the pharmaceutical composition is administered intravenously or intraperitoneally by bolus injection.See, for example, Rahman, et al., U.S. Pat. No. 3,993,754; Sears, U.S. Pat. No. 4,145,410; Papahadjopoulos, et al., U.S. Pat. No. 4,235,871; Schneider, U.S. Pat. No. 4,224,179; Lenk, et al., U.S. Pat. No. 4,522,803; and Fountain, et al., U.S. Pat. No. 4,588,578.

[0150] In other methods, the pharmaceutical preparations of the present invention can be contacted with the target tissue by applying the preparation directly to the tissue. Application can be by topical, "open" or "closed" procedures. "Topical" refers to the direct application of the pharmaceutical preparation to tissues exposed to the environment, such as the skin, oropharynx, ear canal, etc. An "open" procedure is one that involves incising the patient's skin and directly visualizing the underlying tissue to which the pharmaceutical preparation is applied. This is generally accomplished by a surgical procedure, such as a thoracotomy to access the lungs, a laparotomy to access the abdominal viscera, or other direct surgical approach to the target tissue. A "closed" procedure is an invasive procedure in which the internal target tissue is not directly visualized, but is accessed by inserting an instrument through a small wound in the skin. For example, the preparation may be administered to the peritoneum by needle lavage. Similarly, pharmaceutical preparations may be administered to the meninges or spinal cord by lumbar puncture followed by appropriate positioning of the patient and injection therethrough, as is commonly performed for spinal anesthesia or metrazamide imaging of the spinal cord. Alternatively, the preparations may be administered through an endoscopic device.

[0151] Pharmaceutical compositions containing the delivery vehicle of the present invention may be prepared according to standard techniques and may contain water, buffered water, 0.9% saline, 0.3% glycine, 5% dextrose, etc., including glycoproteins, such as albumin, lipoproteins, globulins, etc., to enhance stability. These compositions may be sterilized by conventional, well-known sterilization techniques. The resulting aqueous solutions may be packaged for use or filtered under aseptic conditions, lyophilized, and the lyophilized preparations may be combined with a sterile aqueous solution before administration. The compositions may contain pharma- ceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, etc., as required to approximate physiological conditions, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc. In addition, the delivery vehicle suspension may contain lipid protective agents that protect lipids against free radical and lipid peroxidation damage during storage. Lipophilic free radical quenchers, such as α-tocopherol, and water-soluble iron-specific chelators, such as ferrioxamine, are suitable.

[0152] The concentration of the delivery vehicle in the pharmaceutical formulation can vary widely, such as from less than about 0.05% by weight, usually about 2-5% by weight or at least about 2-5% by weight, to as much as 10-30% by weight, and will be selected primarily by liquid volume, viscosity, etc., according to the particular mode of administration selected. For example, the concentration may be increased to reduce the amount of added liquid associated with the treatment. Alternatively, a delivery vehicle consisting of an irritating lipid may be diluted to a low concentration to reduce inflammation at the site of administration. In the case of diagnosis, the amount of delivery vehicle administered will depend on the particular label used, the disease state being diagnosed, and the judgment of the clinician.

[0153] Preferably, the pharmaceutical compositions of the present invention are administered intravenously. The dosage of the delivery vehicle formulation will depend on the drug to lipid ratio and the opinion of the administering physician based on the age, weight, and condition of the patient.

[0154] In addition to pharmaceutical compositions, formulations suitable for veterinary use may be prepared and administered in a manner suitable for the subject.Preferred veterinary subjects include mammalian species, such as non-human primates, dogs, cats, cows, horses, sheep, and poultry.Subjects may also include laboratory animals, such as rats, rabbits, mice, and guinea pigs, among others.

[0155] When a single composition that contains multiple active agents is included, essentially follow the above procedure.When administering agents in separate delivery vehicle compositions, administration should be timed to maintain desired ratio.Typically, this can be accomplished by simultaneously administering compositions at calculated ratio.

[0156] kit The therapeutic agents in the composition of the present invention may be formulated separately in individual compositions, in which each therapeutic agent is stably associated with a suitable delivery vehicle.These compositions can be administered separately to a subject, as long as the pharmacokinetics of the delivery vehicles are coordinated so that the ratio of the administered therapeutic agents is maintained at the therapeutic target.Therefore, it is useful to construct a kit that comprises a first composition in separate containers, which comprises a delivery vehicle stably associated with at least a first therapeutic agent, and a second composition in a second container, which comprises a delivery vehicle stably associated with at least a second therapeutic agent.These containers can then be packaged into a kit.

[0157] The kit also includes instructions on how to administer the compositions to a subject, including at least a description of the ratio of the amount of each composition to be administered.Alternatively or additionally, the kit is constructed so that the amount of composition in each container is pre-measured so that the combination of the contents of one container with the contents of the other represents the correct ratio.Alternatively or additionally, the containers may be marked with measuring marks that allow the appropriate amount to be dispensed according to visible marks.The containers may themselves be usable for administration, for example, the kit may contain the appropriate amount of each composition in separate syringes.Preformulated formulations with the correct ratio of therapeutic agents may also be packaged in this way, allowing the formulation to be administered directly from a syringe prepackaged in the kit. EXAMPLES

[0158] The following examples illustrate some embodiments of the present invention. The following examples and preparations are provided to enable those skilled in the art to more clearly understand and to practice these and other embodiments of the present invention. They should not be considered as limiting the scope of the invention, but merely as illustrative and representative thereof. Example 1A. General Procedure for Liposome Formulation

[0159] Liposomes containing drug compounds were generated by remote loading of drug compounds into DSPC / cholesterol / PEG-DSG liposomes containing ammonium sulfate (AS), sucrose ammonium octasulfate (NHSOS), sucrose triethylammonium octasulfate (TEASOS), or sucrose triethanolammonium octasulfate (TEA(OH)SOS), or ammonium citrate as a loading aid.

[0160] A. Preparation of Loading Aid A loading aid solution of 250 mM ammonium sulfate was prepared by dissolving solid ammonium sulfate in deionized (DI) water to reach a target concentration of 250 mM. This resulted in a solution with a pH of 5.2. The solution was filtered through a 0.2 μm membrane.

[0161] A loading aid solution of 300 mM ammonium citrate was prepared by dissolving solid ammonium citrate dibasic in DI water to reach a target concentration of 300 mM. This resulted in a solution with a pH of 4.9. The solution was filtered through a 0.2 μm membrane.

[0162] A loading aid solution of 0.5-1.0 N triethylamine-SOS was prepared from potassium SOS. For small batches, 5 g of potassium SOS was dissolved in 12 mL of DI water at 65 °C and filtered through a glass fiber membrane. The warm solution was then loaded onto the top of a Dowex column containing approximately 30 mL of packed 50W-X8 beads pretreated with 3 M HCl and thoroughly washed with DI water. After exhaustion, DI water was loaded to elute the SOS solution. Fractions of the eluate with a conductivity greater than 150 mS / cm were collected through a flow conductivity meter connected to the bottom of the column. The resulting H-SOS solution was immediately titrated with triethylamine until the pH reached 6.5 ± 0.5. The trimethylamine-SOS solution was filtered through a 0.2 μm membrane. The SOS concentration was determined by elemental sulfur analysis on an ICP-OES (5800 VDV, Agilent). The filtered solution was stored at 4°C.

[0163] The 0.5–1.0 N ammonium SOS loading aid was prepared similarly as above, except that the eluted H-SOS solution was immediately titrated with concentrated ammonia to reach a target pH of 6.5 ± 0.5.

[0164] SOS loading aid solutions of 0.5–1.0 N triethanolamine were prepared similarly as above, except that the eluted H-SOS solution was immediately titrated with triethanolamine to reach a target pH of 6.5 ± 0.5.

[0165] Preparations of all SOS solutions can be scaled up proportionally 25-100 fold.

[0166] B. Preparation of Lipid Foams and Extrusion of Liposomes DSPC, cholesterol, and polymer-conjugated lipid were weighed and dissolved in chloroform in a closed glass vial or bottle.

[0167] Upon forming a viscous chloroform solution of concentrated lipids, a lipid foam was generated within seconds by applying vacuum. Chloroform was completely removed by placing the lipid foam in a Buchi vacuum pump system (V-512, Buchi) and heating or leaving overnight at ambient temperature. Dried foam was stored at -20°C if not used immediately.

[0168] The lipid foam was hydrated by adding an aqueous solution of the loading aid that had been preheated to 65 °C in a water bath. The heated solution was added to the lipid foam. Multilamellar vesicles (MLVs) were created by vortexing for 20-30 s cycles followed by heating for 2-3 min.

[0169] Depending on the loading aid used, the 250 mM ammonium sulfate can be replaced with 300 mM ammonium citrate, or 0.5-1 N triethylamine-SOS, or 0.5-1 N ammonium-SOS, or 0.5-1 N triethanolamine-SOS.

[0170] The MLVs were extruded once at 20-100 psi through a 200 nm PCTE (polycarbonate track etched) membrane using a 10 mL jacketed liposome extruder (LIPEX®) maintained at 65° C. The resulting MLVs were then extruded seven times at 50-200 psi through a 100 nm PCTE membrane using a 10 mL jacketed liposome extruder maintained at 65° C. If the liposome formulation requires handling and storage at low temperatures, the liposomes can be extruded using sucrose as a loading aid and cryoprotectant.

[0171] For small-scale volumes (i.e., <20 ml), extruded liposomes containing 250 mM ammonium sulfate or 300 mM ammonium citrate were buffer exchanged into 9% w / v dextrose by loading onto a Sephadex G25 column (PD-10 column, Cytiva) at a bed volume ratio of 1 mL / 8.3 mL and collecting liposome elution fractions at a conductivity of <50 μS / cm, thereby removing unencapsulated loading aid.

[0172] For small-scale volumes, extruded liposomes containing 0.5-1N triethylamine-SOS, or 0.5-1N ammonium-SOS, or 0.5-1N triethanolamine-SOS were buffer exchanged into 9-18% w / v dextrose (depending on the defined concentration of the loading aid to balance the osmotic pressure) by loading the extruded liposomes containing 0.5-1N triethylamine-SOS, or 0.5-1N ammonium-SOS, or 0.5-1N triethanolamine-SOS onto a Sepharose CL4B column at a bed volume ratio of 1 mL / 10 mL and collecting the liposome elution fractions at a conductivity of <20 μS / cm, thereby removing unencapsulated loading aid.

[0173] For larger scale volumes, extruded liposomes containing 250 mM ammonium sulfate, or 300 mM ammonium citrate, or 0.5–1 N triethylamine-SOS, or 0.5–1 N ammonium-SOS, or 0.5–1 N triethanolamine-SOS were buffer exchanged with 18 volume exchanges of 9–18% w / v dextrose using tangential flow filtration (Sartorius Slice 200) on 100 kDa MWCO Hydrosart PES membranes.

[0174] C. Encapsulation of Drug Compounds in Liposomes Drug compounds were dissolved in DMSO at concentrations of 20-100 mg / mL, added dropwise to the prepared liposomes with stirring, and heated to 65°C so that the final drug concentration in the solution was in the range of 0.2-6.0 mg / mL, the final D / L ratio was in the range of 0.1-0.8 mol / mol, and the final organic solvent concentration was 2-30%.

[0175] To facilitate the loading of the drug into the liposomes, the samples were incubated at 65° C. or at room temperature with constant stirring. After loading, the samples were cooled on an ice bath.

[0176] D. Purification of Drug-Loaded Liposomes Purification of the formulations was performed using tangential flow filtration (Slice 200, Sartorius) with 100-300k MWCO (Hydrosart, Sartorius) PES membranes. The formulations were first concentrated to 50 mL and then purified with 12-24 volume exchanges of acidified (0.01 M HCl or MeSO3H) dextrose at a similar concentration as during the loading procedure to remove unencapsulated drug. The formulations were then purified with another 12 volume exchanges of 5% dextrose. Alternatively, sucrose may be used instead of dextrose if low temperature storage of the liposomal formulation is required. Finally, the formulations were concentrated to 10-20 mg / ml lipid, collected, and filtered through a 0.22 μm PES syringe filter.

[0177] Example 1B. General Procedure for Liposome Formulation B Liposomes containing drug compounds were generated by remote loading of drug compounds into DSPC / cholesterol / PEG-DSG liposomes containing ammonium sulfate (AS), sucrose ammonium octasulfate (NHSOS), sucrose triethylammonium octasulfate (TEASOS), or sucrose triethanolammonium octasulfate (TEA(OH)SOS), or ammonium citrate as a loading aid.

[0178] A. Preparation of Loading Aid A loading aid solution of 250 mM ammonium sulfate was prepared by dissolving solid ammonium sulfate in deionized (DI) water to reach a target concentration of 250 mM. This resulted in a solution with a pH of 5.2. The solution was filtered through a 0.2 μm membrane.

[0179] A loading aid solution of 300 mM ammonium citrate was prepared by dissolving solid ammonium citrate dibasic in DI water to reach a target concentration of 300 mM. This resulted in a solution with a pH of 4.9. The solution was filtered through a 0.2 μm membrane.

[0180] A loading aid solution of 0.5-1.0 N triethylamine-SOS was prepared from potassium SOS. For small batches, 5 g of potassium SOS was dissolved in 12 mL of DI water at 65 °C and filtered through a glass fiber membrane. The warm solution was then loaded onto the top of a Dowex column containing approximately 30 mL of packed 50W-X8 beads pretreated with 3 M HCl and thoroughly washed with DI water. After exhaustion, DI water was loaded to elute the SOS solution. Fractions of the eluate with a conductivity greater than 150 mS / cm were collected through a flow conductivity meter connected to the bottom of the column. The resulting H-SOS solution was immediately titrated with triethylamine until the pH reached 6.5 ± 0.5. The trimethylamine-SOS solution was filtered through a 0.2 μm membrane. The SOS concentration was determined by elemental sulfur analysis on an ICP-OES (5800 VDV, Agilent). The filtered solution was stored at 4°C.

[0181] The 0.5–1.0 N ammonium SOS loading aid was prepared similarly as above, except that the eluted H-SOS solution was immediately titrated with concentrated ammonia to reach a target pH of 6.5 ± 0.5.

[0182] SOS loading aid solutions of 0.5–1.0 N triethanolamine were prepared similarly as above, except that the eluted H-SOS solution was immediately titrated with triethanolamine to reach a target pH of 6.5 ± 0.5.

[0183] Preparations of all SOS solutions can be scaled up proportionally 25-100 fold.

[0184] B. Preparation of Lipid Foams and Extrusion of Liposomes DSPC, cholesterol, and polymer-conjugated lipid were weighed and dissolved in chloroform in a closed glass vial or bottle.

[0185] Upon forming a viscous chloroform solution of concentrated lipids, a lipid foam was generated within seconds by applying vacuum. Chloroform was completely removed by placing the lipid foam in a Buchi vacuum pump system (V-512, Buchi) and heating or leaving overnight at ambient temperature. Dried foam was stored at -20°C if not used immediately.

[0186] The lipid foam was hydrated by adding an aqueous solution of the loading aid that had been preheated to 65 °C in a water bath. The heated solution was added to the lipid foam. Multilamellar vesicles (MLVs) were created by vortexing for 20-30 s cycles followed by heating for 2-3 min.

[0187] Depending on the loading aid used, the 250 mM ammonium sulfate can be replaced with 300 mM ammonium citrate, or 0.5-1 N triethylamine-SOS, or 0.5-1 N ammonium-SOS, or 0.5-1 N triethanolamine-SOS.

[0188] The MLVs were extruded once at 20-100 psi through a 200 nm PCTE (polycarbonate track etched) membrane using a 10 mL jacketed liposome extruder (LIPEX®) maintained at 65° C. The resulting MLVs were then extruded seven times at 50-200 psi through a 100 nm PCTE membrane using a 10 mL jacketed liposome extruder maintained at 65° C. If the liposome formulation requires handling and storage at low temperatures, the liposomes can be extruded using sucrose as a loading aid and cryoprotectant.

[0189] For small-scale volumes (i.e., <20 ml), extruded liposomes containing 250 mM ammonium sulfate or 300 mM ammonium citrate were buffer exchanged into 9% w / v dextrose by loading onto a Sephadex G25 column (PD-10 column, Cytiva) at a bed volume ratio of 1 mL / 8.3 mL and collecting liposome elution fractions at a conductivity of <50 μS / cm, thereby removing unencapsulated loading aid.

[0190] For small-scale volumes, extruded liposomes containing 0.5-1N triethylamine-SOS, or 0.5-1N ammonium-SOS, or 0.5-1N triethanolamine-SOS were buffer exchanged into 9-18% w / v dextrose (depending on the defined concentration of the loading aid to balance the osmotic pressure) by loading the extruded liposomes containing 0.5-1N triethylamine-SOS, or 0.5-1N ammonium-SOS, or 0.5-1N triethanolamine-SOS onto a Sepharose CL4B column at a bed volume ratio of 1 mL / 10 mL and collecting the liposome elution fractions at a conductivity of <20 μS / cm, thereby removing unencapsulated loading aid.

[0191] For larger scale volumes, extruded liposomes containing 250 mM ammonium sulfate, or 300 mM ammonium citrate, or 0.5-1 N triethylamine-SOS, or 0.5-1 N ammonium-SOS, or 0.5-1 N triethanolamine-SOS were buffer exchanged with 10-18 volume exchanges of 9-18% w / v dextrose using tangential flow filtration (Sartorius Slice 200) on a 100 kDa MWCO Hydrosart PES membrane. The final conductivity of purified liposomes prepared according to this method is approximately 10-200 μS / cm. Ideally, the final conductivity is approximately 20-100 μS / cm, or approximately 50-100 μS / cm.

[0192] C. Encapsulation of Drug Compounds in Liposomes Drug compounds were dissolved in DMSO at concentrations of 20-100 mg / mL, added dropwise to the prepared liposomes with stirring, and heated to 65°C so that the final drug concentration in the solution was in the range of 0.2-6.0 mg / mL, the final D / L ratio was in the range of 0.1-0.8 mol / mol, and the final organic solvent concentration was 2-30%.

[0193] To facilitate the loading of the drug into the liposomes, the samples were incubated at 65° C. or at room temperature with constant stirring. After loading, the samples were cooled on an ice bath.

[0194] D. Purification of Drug-Loaded Liposomes Purification of the formulations was performed using tangential flow filtration (Slice 200, Sartorius) with 100-300k MWCO (Hydrosart, Sartorius) PES membranes. The formulations were first concentrated to 5 mL or 10 mL and then purified with 12 volume exchanges with acidified sugar solution to remove unencapsulated drug. The acidified sugar solution contained 5 wt% dextrose, 9 wt% dextrose, or 9 wt% sucrose. The acid in the acidified sugar solution was methanesulfonic acid. The concentration of methanesulfonic acid in the acidified sugar solution was 1 mM, 5 mM, or 10 mM. Alternatively, the acidified sugar solution contained 25 mM sodium acetate at a pH of approximately 4.0. The formulations were then purified with another 12 volume exchanges of 5% dextrose or 9% sucrose. Finally, the formulation was concentrated to 10-20 mg / ml lipid, collected, and filtered through a 0.22 μm PES syringe filter.

[0195] Liposomes prepared according to this procedure can be characterized by transmission electron microscopy (TEM), among other standard methods in the field of nanoparticle and liposomal drug delivery.

[0196] Example 1C. General Procedure for Purifying Liposomal Preparations Purification of the formulation is performed using tangential flow filtration (Slice 200, Sartorius) with 100-300k MWCO (Hydrosart, Sartorius) PES membranes. The formulation is first concentrated and then purified with 12-24 volume exchanges of an acidified aqueous solution of similar concentration as during the loading procedure to remove unencapsulated drug. The acidified aqueous solution is dextrose or sucrose. The concentration of the sugar in the acidified aqueous solution is about 5%-20% by weight. The acid in the acidified aqueous solution is hydrochloric acid or methanesulfonic acid. The concentration of the hydrochloric acid or methanesulfonic acid in the acidified aqueous solution is about 1 mM-100 mM. The acidified aqueous solution may alternatively contain sodium acetate at a pH of about 4.0. The concentration of the sodium acetate in the acidified aqueous solution is about 5 mM-100 mM. The formulation is then purified with another 8-24 volume exchanges of the aqueous solution. Finally, the preparation is concentrated to 10-20 mg / ml lipid, collected, and filtered through a 0.22 μm PES syringe filter.

[0197] Liposomes prepared according to this procedure are characterized by transmission electron microscopy (TEM), among other standard methods in the field of nanoparticle and liposomal drug delivery.

[0198] Example 2. Formulation of Compound 9 into Liposomes Liposomes containing compound 9 were generated by remote loading into DSPC / cholesterol / PEG-DSG (molar ratio 3:2:0.3) liposomes containing ammonium sulfate (AS), sucrose ammonium octasulfate (NHSOS), sucrose triethylammonium octasulfate (TEASOS), sucrose triethanolammonium octasulfate (TEA(OH)SOS), or ammonium citrate as loading aids.

[0199] A. Preparation of Loading Aid The loading aid solution was prepared as described in Example 1A.

[0200] B. Preparation of Lipid Foams and Extrusion of Liposomes DSPC / cholesterol / PEG-DSG (molar ratio 3:2:0.3) was weighed and dissolved in chloroform in a closed glass vial or bottle using heat at approximately 60° C. at a concentration of 1 g lipid / 1 mL chloroform.

[0201] Upon forming a viscous chloroform solution of concentrated lipids, a lipid foam was generated within seconds by applying vacuum. Chloroform was completely removed by placing the lipid foam in a Buchi vacuum pump system (V-512, Buchi) and heating for 0.5 h or leaving at ambient temperature overnight. Dried foam was stored at -20°C if not used immediately.

[0202] The lipid foam was hydrated by adding an aqueous solution of 250 mM ammonium sulfate preheated to 65 °C in a water bath. The heated solution was added to the lipid foam at a concentration of 50 mg lipid / 1 mL of 250 mM ammonium sulfate solution. Multilamellar vesicles (MLVs) were created by vortexing for 20-30 s cycles followed by heating for 2-3 min.

[0203] Depending on the loading aid used, the 250 mM ammonium sulfate can be replaced with 300 mM ammonium citrate, or 0.5-1 N triethylamine-SOS, or 0.5-1 N ammonium-SOS, or 0.5-1 N triethanolamine-SOS.

[0204] The MLVs were extruded once at 20-100 psi through a 200 nm PCTE (polycarbonate track etched) membrane using a 10 mL jacketed liposome extruder (LIPEX®) maintained at 65° C. The resulting MLVs were then extruded seven times at 50-200 psi through a 100 nm PCTE membrane using a 10 mL jacketed liposome extruder maintained at 65° C. If the liposome formulation requires handling and storage at low temperatures, the liposomes can be extruded using sucrose as a loading aid and cryoprotectant.

[0205] The extruded liposomes had a mean hydrodynamic diameter of 110 nm±20 nm as measured by dynamic light scattering (Malvern Nano ZS) using 0.9% saline as the diluent.

[0206] For small-scale volumes (i.e., <20 ml), extruded liposomes containing 250 mM ammonium sulfate or 300 mM ammonium citrate were buffer exchanged into 9% w / v dextrose by loading onto a Sephadex G25 column (PD-10 column, Cytiva) at a bed volume ratio of 1 mL / 8.3 mL and collecting liposome elution fractions at a conductivity of <50 μS / cm, thereby removing unencapsulated loading aid.

[0207] For small-scale volumes, extruded liposomes containing 0.5-1N triethylamine-SOS, or 0.5-1N ammonium-SOS, or 0.5-1N triethanolamine-SOS were buffer exchanged into 9-18% w / v dextrose (depending on the defined concentration of the loading aid to balance the osmotic pressure) by loading the extruded liposomes containing 0.5-1N triethylamine-SOS, or 0.5-1N ammonium-SOS, or 0.5-1N triethanolamine-SOS onto a Sepharose CL4B column at a bed volume ratio of 1 mL / 10 mL and collecting the liposome elution fractions at a conductivity of <20 μS / cm, thereby removing unencapsulated loading aid.

[0208] For larger scale volumes, extruded liposomes containing 250 mM ammonium sulfate, or 300 mM ammonium citrate, or 0.5–1 N triethylamine-SOS, or 0.5–1 N ammonium-SOS, or 0.5–1 N triethanolamine-SOS were buffer exchanged with 18 volume exchanges of 9–18% w / v dextrose using tangential flow filtration (Sartorius Slice 200) on 100 kDa MWCO Hydrosart PES membranes.

[0209] C. Encapsulation of Compound 9 in Liposomes When 250 mM ammonium sulfate was the loading aid, compound 9 was dissolved in DMSO at a concentration of 20-100 mg / mL and added dropwise to the prepared liposomes with stirring and heated to 65 °C so that the final drug concentration in the solution was in the range of 0.2-0.8 mg / mL and the final D / L ratio was in the range of 0.1-0.8 mol / mol. The final DMSO concentration was 2-30%.

[0210] To facilitate the loading of the drug into the liposomes, the samples were incubated at 65° C. for 45 min with constant stirring. After loading, the samples were cooled on an ice bath.

[0211] D. Purification of liposomes loaded with compound 9 Purification of the formulations was performed using tangential flow filtration (Slice 200, Sartorius) with 100-300k MWCO (Hydrosart, Sartorius) PES membranes. The formulations were first concentrated to 50 mL and then purified with 12-24 volume exchanges of acidified (0.01 M HCl or MeSO3H) dextrose at a similar concentration as during the loading procedure to remove unencapsulated drug. The formulations were then purified with another 12 volume exchanges of 5% dextrose. Alternatively, sucrose may be used instead of dextrose if low temperature storage of the liposomal formulation is required. Finally, the formulations were concentrated to 10-20 mg / ml lipid, collected, and filtered through a 0.22 μm PES syringe filter.

[0212] Example 3: Drug and lipid assays Liposome formulations containing various drugs were prepared as described in Example 1 A. The lipid content was analyzed using phospholipid C and cholesterol assay kits (Fujifilm) and a UV spectrometer (Cytation 5, BioTek Instruments Inc.).

[0213] To determine drug content, the drug-loaded liposomes were first solubilized in a solubilization mixture containing 70% ethanol and 100 mM HCl. A calibration curve was made using compound 9 powder and the same solubilization mixture. Drug was detected using a UV spectrometer (Cytation 5, BioTek Instruments Inc.) measuring absorbance at 310 nm.

[0214] Drug yield is the percentage of drug recovered after purification of the formulation (i.e., without taking into account lipids). It was calculated as follows: Drug yield (%) = drug concentration in final formulation / drug concentration in control * 100

[0215] The control was the same preparation but not purified (ie, 100% recovery).

[0216] The encapsulation efficiency (EE) is calculated as follows: EE(%)=drug-to-lipid ratio in final formulation / drug-to-lipid ratio in control*100

[0217] For example, if the drug to lipid ratio is 0.10 (control) and the final formulation exhibits a drug to lipid ratio of 0.05 after purification, the EE will be 50% (0.05 / 0.1*100).

[0218] The compositions of these formulations are shown in Table 1. The characterization of these lipids (e.g., encapsulation efficiency, liposome size) is presented below in Table 2. [Table 1] [Table 2]

[0219] Example 4. Formulation and Pharmacokinetic Characterization of Drug-Loaded Liposomes Experimental design Female B6D2F1 / J mice were purchased from Jackson Laboratory and allowed to acclimate for 7 days prior to the start of the study. During the experimental period, mice were housed in autoclaved Allentown ventilated cages with 3 animals per cage. Cages were changed every two weeks. Environmental enrichment provided to the cages was Nestlets from Ancare, clear tinted polycarbonate Mouse Igloos from Bio-Serv on Envigo 7097 1 / 4" corncob bedding. All enrichment was added to the cages prior to autoclaving. Mice were fed Envigo Teklad Global Rodent Diet 2018. This rodent diet was held in a wire-lidded hopper and changed biweekly. Reverse osmosis water was provided at a flow rate of 25-50 ml / min through an Avidity Science automated water valve. Environmental control of lighting and monitoring of temperature, humidity and airflow were performed by WatchDog. The light / dark cycle in the animal housing room was set to 12 hours on and 12 hours off. Temperature, humidity and airflow were maintained and controlled by BCCRC facilities.

[0220] Storage of test / control items All test / control articles (TA / CA) were prepared ready for injection and stored at 2–8°C.

[0221] Dose Administration Based on individual mouse weight, the volume required to administer the prescribed dose (10 mg / kg) to the animal was injected into the mice using a 28 G needle. The injection volume was 200 μL / 20 g mouse. Mice were briefly restrained (less than 30 seconds) during the intravenous injection. Vein dilation was achieved by holding the animals under a heat lamp for 1-2 minutes (current revision of SOP-AF-018).

[0222] Data collection Pharmacokinetic Sampling Mice were individually weighed. Mice were injected with test / control articles and blood was collected according to the study classification table. For blood collection, mice were sacrificed with isoflurane followed by CO2 inhalation (SOP-AF-042). Blood collection was performed by cardiac puncture (current revision of SOP-AF-002). For cardiac puncture, mice were removed from the inhalation chamber at the last breath and approximately 500 μL of blood was collected by cardiac puncture with a 25G needle and placed in an appropriate microtiter tube (K2EDTA). Plasma was separated by centrifuging the samples at 1300g for 15 minutes and then pipetted and placed in a labeled vial. Plasma was frozen at -20°C and the remaining dosage material was stored at approximately 4°C until the samples were shipped.

[0223] Determination of drug plasma concentrations Plasma samples were diluted with acidified methanol and vortexed vigorously. The plasma / methanol samples were then centrifuged at 10,000 rcf for 10 min at 8°C. The supernatant was injected onto a Phenomenex Synergi Fusion-RP 2.5 μm 50 × 3 mm column and run with a gradient of 10 mM ammonium acetate buffer and acetonitrile. Drug was detected at a wavelength of 300 nm using a diode array detector. Plasma drug concentrations were calculated against a linear calibration curve ranging from 1 to 40 μg / mL.

[0224] Liposomal formulation All liposomes used in the pharmacokinetic studies described herein contained DPSC / cholesterol / PEGylation (56 / 38 / 5.6 mol%) and were prepared according to Examples 1A, 1B, and 2. [Table 3] [Table 4]

[0225] Example 5. Determination of Plasma Stability of Liposomal Formulations of Compound 2 Plasma stability was determined by measuring the concentration of Compound 2 at various time points in plasma collected as described in Example 4. All liposomal formulations contained DSPC / cholesterol / PEGylation (56 / 38 / 5.6 mol%) as described in Examples 1A, 1B, and 2. Formulations 1-4 were prepared using 250 mM AS as a loading aid. Formulation 5 was prepared using 500 mM AS as a loading aid. Compound 2 was loaded into liposomes of formulations 1, 4, and 5 using 2% DMSO. Compound 2 was loaded into liposomes of formulations 2 and 3 using 10% DMSO.

[0226] FIG. 1A shows the time course of normalized plasma concentrations of Compound 2 using various liposomal formulations compared to using the free drug.

[0227] FIG. 1B shows the change over time in normalized plasma concentrations of compound 2 using an exemplary liposomal formulation of the present disclosure compared to using a liposomal formulation prepared according to a previously published method, and compared to using free drug. The formulation labeled "Merrimack" contained liposomes containing DSPC / cholesterol / PEGylated (56 / 38 / 5.6 mol%) that were loaded without DMSO using TEASOS (0.43N) as a loading aid, according to the procedures described in WO2017 / 123616. In contrast to formulations 1-4, the "Merrimack" formulation was not purified with acidified aqueous solution.

[0228] Example 6. Determination of Plasma Stability of Liposomal Formulations of Compound 9 Plasma stability was determined by measuring the concentration of Compound 9 at various time points in plasma collected as described in Example 4. All liposome formulations contained DSPC / cholesterol / PEGylation (56 / 38 / 5.6 mol%) as described in Examples 1A, 1B, and 2. Formulation 1 used 250 mM AS as a loading aid. Formulation 2 used 500 mM AS as a loading aid. Formulation 3 used 0.5 N TEASOS as a loading aid. Formulation 4 used 0.5 N NH4SOS as a loading aid.

[0229] FIG. 2A shows the normalized plasma concentration of compound 9 over time using various liposomal formulations.

[0230] FIG. 2B shows the change over time in normalized plasma concentrations of compound 9 using an exemplary liposomal formulation of the present disclosure compared to using a liposomal formulation prepared according to previously published methods, and compared to using free drug. The formulation labeled "Merrimack" contained liposomes containing DSPC / cholesterol / PEGylated (56 / 38 / 5.6 mol%) that were loaded without DMSO using TEASOS (0.43N) as a loading aid, according to the procedures described in WO2017 / 123616. In contrast to formulations 1-4, the "Merrimack" formulation was not purified with acidified aqueous solution.

[0231] Example 7: Removal of free drug during purification with acidified sugar solutions Liposomal formulations containing compound 9 were prepared and purified as described in Examples 1A, 1B, and 2. The liposomal formulation contained DSPC / cholesterol / PEGylation (58 / 40 / 2 mol%) and 250 mM AS was used as a loading aid.

[0232] During purification of the formulation using tangential flow filtration, the concentration of compound 9 in the permeate was measured, as shown below in Table 7. Figure 3 shows the concentration of compound 9 in the permeate versus wash volume. TIFF2024526791000113.tif65165

[0233] Example 8. Effect of Acid Wash Purification on Plasma Stability of Liposomal Formulations of Compound 7 Plasma stability was determined by measuring the concentration of compound 7 at various time points in plasma collected as described in Example 4. Both liposomal formulations contained DSPC / cholesterol / PEGylation (59 / 39 / 2 mol%) as described in Examples 1A and 1B. The loading aid was 500 mM AS, and the D / L ratio was 0.4 for both liposomal formulations.

[0234] The acid washed formulations were purified according to the procedures generally described in Examples 1A and 1B, with the specific parameters described herein. Purification of the formulations was performed using tangential flow filtration (Slice 200, Sartorius) with a 100-300k MWCO (Hydrosart, Sartorius) PES membrane. The formulations were first concentrated to 50 mL and then purified with 12 volume exchanges of 10 mM methanesulfonic acid in 5 wt% dextrose in water. The formulations were then purified with another 12 volume exchanges of 5% dextrose. Finally, the formulations were concentrated to 10-20 mg / ml lipid, collected, and filtered through a 0.22 μm PES syringe filter.

[0235] Non-acid washed formulations were purified using tangential flow filtration (Slice 200, Sartorius) with 100-300k MWCO (Hydrosart, Sartorius) PES membranes. Formulations were first concentrated to 50 mL and then purified with 12 volume exchanges of 5% dextrose to 10-20 mg / ml lipid, collected, and filtered through a 0.22 μm PES syringe filter.

[0236] Figure 4 shows the normalized plasma concentration of compound 7 over time using exemplary liposomal formulations of the present disclosure prepared with or without an acid wash step. Figure 4 shows that liposomes with an acid wash purification step exhibit better pharmacokinetic properties (e.g., slower release of compound 7) than liposomes without an acid wash purification step. As shown in Figure 4, liposomes without an acid wash purification step resulted in a burst release of drug, as seen by more than 50% drug loss in the first time period. In contrast, liposomes pre-washed with acid exhibited the desired sustained release of drug over time.

[0237] Example 9. Effect of different acids on the preparation of liposomal formulations Liposomal formulations containing compound 9 were prepared as described in Examples 1A, 1B, and Steps A-C of 2. All liposomal formulations contained DSPC / cholesterol / PEGylation (58 / 40 / 2 mol%) and 250 mM AS was used as a loading aid. In this example, purification of drug-loaded liposomes was performed according to the parameters described herein.

[0238] Purification of the formulations was performed using tangential flow filtration (Slice 200, Sartorius) with 100-300k MWCO (Hydrosart, Sartorius) PES membranes. The formulations were first concentrated to 5 mL and then purified with 12 volume exchanges of acidified sugar (dextrose or sucrose) solution. The acid in the acidified sugar solution was methanesulfonic acid. The concentration of methanesulfonic acid in the acidified sugar solution was 1 mM or 5 mM. The acidified sugar solution alternatively contained sodium acetate at a pH of approximately 4.0. The concentration of sodium acetate in the acidified sugar solution was approximately 25 mM. The formulations were then purified with another 12 volume exchanges of 9% dextrose.

[0239] Specific purification parameters (eg, composition of the acidified sugar solution) and characterization of liposome samples prepared according to this example are shown in Table 5 below. TIFF2024526791000114.tif71165

[0240] Example 10. Effect of different buffers on the preparation of liposomal formulations Liposomal formulations containing compound 9 were prepared as described in Examples 1A, 1B, and Steps A-C of 2. All liposomal formulations contained DSPC / cholesterol / PEGylation (58 / 40 / 2 mol%) and 250 mM AS was used as a loading aid. In this example, purification of drug-loaded liposomes was performed according to the parameters described herein.

[0241] Purification of the formulations was performed using tangential flow filtration (Slice 200, Sartorius) with 100-300k MWCO (Hydrosart, Sartorius) PES membranes. The formulations were first concentrated to 10 mL and then purified with 12 volume exchanges of acidified sugar (dextrose or sucrose) solution. The acid in the acidified sugar solution was 10 mM methanesulfonic acid. The formulations were then purified with another 8 volume exchanges of 5% dextrose. Finally, the formulations were concentrated to 5 mL, collected, and filtered through a 0.22 μm PES syringe filter.

[0242] Specific purification parameters (eg, composition of the acidified sugar solution) and characterization of liposome samples prepared according to this example are shown in Table 6 below. TIFF2024526791000115.tif66165

Claims

**Claim 1** A method for producing a purified liposome composition comprising liposomes, wherein the liposomes comprise (a) a lipid bilayer, (b) an internal medium, and (c) a first compartment of a therapeutic agent encapsulated in the internal medium of the liposome, the therapeutic agent has low water solubility and can be protonated into a protonated form, a second compartment of the therapeutic agent is entrained in the lipid bilayer, the method comprising (i) providing a crude liposome composition, and (ii) purifying the crude liposome composition using an acidified aqueous solution. **Claim 2** The method according to claim 1, wherein the second compartment of the therapeutic agent is protonated and released from the lipid bilayer. **Claim 3** The method according to claim 2, further comprising separating the released protonated therapeutic agent from the liposome, thereby purifying the purified liposome composition. **Claim 4** The lipid bilayer comprises a first lipid and a first sterol, and the internal medium comprises a first loading aid. **Claim 5** The first lipid is selected from the group consisting of 1,2-distearoyl-rac-glycero-3-methoxypoly(ethylene glycol), 1,2-dimyristoyl-rac-glycero-3-methoxypoly(ethylene glycol), 1,2-dipalmitoyl-rac-glycero-3-methoxypoly(ethylene glycol), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol), DSG-PEG2000, DMG-PEG2000, DPG-PEG2000, and DSPE-PEG2000. **Claim 6** The lipid bilayer further comprises a second lipid: the second lipid is a phospholipid; or the second lipid is distearoyl phosphatidylcholine (DSPC) or hydrogenated sphingomyelin. **Claim 7** The internal medium is an aqueous internal medium; the aqueous internal medium is an acidic aqueous internal medium. **Claim 8** The internal medium further comprises an additional solvent; the additional solvent is an organic solvent; or the additional solvent is dimethyl sulfoxide (DMSO). **Claim 9** The method according to claim 4, wherein the first loading aid is an ionic loading aid selected from the group consisting of ammonium sulfate (AS), ammonium sucrose octasulfate (NH4SOS), potassium sucrose octasulfate (KSOS), triethanolammonium sucrose octasulfate (TEA(OH)SOS), triethylammonium sucrose octasulfate (TEASOS), and sodium citrate.

10. The method according to claim 4, wherein the internal medium further comprises a second loading aid; and the second loading aid is an ionic loading aid selected from the group consisting of ammonium sulfate (AS), ammonium sucrose octasulfate (NH4SOS), potassium sucrose octasulfate (KSOS), triethylammonium sucrose octasulfate (TEASOS), and sodium citrate.

11. The method according to claim 4, wherein the first sterol is cholesterol or β-sitosterol.

12. The method according to claim 1, wherein the therapeutic agent has a cLogP greater than about 2; and the protonated form has a pKa greater than about 2.

13. The method according to claim 12, wherein the therapeutic agent is selected from the group consisting of an anti-angiogenic agent, an antimetabolite, an apoptosis inducer, a cell cycle inhibitor, a cell cycle control inhibitor, a checkpoint inhibitor, a cyclin-dependent kinase inhibitor, a cytotoxic agent, a DNA-damaging agent, a DNA repair inhibitor, a mitochondrial poison, a telomerase inhibitor, a signal transduction inhibitor, a transcription inhibitor, a Bcl inhibitor, a PARP inhibitor, a PI3K inhibitor, an HSP90 inhibitor, a JAK inhibitor, an ATR inhibitor, an HDAC inhibitor, a tyrosine kinase inhibitor, a receptor tyrosine kinase inhibitor, a BTK inhibitor, an alkylating agent, an SMO inhibitor, an anti-tubulin agent, a MEK inhibitor, a topoisomerase inhibitor, a RAF inhibitor, a BRAF inhibitor, and a proteasome inhibitor.

14. the therapeutic agent is a Bcl inhibitor selected from the group consisting of a Bcl-2 inhibitor, a Bcl-X Ｌ inhibitor, and a Bcl-2 / Bcl-X Ｌ dual inhibitor; or the Bcl inhibitor is 【Chemical 1】 The method according to claim 13, which is as follows.

15. The liposome further comprises an additional therapeutic agent encapsulated in the liposome; the additional therapeutic agent is selected from an anti-angiogenic agent, an antimetabolite, an apoptosis inducer, a cell cycle inhibitor, a cell cycle control inhibitor, a checkpoint inhibitor, a cyclin-dependent kinase inhibitor, a cytotoxic agent, a DNA-damaging agent, a DNA repair inhibitor, a mitochondrial poison, a telomerase inhibitor, a signal transduction inhibitor, a transcription inhibitor, a Bcl inhibitor, a PARP inhibitor, a PI3K inhibitor, an HSP90 inhibitor, a JAK inhibitor, an ATR inhibitor, an HDAC inhibitor, a tyrosine kinase inhibitor, a receptor tyrosine kinase inhibitor, a BTK inhibitor, an alkylating agent, an SMO inhibitor, an anti-tubulin agent, a MEK inhibitor, a topoisomerase inhibitor, a RAF inhibitor, a BRAF inhibitor, and a proteasome inhibitor, the method according to claim 1.

16. The method according to claim 1, wherein the acidified aqueous solution comprises sugar, dextrose, or sucrose.

17. The method according to claim 1, wherein the acidified aqueous solution contains an acid at a concentration of about 1 mM to 100 mM; the acidified aqueous solution contains methanesulfonic acid.

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