Liposomal formulation of BCL inhibitors

Abstract

Provided herein are liposomes comprising B-cell lymphoma (Bcl) protein inhibitors, compositions comprising such liposomes, and methods of using such formulations to treat hyperproliferative disorders.

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 liposomes comprising B-cell lymphoma (Bcl) protein inhibitors, compositions comprising such liposomes, and methods of using such formulations to treat hyperproliferative disorders. [Background technology]

[0003] Apoptosis is recognized as a biological process essential for tissue homeostasis in all species. Particularly in mammals, it has been shown to regulate early embryonic development. Later in life, cell death is the default mechanism by which potentially dangerous cells (e.g., cells with cancerous defects) are eliminated. Several apoptotic pathways have been defined, but one of the most important involves the B-cell lymphoma 2 (Bcl-2) family of proteins, which are key regulators of the mitochondrial (also called "intrinsic") pathway of apoptosis. See Danial, NN and Korsmeyer, SJ Cell (2004) 116, 205-219. Cancer targeted therapeutic studies have been reported against members of the Bcl-2 protein family, which are central regulators of programmed cell death. For example, pro-apoptotic proteins (Bax, Bad, Bid, Bim, Bik, Puma, Noxa, etc.) and anti-apoptotic proteins (Bcl-2 family: Bcl-2, Bcl-X, etc.) have been reported. L , Mcl-1, etc.) are thought to play a role in cell survival and death.

[0004] Bcl-2 is a good target in hematological cancers, whereas Bcl-X Lis believed to be a target in solid tumors. The observed thrombocytopenia may be due to Bcl-X L It was speculated that this is caused by the inhibition of apoptosis (Cell, 2007, 128, 1173-1186). Bcl-2 family members that inhibit apoptosis are overexpressed in cancer and contribute to tumorigenesis. Bcl-2 expression is strongly correlated with resistance to cancer therapy and reduced survival. Proteins of the Bcl-2 family can be further classified into three subfamilies depending on how many homology domains each protein contains and its biological activity (i.e., whether it has a pro-apoptotic or anti-apoptotic function). The first subgroup contains proteins with all four homology domains, i.e., BH1, BH2, BH3, and BH4. Their general action is anti-apoptotic, i.e., protecting cells from the initiation of the cell death process. For example, Bcl-2, Bcl-X L Proteins such as Mcl-1 and Mcl-2 are members of this first subgroup.

[0005] Bcl-2 family inhibitor compounds (e.g., Bcl-2 and Bcl-X) are used for the treatment of cancer, either as single agents or as part of a combination therapy (e.g., in combination with chemotherapy and / or radiation therapy). L There remains a need for the development of therapies that deliver both inhibitors of Bcl-2 family and Bcl-2 inhibitors to a subject. In particular, there remains a need for therapeutically effective Bcl-2 family inhibitor compositions that are not associated with dose-limiting thrombocytopenia (e.g., acceptably high platelet counts following administration to a subject) and that do not induce tumor lysis syndrome or other unacceptable side effects.

[0006] 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.

[0007] 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.

[0008] 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).

[0009] It is believed that hydrophobic drugs can be loaded into liposomes primarily 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 achieved by adding the drug to preformed liposomes" (US2009 / 0028931). However, such loading relies on the hydrophobic drug associating with or being trapped 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.

[0010] Remote loading of poorly soluble drugs into liposomes under conditions where the drug is in the form of a precipitate beyond its solubility limit is an unexpected event. D. Zucker et al., Journal of Controlled Release (2009) 139:73-80, states that "hydrophobic molecules can aggregate, and these aggregates have low permeability through the liposome membrane. Thus, to achieve high loading, it is necessary for the drug to have reasonable solubility (>1.9 mM), since only soluble uncharged molecules can enter liposomes when the non-polar / polar surface area ratio is >2.31 (see Figure 4 in Zucker et al. Journal of Controlled Release (2009) 139:73-80)." (D. Zucker et al., Journal of Controlled Release (2009) 139:73-80).

[0011] Only recently have methods been developed for active loading of poorly water-soluble drugs from precipitates into the aqueous core of liposomes (see, e.g., Szoka et al. WO2014121211 and Li et al., Pharmaceutics (2019), 11, 465). [Prior art documents] [Patent documents]

[0012] [Patent Document 1] US2009 / 0028931 [Patent Document 2] WO2014121211 [Non-patent literature]

[0013] [Non-Patent Document 1] Danial, NNand Korsmeyer, SJCell (2004) 116, 205-219 [Non-Patent Document 2] Cell,2007,128,1173-1186 [Non-Patent Document 3] D. Zucker et al., Journal of Controlled Release (2009) 139:73-80 [Non-Patent Document 4] PRCullis et al., Biochimica et Biophysica Acta, (1997) 1331:187-211 [Non-Patent Document 5] Li et al.,Pharmaceutics(2019),11,465 Summary of the Invention

[0014] The present disclosure relates to compositions and methods for administering an effective amount of a Bcl inhibitor using liposomes that encapsulate the Bcl inhibitor.

[0015] In one aspect, provided is a liposome composition comprising a liposome, the liposome comprising: (a) a lipid bilayer comprising a first lipid and a first sterol; (b) an inner medium comprising a first loading aid; and (c) a Bcl inhibitor encapsulated in the internal medium of said liposome. The liposome composition comprises:

[0016] In another aspect, provided is a liposome composition comprising a liposome and a therapeutic agent present outside the liposome, the liposome comprising: (a) a lipid bilayer comprising a first lipid and a first sterol; (b) an inner medium comprising a first loading aid; and (c) a Bcl inhibitor encapsulated in the internal medium of said liposome. The liposome composition comprises:

[0017] In another embodiment, provided is a method for delivering a therapeutically effective amount of a Bcl inhibitor, comprising administering to a subject a liposomal composition comprising a liposome, the liposome comprising: (a) a lipid bilayer comprising a first lipid and a first sterol; (b) an inner medium comprising a first loading aid; and (c) a Bcl inhibitor encapsulated in the internal medium of said liposome. The method includes:

[0018] In another aspect, provided is a method for treating a hyperproliferative disorder, comprising administering to a subject a liposomal composition comprising a liposome, the liposome comprising: (a) a lipid bilayer comprising a first lipid and a first sterol; (b) an inner medium comprising a first loading aid; and (c) a Bcl inhibitor encapsulated in the internal medium of said liposome. The method includes:

[0019] In another aspect, provided is a method for reducing thrombocytopenia associated with administration of a therapeutically effective amount of a Bcl inhibitor in a non-liposomal form, comprising administering to a subject a liposomal composition comprising liposomes, the liposomes comprising: (a) a lipid bilayer comprising a first lipid and a first sterol; (b) an inner medium comprising a first loading aid; and (c) a Bcl inhibitor encapsulated in the internal medium of said liposome. Includes; the total amount of the Bcl inhibitor in the liposomal composition is equal to the therapeutically effective amount of the Bcl inhibitor when administered in a non-liposomal form; This is the method.

[0020] 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.

[0021] 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), hydrogenated sphingomyelin (dihydrosphingomyelin), or egg sphingomyelin. In some embodiments, the second lipid is distearoylphosphatidylcholine (DSPC) or hydrogenated sphingomyelin (dihydrosphingomyelin). In some embodiments, the second lipid is distearoylphosphatidylcholine (DSPC). In some embodiments, the second lipid is hydrogenated sphingomyelin (dihydrosphingomyelin). In some embodiments, the second lipid is egg sphingomyelin.

[0022] 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).

[0023] 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), ammonium sucrose octasulfate (NHSOS), potassium sucrose octasulfate (KSOS), triethanolammonium sucrose octasulfate (TEA(OH)SOS), triethylammonium sucrose octasulfate (TEASOS), 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 triethylammonium sucrose octasulfate (TEASOS).

[0024] 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.

[0025] In some embodiments, the first loading aid is potassium sucrose octasulfate (KSOS) and the second loading aid is sodium citrate.

[0026] In some embodiments, the first sterol is cholesterol or β-sitosterol. [Brief description of the drawings]

[0027] [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. [Figure 3A] The change in mean platelet count over time following administration of various doses of free drug or various doses of a liposomal formulation containing compound 9 encapsulated in the internal medium of liposomes is compared. [Figure 3B] The time course of normalized platelet counts following administration of various doses of free drug or various doses of liposomal formulations containing compound 9 encapsulated in the internal medium of liposomes are compared. [Figure 4A] 1 compares the change in normalized platelet count over time following administration of various doses of free Compound 9. [Figure 4B] 1 compares the change over time in normalized platelet counts following administration of various doses of liposomal formulations containing compound 9 encapsulated in the internal medium of liposomes. [Figure 5A] FIG. 1 shows the normalized plasma concentration of Compound 9 (Bcl-22B) over time using various liposomal formulations. [Figure 5B] 1 shows the change in drug to lipid ratio over time for compound 9 (Bcl-22B) using various liposomal formulations. [Figure 6A] FIG. 11 compares the time course of normalized platelet counts following administration of liposomal formulations containing compound 9 encapsulated in the internal medium of liposomes of different lipid composition. [Figure 6B]FIG. 11 compares the change over time in normalized platelet count following administration of liposomal formulations containing compound 9 encapsulated in the internal medium of liposomes containing various amounts of PEG-DSG. [Figure 6C] FIG. 11 compares the change over time in normalized platelet count following administration of liposomal formulations containing compound 9 encapsulated in the internal medium of liposomes containing various amounts of PEG-DMG. [Figure 6D] FIG. 10 compares the change over time in normalized platelet counts following administration of liposomal formulations containing compound 9 encapsulated in the internal medium of liposomes containing 1% PEG-DMG or 1% PEG-DSG. [Figure 6E] FIG. 10 compares the change over time in normalized platelet counts following administration of liposomal formulations containing compound 9 encapsulated in the internal medium of liposomes containing 0.5% PEG-DSG or 0.5% PEG-DMG. [Figure 7A] Compare the change over time in normalized platelet counts following administration of a liposomal formulation dosed at 1 mg / kg containing compound 9 encapsulated in the internal medium of liposomes containing low doses of ammonium sulfate (LAS) or triethylammonium sucrose octasulfate (TEA-SOS), or free drug at 0.1 mg / kg. [Figure 7B] Compare the change in normalized platelet count over time following administration of a liposomal formulation dosed at 2 mg / kg containing compound 9 encapsulated in the internal medium of liposomes containing low doses of ammonium sulfate (LAS) or triethylammonium sucrose octasulfate (TEA-SOS), or 0.2 mg / kg free drug. [Figure 7C] Compare the change over time in normalized platelet counts following administration of a liposomal formulation dosed at 3 mg / kg containing compound 9 encapsulated in the internal medium of liposomes containing low doses of ammonium sulfate (LAS) or triethylammonium sucrose octasulfate (TEA-SOS), or 0.3 mg / kg of free drug. [Figure 7D]Compare the change in normalized platelet count over time following administration of a liposomal formulation dosed at 2 mg / kg containing compound 9 encapsulated in the internal medium of liposomes containing low doses of ammonium sulfate (LAS) or triethylammonium sucrose octasulfate (TEA-SOS), or free drug at 0.1 mg / kg. [Figure 7E] The change in normalized platelet count over time following administration of liposomes containing DHSM:cholesterol:PEG (53:45:2) and compound 9 ("22B") (drug:lipid ratio 0.1) dosed at 3 mg / kg, liposomes containing DSPC:cholesterol:PEG (57:38:5.6) and compound 9 ("22B") (drug:lipid ratio 0.1) dosed at 3 mg / kg, or free drug dosed at 3 mg / kg is compared. [Figure 7F] The time course of normalized platelet counts following administration of various doses of liposomal formulations containing compound 7 encapsulated in the internal medium of liposomes or free drug are compared. [Figure 7G] The time course of normalized platelet counts following administration of a liposomal formulation containing Compound 2 encapsulated in the internal medium of DSPC-containing liposomes or free drug is compared. [Figure 8] 1 shows the change over time in normalized plasma concentrations of compound 7 using exemplary liposomal formulations of the present disclosure prepared with and without an acid wash step. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] Unless otherwise defined, all technical terms, notations, and other scientific or technical terms used herein shall have the same meaning as commonly understood by one 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, but the inclusion of such definitions herein should not necessarily be interpreted as representing a substantial difference from what is commonly understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly used using conventional methodology by those skilled in the art. 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.

[0029] definition As used herein, the use of the terms "a," "an," and the like, refers to one or more, unless otherwise specified.

[0030] 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."

[0031] The terms "therapeutic agent" or "drug," as used herein, refer to chemical moieties used in a variety of therapeutic applications, including pharmaceutical applications.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] "Treatment" (and related terms, such as "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, and situations in which a disease or disorder is not currently experienced but is expected to occur. The term encompasses both complete and partial reduction or prevention of a condition or disorder, as well as complete or partial reduction 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 reduce 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.

[0036] In one aspect, provided is a liposome composition comprising a liposome, the liposome comprising: (a) a lipid bilayer comprising a first lipid and a first sterol; (b) an inner medium comprising a first loading aid; and (c) a Bcl inhibitor encapsulated in the internal medium of said liposome. The liposome composition comprises:

[0037] In another aspect, provided is a liposome composition comprising a liposome and a therapeutic agent present outside the liposome, the liposome comprising: (a) a lipid bilayer comprising a first lipid and a first sterol; (b) an inner medium comprising a first loading aid; and (c) a Bcl inhibitor encapsulated in the internal medium of said liposome. The liposome composition comprises:

[0038] In another embodiment, provided is a method for delivering a therapeutically effective amount of a Bcl inhibitor, comprising administering to a subject a liposomal composition comprising a liposome, the liposome comprising: (a) a lipid bilayer comprising a first lipid and a first sterol; (b) an inner medium comprising a first loading aid; and (c) a Bcl inhibitor encapsulated in the internal medium of said liposome. The method includes:

[0039] In another aspect, provided is a method for treating a hyperproliferative disorder, comprising administering to a subject a liposomal composition comprising a liposome, the liposome comprising: (a) a lipid bilayer comprising a first lipid and a first sterol; (b) an inner medium comprising a first loading aid; and (c) a Bcl inhibitor encapsulated in the internal medium of said liposome. The method includes:

[0040] In another aspect, provided is a method for reducing thrombocytopenia associated with administration of a therapeutically effective amount of a Bcl inhibitor in a non-liposomal form, comprising administering to a subject a liposomal composition comprising liposomes, the liposomes comprising: (a) a lipid bilayer comprising a first lipid and a first sterol; (b) an inner medium comprising a first loading aid; and (c) a Bcl inhibitor encapsulated in the internal medium of said liposome. Includes; the total amount of the Bcl inhibitor in the liposomal composition is equal to the therapeutically effective amount of the Bcl inhibitor when administered in a non-liposomal form; This is the method.

[0041] In another embodiment, provided is a method for reducing the total dose of a Bcl inhibitor administered to a subject to achieve a therapeutic effect, comprising administering to the subject a liposomal 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 (c) a Bcl inhibitor encapsulated in the internal medium of said liposome. Includes; the total amount of Bcl inhibitor in the liposomal composition required to achieve a therapeutic effect is less than the total amount of Bcl inhibitor required to achieve the same therapeutic effect when administered in a non-liposomal form; This is the method.

[0042] In another embodiment, provided is a method for reducing the total dose of a Bcl inhibitor administered to a subject to achieve a therapeutic effect in a hyperproliferative disorder, comprising administering to the subject a liposomal 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 (c) a Bcl inhibitor encapsulated in the internal medium of said liposome. Includes; the total amount of Bcl inhibitor in the liposomal composition required to achieve a therapeutic effect is less than the total amount of Bcl inhibitor required to achieve the same therapeutic effect when administered in a non-liposomal form; This is the method.

[0043] In another embodiment, provided is a method for reducing inter-subject variability in exposure to a Bcl inhibitor administered to a subject in need thereof, comprising administering to the subject a liposomal 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 (c) a Bcl inhibitor encapsulated in the internal medium of said liposome. Includes; the inter-subject variability of exposure to the Bcl inhibitor in the administered liposomal composition is less than the inter-subject variability of exposure to the Bcl inhibitor when administered in a non-liposomal form; the total amount of the Bcl inhibitor in the liposomal composition is equal to the therapeutically effective amount of the Bcl inhibitor when administered in a non-liposomal form; This is the method.

[0044] In another embodiment, provided is a method for increasing the half-life of a Bcl inhibitor in a subject, comprising administering to the subject a liposomal 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 (c) a Bcl inhibitor encapsulated in the internal medium of said liposome. Includes; the half-life of the Bcl inhibitor when administered in a liposomal composition is longer than the half-life of the Bcl inhibitor when administered in a non-liposomal form; This is the method.

[0045] In another embodiment, provided is a method for reducing dosing frequency of a Bcl inhibitor in a subject, comprising administering to the subject a liposomal 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 (c) a Bcl inhibitor encapsulated in the internal medium of said liposome. Includes; the frequency of dosing of the Bcl inhibitor administered in the liposomal composition is less than the frequency of dosing of the Bcl inhibitor when administered in a non-liposomal form; the total amount of the Bcl inhibitor in the liposomal composition is equal to the therapeutically effective amount of the Bcl inhibitor when administered in a non-liposomal form; This is the method.

[0046] In another embodiment, provided is a method for reducing the incidence or severity of one or more adverse events associated with administration of a therapeutically effective amount of a Bcl inhibitor, comprising administering to a subject a liposomal 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 (c) a Bcl inhibitor encapsulated in the internal medium of said liposome. Includes; the incidence or severity of said one or more adverse events incurred by administering the Bcl inhibitor in a liposomal composition is less than the incidence or severity of said one or more adverse events incurred by administering the Bcl inhibitor in a non-liposomal form; the total amount of the Bcl inhibitor in the liposomal composition is equal to the therapeutically effective amount of the Bcl inhibitor when administered in a non-liposomal form; This is the method.

[0047] In one embodiment, provided is a liposome comprising: (a) a lipid bilayer comprising a first lipid and a first sterol; (b) an internal medium comprising a first loading aid, a first solvent, and a second solvent; and (c) a Bcl inhibitor encapsulated in the internal medium of said liposome. The liposome is a liposome comprising:

[0048] In another embodiment, provided is a 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) a Bcl inhibitor encapsulated in the internal medium of said liposome. The liposome composition comprises:

[0049] In another embodiment, provided is a pharmaceutical composition comprising one or more liposomes and a therapeutic agent present outside the one or more liposomes, 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) a Bcl inhibitor encapsulated in the internal medium of said liposome. A pharmaceutical composition comprising:

[0050] In another embodiment, provided is a method for delivering a therapeutically effective amount of a Bcl inhibitor to a subject, comprising administering to the subject a liposomal 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) a Bcl inhibitor encapsulated in the internal medium of said liposome. The method includes:

[0051] In another embodiment, provided is a method for treating a hyperproliferative disorder in a subject in need thereof, comprising administering to the subject a liposomal 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) a Bcl inhibitor encapsulated in the internal medium of said liposome. The method includes:

[0052] In another embodiment, provided is a method for reducing the incidence or severity of thrombocytopenia associated with administration of a therapeutically effective amount of a Bcl inhibitor, comprising administering to a subject a liposomal 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) a Bcl inhibitor encapsulated in the internal medium of said liposome. Includes; the incidence or severity of thrombocytopenia incurred by administering the Bcl inhibitor in a liposomal composition is less than the incidence or severity of thrombocytopenia incurred by administering the Bcl inhibitor in a non-liposomal form; the total amount of the Bcl inhibitor in the liposomal composition is equal to the therapeutically effective amount of the Bcl inhibitor when administered in a non-liposomal form; This is the method.

[0053] In another embodiment, provided is a method for reducing the total dose of a Bcl inhibitor administered to a subject to achieve a therapeutic effect, comprising administering to the subject a liposomal 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) a Bcl inhibitor encapsulated in the internal medium of said liposome. Including, the total amount of Bcl inhibitor in the liposomal composition required to achieve a therapeutic effect is less than the total amount of Bcl inhibitor required to achieve the same therapeutic effect when administered in a non-liposomal form; This is the method.

[0054] In another embodiment, provided is a method for reducing the total dose of a Bcl inhibitor administered to a subject to achieve a therapeutic effect in a hyperproliferative disorder, comprising administering to the subject a liposomal 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) a Bcl inhibitor encapsulated in the internal medium of said liposome. Including, the total amount of Bcl inhibitor in the liposomal composition required to achieve a therapeutic effect is less than the total amount of Bcl inhibitor required to achieve the same therapeutic effect when administered in a non-liposomal form; This is the method.

[0055] In another embodiment, provided is a method for reducing inter-subject variability in exposure to a Bcl inhibitor administered to a subject in need thereof, comprising administering to the subject a liposomal 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) a Bcl inhibitor encapsulated in the internal medium of said liposome. Includes; the inter-subject variability of exposure to the Bcl inhibitor in the administered liposomal composition is less than the inter-subject variability of exposure to the Bcl inhibitor when administered in a non-liposomal form; the total amount of the Bcl inhibitor in the liposomal composition is equal to the therapeutically effective amount of the Bcl inhibitor when administered in a non-liposomal form; This is the method.

[0056] In another embodiment, provided is a method for increasing the half-life of a Bcl inhibitor in a subject, comprising administering to the subject a liposomal 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) a Bcl inhibitor encapsulated in the internal medium of said liposome. Includes; the half-life of the Bcl inhibitor when administered in a liposomal composition is longer than the half-life of the Bcl inhibitor when administered in a non-liposomal form; This is the method.

[0057] In another embodiment, provided is a method for reducing dosing frequency of a Bcl inhibitor in a subject, comprising administering to the subject a liposomal 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) a Bcl inhibitor encapsulated in the internal medium of said liposome. Includes; the frequency of dosing of the Bcl inhibitor administered in the liposomal composition is less than the frequency of dosing of the Bcl inhibitor when administered in a non-liposomal form; the total amount of the Bcl inhibitor in the liposomal composition is equal to the therapeutically effective amount of the Bcl inhibitor when administered in a non-liposomal form; This is the method.

[0058] In another embodiment, provided is a method for reducing the incidence or severity of one or more adverse events associated with administration of a therapeutically effective amount of a Bcl inhibitor, comprising administering to a subject a liposomal 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) a Bcl inhibitor encapsulated in the internal medium of said liposome. Includes; the incidence or severity of said one or more adverse events incurred by administering the Bcl inhibitor in a liposomal composition is less than the incidence or severity of said one or more adverse events incurred by administering the Bcl inhibitor in a non-liposomal form; the total amount of the Bcl inhibitor in the liposomal composition is equal to the therapeutically effective amount of the Bcl inhibitor when administered in a non-liposomal form; This is the method.

[0059] 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 sphingomyelin. In some embodiments, the second lipid is distearoylphosphatidylcholine (DSPC), hydrogenated sphingomyelin (dihydrosphingomyelin), or egg sphingomyelin. In some embodiments, the second lipid is distearoylphosphatidylcholine (DSPC) or hydrogenated sphingomyelin (dihydrosphingomyelin). In some embodiments, the second lipid is distearoylphosphatidylcholine (DSPC). In some embodiments, the second lipid is hydrogenated sphingomyelin (dihydrosphingomyelin).In some embodiments, the second lipid is egg sphingomyelin.

[0060] 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. 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).

[0061] In some embodiments, the lipid bilayer comprises dihydrosphingomyelin (DHSM), cholesterol, and DSG-PEG2000. In some embodiments, the lipid bilayer comprises DHSM / cholesterol / DSG-PEG2000 in a molar ratio of about 53:45:2. In some embodiments, the lipid bilayer comprises distearoylphosphatidylcholine (DSPC), cholesterol, and DSG-PEG2000. In some embodiments, the lipid bilayer comprises DSPC / cholesterol / DSG-PEG2000 in a molar ratio of about 57:38:5.6.

[0062] 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.

[0063] 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).

[0064] In some embodiments of all the foregoing aspects, the first loading aid is an ionic loading aid. In some embodiments, the first loading aid is sulfate, sucrose octasulfate, or citrate. In some embodiments, the first loading aid is selected from the group consisting of ammonium sulfate (AS), sucrose ammonium octasulfate (NHSOS), sucrose potassium octasulfate (KSOS), sucrose triethanolammonium octasulfate (TEA(OH)SOS), sucrose triethylammonium octasulfate (TEASOS), ammonium citrate, and sodium citrate. In some embodiments, the first loading aid is selected from the group consisting of ammonium sulfate (AS), sucrose ammonium octasulfate (NHSOS), sucrose potassium 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.

[0065] In some embodiments of all the above aspects, the first sterol is cholesterol, a cholesterol derivative, 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.

[0066] In some embodiments of all the foregoing aspects, the Bcl inhibitor is a Bcl-2 inhibitor, Bcl-X L Inhibitors and Bcl-2 / Bcl-X L In some embodiments of all the foregoing aspects, 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, the Bcl inhibitor is selected from the group consisting of: [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 venetoclax. In some embodiments, the Bcl inhibitor is navitoclax.

[0067] In some embodiments of all the foregoing aspects, the liposome further 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, the additional therapeutic agent has a cLogP greater than about 2. In some embodiments, the additional therapeutic agent has a cLogP between about -6 and about 12. In some embodiments, the additional therapeutic agent has a cLogP between about -6 and about 0. In some embodiments, the additional therapeutic agent has a cLogP between about -3 and about 0. In some embodiments, the additional therapeutic agent has a cLogP between about 0 and about 2. In some embodiments, the additional therapeutic agent has a cLogP between about -1 and about 12. In some embodiments, the additional therapeutic agent has a cLogP between about 3 and about 12. In some embodiments, the additional therapeutic agent has a cLogP between about -6 and about -1. In some embodiments, the additional therapeutic agent has a cLogP between about -1 and about 3. In some embodiments, the additional therapeutic agent has a cLogP between about 2 and about 12. In some embodiments, the additional therapeutic agent has a cLogP between about 2 and about 4. In some embodiments, the additional therapeutic agent has a cLogP between about 2 and about 8. In some embodiments, the additional therapeutic agent has a cLogP between about 4 and about 12. In some embodiments, the additional therapeutic agent has a cLogP between about 4 and about 8. In some embodiments, the additional therapeutic agent has a cLogP between about 8 and about 12. In some embodiments, the additional therapeutic agent has a cLogP between about 10 and about 12. In some embodiments, the additional therapeutic agent can be protonated, and the protonated form has a pK greater than about 2. 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 between about -6 and about 11 in its protonated form. a In some embodiments, the additional therapeutic agent can be protonated and has a pK of between about 3 and about 11. a In some embodiments, the additional therapeutic agent can be protonated and has a pK of between about 6 and about 11. a In some embodiments, the additional therapeutic agent can be protonated and has a pK of between about 8 and about 11. a In some embodiments, the additional therapeutic agent can be protonated and has a pK of between about -6 and about 3 in its protonated form. a In some embodiments, the additional therapeutic agent can be protonated and has a pK of between about 3 and about 6. a In some embodiments, the additional therapeutic agent can be protonated and has a pK of between about 6 and about 8. a In some embodiments, the additional therapeutic agent can be protonated and has a pK of between about -6 and about 8. a In some embodiments, the additional therapeutic agent can be protonated and has a pK of between about 3 and about 8. a In some embodiments, the additional therapeutic agent can be protonated and has a pK of between about -6 and about 6 in its protonated form. a In some embodiments, the additional therapeutic agent can be protonated and has a pK of between about 2 and about 12 in its protonated form. a In some embodiments, the additional therapeutic agent can be protonated and has a pK of between about 2 and about 8. a In some embodiments, the additional therapeutic agent can be protonated and has a pK of between about 2 and about 4. aIn some embodiments, the additional therapeutic agent can be protonated and has a pK of between about 4 and about 12 in its protonated form. a In some embodiments, the additional therapeutic agent can be protonated and has a pK of between about 4 and about 8. a In some embodiments, the additional therapeutic agent can be protonated and has a pK of between about 8 and about 12 in its protonated form. a In some embodiments, the additional therapeutic agent can be protonated and has a pK of between about 10 and about 12 in its protonated form. a has.

[0068] In some embodiments of all the above aspects, the additional therapeutic agent is an anti-neoplastic agent. In some embodiments, the additional therapeutic agent is selected from an alkylating agent, 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 receptor tyrosine kinase inhibitor, 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.

[0069] In some embodiments, the additional therapeutic agent is a second Bcl inhibitor, such as those described above.

[0070] In some embodiments, the additional therapeutic agent is an HSP90 inhibitor. In some embodiments, the HSP90 inhibitor is luminespib.

[0071] In some embodiments, the additional therapeutic agent is an alkylating agent selected from the group consisting of bendamustine and chlorambucil.

[0072] 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.

[0073] In some embodiments, the additional therapeutic agent is an ATR inhibitor.

[0074] 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.

[0075] In some embodiments, the additional therapeutic agent is a BTK inhibitor. In some embodiments, the BTK inhibitor is ibrutinib.

[0076] In some embodiments, the additional therapeutic agent is an HDAC inhibitor, hi some embodiments, the HDAC inhibitor is panobinostat.

[0077] In some embodiments, the additional therapeutic agent is a JAK inhibitor. In some embodiments, the JAK inhibitor is ruxolitinib.

[0078] 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.

[0079] 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.

[0080] In some embodiments, the additional therapeutic agent is a PI3K inhibitor. In some embodiments, the PI3K inhibitor is idelalisib.

[0081] In some embodiments, the additional therapeutic agent is a proteasome inhibitor. In some embodiments, the proteasome inhibitor is carfilzomib.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] In some embodiments, the additional therapeutic agent is a Bcl inhibitor, such as those described above.

[0086] In some embodiments of all the foregoing aspects, 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 50 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 50 nm and about 100 nm. In some embodiments, the liposomes have an average diameter of about 80 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 of about 120 nm.

[0087] In some embodiments of all the foregoing aspects, the liposome composition further comprises a dispersion medium. In some embodiments, the 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.

[0088] In some embodiments of all the foregoing aspects, the liposome composition has a polydispersity index (PDI) between about 0.001 and about 0.5. In some embodiments, the liposome composition has a polydispersity index (PDI) between about 0.001 and about 0.3. In some embodiments, the liposome composition has a polydispersity index (PDI) between about 0.001 and about 0.1. In some embodiments, the liposome composition has a polydispersity index (PDI) between about 0.1 and about 0.3. In some embodiments, the liposome composition has a polydispersity index (PDI) between about 0.1 and about 0.5. In some embodiments, the liposome composition has a polydispersity index (PDI) between about 0.3 and about 0.5.

[0089] In some embodiments of all the foregoing aspects, the liposome composition further comprises a therapeutic agent external to the one or more liposomes.

[0090] Liposomes and liposomal compositions 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.

[0091] 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.

[0092] 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 greater than 200 nm.

[0093] 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) between about 0.001 and about 0.5. In some embodiments, the liposome composition has a polydispersity index (PDI) between about 0.001 and about 0.4. In some embodiments, the liposome composition has a polydispersity index (PDI) between about 0.001 and about 0.3. In some embodiments, the liposome composition has a polydispersity index (PDI) between about 0.005 and about 0.5. In some embodiments, the liposome composition has a polydispersity index (PDI) between about 0.005 and about 0.4. In some embodiments, the liposome composition has a polydispersity index (PDI) between about 0.005 and about 0.3. In some embodiments, the liposome composition has a polydispersity index (PDI) between about 0.001 and about 0.2. In some embodiments, the liposome composition has a polydispersity index (PDI) between about 0.001 and about 0.1. In some embodiments, the liposome composition has a polydispersity index (PDI) between about 0.005 and about 0.2. In some embodiments, the liposome composition has a polydispersity index (PDI) between about 0.005 and about 0.1. In some embodiments, the liposome composition has a polydispersity index (PDI) between about 0.01 and about 0.5. In some embodiments, the liposome composition has a polydispersity index (PDI) between about 0.01 and about 0.4. In some embodiments, the liposome composition has a polydispersity index (PDI) between about 0.01 and about 0.2. In some embodiments, the liposome composition has a polydispersity index (PDI) between about 0.01 and about 0.1. In some embodiments, the liposome composition has a polydispersity index (PDI) between about 0.1 and about 0.5. In some embodiments, the liposome composition has a polydispersity index (PDI) between about 0.1 and about 0.3. In some embodiments, the liposome composition has a polydispersity index (PDI) between about 0.3 and about 0.5.

[0094] 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.

[0095] Therapeutic Agents 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.

[0096] In some embodiments of all the foregoing aspects, 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] It is.

[0097] 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.

[0098] 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.

[0099] 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), 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] Selected from the group consisting of venetoclax (ABT-199), and navitoclax (ABT-263). 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: [ka] In some embodiments, the Bcl inhibitor is venetoclax. In some embodiments, the Bcl inhibitor is navitoclax.

[0100] 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 a hydrophilic therapeutic agent. In some embodiments, the additional therapeutic agent is encapsulated in the liposome. In some embodiments, the additional therapeutic agent is present 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 is present external to 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 between about -6 and about 12. In some embodiments, the additional therapeutic agent has a cLogP between about -6 and about 0. In some embodiments, the additional therapeutic agent has a cLogP between about -3 and about 0. In some embodiments, the additional therapeutic agent has a cLogP between about 0 and about 2. In some embodiments, the additional therapeutic agent has a cLogP between about -1 and about 12. In some embodiments, the additional therapeutic agent has a cLogP of between about 3 and about 12. In some embodiments, the additional therapeutic agent has a cLogP of between about -6 and about -1. In some embodiments, the additional therapeutic agent has a cLogP of between about -1 and about 3. In some embodiments, the additional therapeutic agent has a cLogP of between about 2 and about 12. In some embodiments, the additional therapeutic agent has a cLogP of between about 2 and about 4. In some embodiments, the additional therapeutic agent has a cLogP of between about 2 and about 8. In some embodiments, the additional therapeutic agent has a cLogP of between about 4 and about 12. In some embodiments, the additional therapeutic agent has a cLogP of between about 4 and about 8. In some embodiments, the additional therapeutic agent has a cLogP of between about 8 and about 12. In some embodiments, the additional therapeutic agent has a cLogP of between about 10 and about 12. In some embodiments, the additional therapeutic agent can be protonated, and the protonated form has a pK greater than about 2. aIn some embodiments, the additional therapeutic agent can be protonated and has a pK of between about -6 and about 12 in its protonated form. a In some embodiments, the additional therapeutic agent can be protonated and has a pK between about -6 and about 11 in its protonated form. a In some embodiments, the additional therapeutic agent can be protonated and has a pK of between about 3 and about 11. a In some embodiments, the additional therapeutic agent can be protonated and has a pK of between about 6 and about 11. a In some embodiments, the additional therapeutic agent can be protonated and has a pK of between about 8 and about 11. a In some embodiments, the additional therapeutic agent can be protonated and has a pK of between about -6 and about 3 in its protonated form. a In some embodiments, the additional therapeutic agent can be protonated and has a pK of between about 3 and about 6. a In some embodiments, the additional therapeutic agent can be protonated and has a pK of between about 6 and about 8. a In some embodiments, the additional therapeutic agent can be protonated and has a pK of between about -6 and about 8. a In some embodiments, the additional therapeutic agent can be protonated and has a pK of between about 3 and about 8. a In some embodiments, the additional therapeutic agent can be protonated and has a pK of between about -6 and about 6 in its protonated form. a In some embodiments, the additional therapeutic agent can be protonated and has a pK of between about 2 and about 12 in its protonated form. a In some embodiments, the additional therapeutic agent can be protonated and has a pK of between about 2 and about 8. aIn some embodiments, the additional therapeutic agent can be protonated and has a pK of between about 2 and about 4. a In some embodiments, the additional therapeutic agent can be protonated and has a pK of between about 4 and about 12 in its protonated form. a In some embodiments, the additional therapeutic agent can be protonated and has a pK of between about 4 and about 8. a In some embodiments, the additional therapeutic agent can be protonated and has a pK of between about 8 and about 12 in its protonated form. a In some embodiments, the additional therapeutic agent can be protonated and has a pK of between about 10 and about 12 in its protonated form. a has.

[0101] In some embodiments of all the above aspects, the additional therapeutic agent is an anti-neoplastic agent.The additional therapeutic agent is selected from alkylating agents, antiangiogenic agents, antimetabolites, apoptosis inducers, cell cycle inhibitors, cell cycle control inhibitors, checkpoint inhibitors, cyclin-dependent kinase inhibitors, cytotoxic agents, DNA damaging agents, DNA repair inhibitors, mitochondrial poisons, receptor tyrosine kinase inhibitors, telomerase inhibitors, signal transduction inhibitors, transcription inhibitors, Bcl inhibitors, PARP inhibitors, PI3K inhibitors, HSP90 inhibitors, JAK inhibitors, ATR inhibitors, HDAC inhibitors, tyrosine kinase inhibitors, receptor tyrosine kinase inhibitors, BTK inhibitors, alkylating agents, SMO inhibitors, antitubulin agents, MEK inhibitors, topoisomerase inhibitors, RAF inhibitors, BRAF inhibitors, or proteasome inhibitors.

[0102] In some embodiments, the additional therapeutic agent is a Bcl inhibitor, such as those described above.

[0103] In some embodiments, the additional therapeutic agent is an HSP90 inhibitor. In some embodiments, the HSP90 inhibitor is luminespib.

[0104] In some embodiments, the additional therapeutic agent is an alkylating agent selected from the group consisting of bendamustine and chlorambucil.

[0105] 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.

[0106] In some embodiments, the additional therapeutic agent is an ATR inhibitor.

[0107] 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.

[0108] In some embodiments, the additional therapeutic agent is a BTK inhibitor. In some embodiments, the BTK inhibitor is ibrutinib.

[0109] In some embodiments, the additional therapeutic agent is an HDAC inhibitor, hi some embodiments, the HDAC inhibitor is panobinostat.

[0110] In some embodiments, the additional therapeutic agent is a JAK inhibitor. In some embodiments, the JAK inhibitor is ruxolitinib.

[0111] 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.

[0112] 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.

[0113] In some embodiments, the additional therapeutic agent is a PI3K inhibitor. In some embodiments, the PI3K inhibitor is idelalisib.

[0114] In some embodiments, the additional therapeutic agent is a proteasome inhibitor. In some embodiments, the proteasome inhibitor is carfilzomib.

[0115] 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.

[0116] 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.

[0117] 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.

[0118] 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.

[0119] The mechanism of action of one or more of the agents may be unknown or imprecisely specified.

[0120] 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.

[0121] Methods for Making Liposomes and Liposome 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.

[0122] Lipids The 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. The liposomes may also contain phospholipids, such as phosphatidylcholine lipids, phosphatidylethanolamine lipids, phosphatidylserine lipids, and phosphatidylglycerol lipids. The liposomes may be prepared to contain phosphatidylcholine lipids, such as distearoylphosphatidylcholine (DSPC). The 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).

[0123] 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.

[0124] The incorporation of negatively charged lipids such as phosphatidylglycerol (PG) and phosphatidylinositol (PI) can also be added to the liposomal formulation to increase the circulation life of the carrier. These lipids can be used in place of or in combination with hydrophilic polymer-lipid conjugates as surface stabilizers.

[0125] Sterols The liposomes may also contain a sterol, such as cholesterol, a cholesterol derivative, or a plant sterol, such as β-sitosterol.

[0126] Methods for loading liposomes A variety of methods can be used 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, or by equilibrium 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.

[0127] 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.

[0128] 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. The passive method of encapsulating active agents into liposomes includes 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 involves passive equilibration after formation of the liposomes. This method 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.

[0129] Active methods of encapsulation include the pH gradient loading technique described in US 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 US 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.

[0130] 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.

[0131] Passive and active entrapment methods can also be combined to prepare liposomal formulations containing two or more encapsulated drugs.

[0132] Loading Aids In some embodiments, the loading aid may be an ionic loading aid. In some embodiments, the loading aid is sulfate, sucrose octasulfate, or 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), ammonium sucrose octasulfate (TEA(OH)SOS), triethylammonium sucrose octasulfate (TEASOS), 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), ammonium sucrose octasulfate (TEASOS), triethanolammonium sucrose octasulfate (TEA(OH)SOS), ammonium citrate, and sodium citrate, or mixtures thereof. In some embodiments, the loading aid is selected from the group consisting of ammonium sulfate (AS), ammonium sucrose octasulfate (NHSOS), potassium sucrose octasulfate (KSOS), triethylammonium sucrose octasulfate (TEASOS), sodium citrate, and the like, or mixtures thereof. In some embodiments, the 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 loading aid is ammonium sulfate (AS). In some embodiments, the loading aid is ammonium sucrose octasulfate (NHSOS). In some embodiments, the loading aid is potassium sucrose octasulfate (KSOS). In some embodiments, the loading aid is triethylammonium sucrose octasulfate (TEASOS). In some embodiments, the loading aid is sodium citrate. In some embodiments, a second loading aid is used.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 (NH4SOS), potassium sucrose octasulfate (KSOS), sucrose triethylammonium octasulfate (TEASOS), sucrose triethanolammonium octasulfate (TEA(OH)SOS), ammonium citrate, sodium citrate, and the like, or mixtures thereof. In some embodiments, the second loading aid is selected from the group consisting of ammonium sulfate (AS), potassium sucrose octasulfate (KSOS), sucrose triethylammonium octasulfate (TEASOS), sodium citrate, and the like, or mixtures thereof. In some embodiments, the second loading aid is selected from the group consisting of ammonium sulfate (AS), potassium sucrose octasulfate (KSOS), triethylammonium sucrose octasulfate (TEASOS), 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.

[0133] Loading Solvent In some embodiments, the loading solvent is an organic solvent. In some embodiments, the organic solvent is an aprotic organic solvent. In some embodiments, the loading solvent is dimethylsulfoxide (DMSO).

[0134] How to use Also provided is a method for delivering a therapeutically effective amount of a Bcl inhibitor, comprising administering to a subject a liposomal 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) a Bcl inhibitor encapsulated in the internal medium of said liposome. The method includes:

[0135] Also provided is a method for treating a hyperproliferative disorder, comprising administering to a subject a liposomal 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) a Bcl inhibitor encapsulated in the internal medium of said liposome. The method includes:

[0136] In some embodiments, the hyperproliferative disorder is cancer. In some embodiments, the hyperproliferative disorder is a solid cancer or a hematological cancer. In some embodiments, the hyperproliferative disorder is a solid cancer. In some embodiments, the solid cancer is colorectal cancer or small cell lung cancer. In some embodiments, the solid cancer is colorectal cancer. In some embodiments, the solid cancer is small cell lung cancer. In some embodiments, the hyperproliferative disorder is a hematological cancer. In some embodiments, the hematological cancer is selected from the group consisting of multiple myeloma (MM), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), blastic plasmacytoid dendritic cell neoplasm (BPDCN), T-cell prolymphocytic leukemia, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), myeloproliferative neoplasm (MPN), and lymphoma. In some embodiments, the hematological cancer is multiple myeloma (MM). In some embodiments, the hematological cancer is acute lymphoblastic leukemia (ALL). In some embodiments, the hematological cancer is acute myeloid leukemia (AML). In some embodiments, the hematological cancer is blastic plasmacytoid dendritic cell neoplasm (BPDCN). In some embodiments, the hematological cancer is T-cell prolymphocytic leukemia. In some embodiments, the hematological cancer is chronic lymphocytic leukemia (CLL). In some embodiments, the hematological cancer is chronic myelogenous leukemia (CML). In some embodiments, the hematological cancer is myeloproliferative neoplasm (MPN). In some embodiments, the hematological cancer is lymphoma. In some embodiments, the lymphoma is non-Hodgkin's lymphoma (NHL). In some embodiments, the non-Hodgkin's lymphoma (NHL) is selected from the group consisting of cutaneous B-cell lymphoma (CBCL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), and follicular lymphoma (FL). In some embodiments, the non-Hodgkin's lymphoma (NHL) is cutaneous B-cell lymphoma (CBCL). In some embodiments, the non-Hodgkin's lymphoma (NHL) is small lymphocytic lymphoma (SLL).In some embodiments, the non-Hodgkin's lymphoma (NHL) is mantle cell lymphoma (MCL). In some embodiments, the non-Hodgkin's lymphoma (NHL) is diffuse large B-cell lymphoma (DLBCL). In some embodiments, the non-Hodgkin's lymphoma (NHL) is follicular lymphoma (FL).

[0137] Also provided is a method for reducing the incidence or severity of thrombocytopenia associated with administration of a therapeutically effective amount of a Bcl inhibitor, comprising administering to a subject a liposomal 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) a Bcl inhibitor encapsulated in the internal medium of said liposome. Includes; the incidence or severity of thrombocytopenia incurred by administering the Bcl inhibitor in a liposomal composition is less than the incidence or severity of thrombocytopenia incurred by administering the Bcl inhibitor in a non-liposomal form; the total amount of the Bcl inhibitor in the liposomal composition is equal to the therapeutically effective amount of the Bcl inhibitor when administered in a non-liposomal form; This is the method.

[0138] Also provided is a method for reducing the total dose of a Bcl inhibitor administered to a subject to achieve a therapeutic effect, comprising administering to the subject a liposomal 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) a Bcl inhibitor encapsulated in the internal medium of said liposome. Includes; the total amount of Bcl inhibitor in the liposomal composition required to achieve a therapeutic effect is less than the total amount of Bcl inhibitor required to achieve the same therapeutic effect when administered in a non-liposomal form; This is the method.

[0139] Also provided is a method for reducing the total dose of a Bcl inhibitor administered to a subject to achieve a therapeutic effect in a hyperproliferative disorder, comprising administering to the subject a liposomal 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) a Bcl inhibitor encapsulated in the internal medium of said liposome. Includes; the total amount of Bcl inhibitor in the liposomal composition required to achieve a therapeutic effect is less than the total amount of Bcl inhibitor required to achieve the same therapeutic effect when administered in a non-liposomal form; This is the method.

[0140] Also provided is a method for reducing inter-subject variability in exposure to a Bcl inhibitor administered to a subject in need thereof, comprising administering to the subject a liposomal 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) a Bcl inhibitor encapsulated in the internal medium of said liposome. Includes; the inter-subject variability of exposure to the Bcl inhibitor in the administered liposomal composition is less than the inter-subject variability of exposure to the Bcl inhibitor when administered in a non-liposomal form; the total amount of the Bcl inhibitor in the liposomal composition is equal to the therapeutically effective amount of the Bcl inhibitor when administered in a non-liposomal form; This is the method.

[0141] Also provided is a method for increasing the half-life of a Bcl inhibitor in a subject, comprising administering to the subject a 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) a Bcl inhibitor encapsulated in the internal medium of said liposome. Includes; the half-life of the Bcl inhibitor when administered in a liposomal composition is longer than the half-life of the Bcl inhibitor when administered in a non-liposomal form; This is the method.

[0142] Also provided is a method for reducing dosing frequency of a Bcl inhibitor in a subject, comprising administering to the subject a 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) a Bcl inhibitor encapsulated in the internal medium of said liposome. Includes; the frequency of dosing of the Bcl inhibitor administered in the liposomal composition is less than the frequency of dosing of the Bcl inhibitor when administered in a non-liposomal form; the total amount of the Bcl inhibitor in the liposomal composition is equal to the therapeutically effective amount of the Bcl inhibitor when administered in a non-liposomal form; This is the method.

[0143] Also provided is a method for reducing the incidence or severity of one or more adverse events associated with administration of a therapeutically effective amount of a Bcl inhibitor, comprising administering to a subject a liposomal 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) a Bcl inhibitor encapsulated in the internal medium of said liposome. Includes; the incidence or severity of said one or more adverse events incurred by administering the Bcl inhibitor in a liposomal composition is less than the incidence or severity of said one or more adverse events incurred by administering the Bcl inhibitor in a non-liposomal form; the total amount of the Bcl inhibitor in the liposomal composition is equal to the therapeutically effective amount of the Bcl inhibitor when administered in a non-liposomal form; This is the method.

[0144] 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.

[0145] 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.

[0146] 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 injection during lumbar puncture followed by appropriate positioning of the patient, as is commonly performed for spinal anesthesia or metrazamide imaging of the spinal cord. Alternatively, the preparations may be administered through an endoscopic device.

[0147] 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 and lyophilized, and the lyophilized preparations are 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.

[0148] 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, and the like, 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.

[0149] 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.

[0150] 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.

[0151] When a single composition that contains two or more active agents is included, follow the above procedure essentially.When the agents are administered in separate delivery vehicle compositions, administration should be timed so that desired ratio is maintained.Typically, this can be accomplished by simultaneously administering the compositions in calculated ratio.

[0152] kit The therapeutic agents in the compositions 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 includes 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.

[0153] 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 pre-packaged in the kit. EXAMPLES

[0154] 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.

[0155] Example 1. General Procedure for Liposome Formulation 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.

[0156] 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.

[0157] 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.

[0158] 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.

[0159] 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.

[0160] 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.

[0161] Preparations of all SOS solutions can be scaled up proportionally 25-100 fold.

[0162] 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.

[0163] 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.

[0164] 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.

[0165] 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.

[0166] 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.

[0167] 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, thereby removing unencapsulated loading aid, 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.

[0168] For small-scale volumes, extruded liposomes containing 0.5–1 N triethylamine-SOS, or 0.5–1 N ammonium-SOS, or 0.5–1 N triethanolamine-SOS were buffer exchanged into 9–18% w / v dextrose (depending on the prescribed concentration of the loading aid to balance the osmotic pressure) by loading the extruded liposomes at a bed volume ratio of 1 mL / 10 mL onto a Sepharose CL4B column and collecting the liposome elution fractions at a conductivity of <20 μS / cm, thereby removing unencapsulated loading aid.

[0169] 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.

[0170] 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 between 2-30%.

[0171] 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.

[0172] 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 similar concentrations 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 formulations is required. Finally, the formulations were concentrated to 10-20 mg / ml lipid, collected, and filtered through a 0.22 μm PES syringe filter.

[0173] 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.

[0174] A. Preparation of Loading Aid The loading aid solution was prepared as described in Example 1.

[0175] 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.

[0176] 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.

[0177] 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.

[0178] 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.

[0179] 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.

[0180] 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.

[0181] 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, thereby removing unencapsulated loading aid, 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.

[0182] For small-scale volumes, extruded liposomes containing 0.5–1 N triethylamine-SOS, or 0.5–1 N ammonium-SOS, or 0.5–1 N triethanolamine-SOS were buffer exchanged into 9–18% w / v dextrose (depending on the prescribed concentration of the loading aid to balance the osmotic pressure) by loading the extruded liposomes at a bed volume ratio of 1 mL / 10 mL onto a Sepharose CL4B column and collecting the liposome elution fractions at a conductivity of <20 μS / cm, thereby removing unencapsulated loading aid.

[0183] 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.

[0184] 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, the final D / L ratio was in the range of 0.1-0.8 mol / mol, and the final DMSO concentration was between 2-30%.

[0185] 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.

[0186] 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 similar concentrations 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 formulations is required. Finally, the formulations were concentrated to 10-20 mg / ml lipid, collected, and filtered through a 0.22 μm PES syringe filter.

[0187] Example 3: Drug and lipid assays Liposome formulations containing various drugs were prepared as described in Examples 1 and 2. Lipid content was analyzed using phospholipid C and cholesterol assay kits (Fujifilm) and a UV spectrometer (Cytation 5, BioTek Instruments Inc.).

[0188] To determine the 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. The drug was detected using a UV spectrometer (Cytation 5, BioTek Instruments Inc.) measuring absorbance at 310 nm.

[0189] 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

[0190] The control was the same preparation but not purified (ie, 100% recovery).

[0191] The encapsulation efficiency (EE) is calculated as follows: EE(%)=drug-to-lipid ratio in final formulation / drug-to-lipid ratio in control*100

[0192] 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).

[0193] 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 1-1] [Table 1-2] [Table 2]

[0194] 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 consisted of Nestlets from Ancare, Envigo 7097 1 The cages were housed in Bio-Serv clear tinted polycarbonate Mouse Igloos on 1 / 4" corn cob bedding. All enrichment was added to the cages prior to autoclaving the cages. Mice were fed Envigo Teklad Global Rodent Diet 2018. This rodent food 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 automatic 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 at 12 hours on and 12 hours off. Temperature, humidity and airflow were maintained and controlled by the BCCRC facility.

[0195] Storage of test / control items All test / control articles (TA / CA) were prepared ready for injection and stored at 2–8°C.

[0196] 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 intravenous injection. Vein dilation was achieved by holding the animals under a heat lamp for 1-2 minutes (current revision of SOP-AF-018).

[0197] 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 dosing material was stored at approximately 4°C until shipping of the samples.

[0198] 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.

[0199] Liposomal formulation All liposomes used in the pharmacokinetic studies described herein contained DSPC / cholesterol / PEGylation (56 / 38 / 5.6 mol%) and were prepared according to Examples 1-2. [Table 3] [Table 4]

[0200] 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 liposome formulations contained DSPC / cholesterol / PEGylation (56 / 38 / 5.6 mol%) as described in Examples 1-2. 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.

[0201] FIG. 1A shows the time course of normalized plasma concentrations of Compound 2 using various liposomal formulations compared to using the free drug.

[0202] 1B shows the normalized plasma concentration of Compound 2 over time 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 the free drug. The formulation labeled "Merrimack" contained liposomes containing DSPC / cholesterol / PEGylation (56 / 38 / 5.6 mol%) loaded without DMSO using TEASOS (0.43N) as a loading aid according to the procedure described in WO2017 / 123616.

[0203] 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 1-2. 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.

[0204] FIG. 2A shows the normalized plasma concentration of compound 9 over time using various liposomal formulations.

[0205] 2B shows the normalized plasma concentration of Compound 9 over time 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 the free drug. The formulation labeled "Merrimack" contained liposomes containing DSPC / cholesterol / PEGylation (56 / 38 / 5.6 mol%) loaded without DMSO using TEASOS (0.43N) as a loading aid according to the procedure described in WO2017 / 123616.

[0206] Example 7: Effect of liposomal encapsulation on the thrombocytopenic activity of compound 9 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 consisted of Nestlets from Ancare, Envigo 7097 1 The cages were housed in Bio-Serv clear tinted polycarbonate Mouse Igloos on 1 / 4" corn cob bedding. All enrichment was added to the cages prior to autoclaving the cages. Mice were fed Envigo Teklad Global Rodent Diet 2018. This rodent food 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 automatic 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 at 12 hours on and 12 hours off. Temperature, humidity and airflow were maintained and controlled by the BCCRC facility.

[0207] Storage of test / control items All test / control articles (TA / CA) were prepared ready for injection and stored at 2–8°C.

[0208] 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 intravenous injection. Vein dilation was achieved by holding the animals under a heat lamp for 1-2 minutes (current revision of SOP-AF-018).

[0209] Data collection Blood collection Mice were weighed and randomized into their test groups. Mice were restrained in Falcon tubes during saphenous vein blood collection. Vein distention was achieved by holding the animal under a heat lamp for 1-2 min, and the legs were shaved and wiped with isopropanol. Vaseline was applied to the saphenous vein to occlude it. The vein was pierced using a 25G needle at a 90 degree angle, and 50-75 μL of blood was collected in a Microvette® CB 300 K2 EDTA tube, mixed 8-10 times with an EDTA pipette tip to ensure the absence of clots, and placed on ice. The vein was clamped to achieve hemostasis (SOP-AF-001) before the animal was returned to its cage.

[0210] For terminal 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 time of last breath and approximately 500 μL of blood was collected by cardiac puncture with a 25G needle and placed into appropriate microtiter tubes (K2EDTA).

[0211] For hematology, whole blood was placed into K2 EDTA tubes and gently inverted a minimum of 8-10 times. Samples were inverted until certain that the sample was not clotted and placed on ice. Samples were sent for complete blood count (CBC) analysis within 30 minutes of collection. CBC analysis was completed within 2 hours of sample collection on the Element HT5. Samples were run in duplicate. 20 μL of whole blood was diluted with 480 μL of V-52 D Diluent, allowed to stand for 3 minutes, and run on the Element HT5. For complete instructions on running samples, please refer to the work instructions "Element HT5 Auto Hematology Analyzer".

[0212] 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.

[0213] Liposomal formulation All liposomes used in the pharmacokinetic studies described herein contained DPSC / cholesterol / PEGylated (56 / 38 / 5.6 mol%) and compound 9 and were prepared according to Examples 1-2.

[0214] FIG. 3A compares the change in mean platelet count over time following administration of various doses of free drug or various doses of a liposomal formulation containing compound 9 encapsulated in the internal medium of liposomes.

[0215] FIG. 3B compares the change in normalized platelet count over time following administration of various doses of free drug or various doses of liposomal formulations containing compound 9 encapsulated in the internal medium of liposomes.

[0216] FIG. 4A compares the change in normalized platelet count over time following administration of various doses of free Compound 9.

[0217] FIG. 4B compares the change in normalized platelet count over time following administration of various doses of liposomal formulations containing compound 9 encapsulated in the internal medium of the liposomes.

[0218] Example 8: Effect of liposome composition on drug retention Experimental design Liposomes containing DHSM:cholesterol:PEG 53:45:2 (drug:lipid ratio 0.1) were produced using the method described above with DMSO loading aid and showed encapsulation efficiency of over 90%. Liposomes of DSPC:cholesterol:PEG 57:38:5.6 (drug:lipid ratio 0.1) were also produced using the method described above with DMSO loading aid and showed encapsulation efficiency of over 90%. Pharmacokinetic and drug retention studies were performed as described previously. DHSM: dihydrosphingomyelin; PEG: 1,2-distearoyl-rac-glycero-3-methoxypoly(ethylene glycol-2000) (also known as DSG-PEG2000 and PEG(2000)-distearoylglycerol)

[0219] FIG. 5A shows the normalized plasma concentration of compound 9 (Bcl-22B) over time using various liposomal formulations.

[0220] FIG. 5B shows the change in drug-to-lipid ratio over time for compound 9 (Bcl-22B) using various liposomal formulations.

[0221] Example 9: Effect of liposomal PEG on thrombocytopenia Various liposomes were prepared using a method similar to that described above. Different liposome compositions included: DSPC / Chol / β-sitosterol / 5.6%PEG-DSG; DSPC / Chol / 2%PEG-DMG, DSPC / Chol / 5.6%PEG-DSG, EPC / Chol / 5.6%PEG-DSG, and DSPC / Chol / 5.6%PEG-DSG / TEASOS. Compound 9 was loaded into these liposomes as described above.

[0222] FIG. 6A compares the time course of normalized platelet counts following administration of liposomal formulations containing compound 9 encapsulated in the internal medium of liposomes of different lipid composition.

[0223] As shown in FIG. 6B, lowering the amount of PEG-DSG in the liposomes reduces platelet count reduction.

[0224] As shown in FIG. 6C, lowering the amount of PEG-DMG in the liposomes significantly reduced the platelet count reduction.

[0225] The graph in FIG. 6D shows that liposomes containing 1% PEG-DMG produced a greater amount of platelet reduction than liposomes containing 1% PEG-DSG.

[0226] The graph in FIG. 6E shows that liposomes containing 0.5% PEG-DSG and liposomes containing 0.5% PEG-DMG produced similar platelet reductions.

[0227] Example 10. Comparison of free vs. liposomal BCL inhibitors for platelet protection Liposomes were produced using various loading aids, including low dose ammonium sulfate (LAS) and triethylammonium sucrose octasulfate (TEA-SOS), and loaded with compound 2 (11B), compound 7 (11G), or compound 9 (22B). Platelet protection studies were performed to compare the effects of these liposomal formulations against the corresponding free drug (FD). In Figures 7A-7G, as follows:

[0228] As shown in FIG. 7A, liposomal formulations dosed at 1 mg / kg containing compound 9 encapsulated in the internal medium of liposomes containing low doses of ammonium sulfate (LAS) or triethylammonium sucrose octasulfate (TEA-SOS) provided more than 10-fold greater platelet protection when compared to 0.1 mg / kg of free drug (FD).

[0229] As shown in FIG. 7B, liposomal formulations dosed at 2 mg / kg containing compound 9 encapsulated in the internal medium of liposomes containing low doses of ammonium sulfate (LAS) or triethylammonium sucrose octasulfate (TEA-SOS) provided more than 10-fold greater platelet protection when compared to free drug (FD) at 0.2 mg / kg.

[0230] As shown in FIG. 7C, liposomal formulations dosed at 3 mg / kg containing compound 9 encapsulated in the internal medium of liposomes containing low doses of ammonium sulfate (LAS) or triethylammonium sucrose octasulfate (TEA-SOS) provided more than 10-fold the amount of platelet protection when compared to free drug at 0.3 mg / kg.

[0231] As shown in FIG. 7D, liposomal formulations containing compound 9 encapsulated in the internal medium of liposomes containing low doses of ammonium sulfate (LAS) or triethylammonium sucrose octasulfate (TEA-SOS) exhibited approximately 20-fold platelet protection when compared to the free drug.

[0232] The graph in FIG. 7E shows that liposomes containing DHSM:cholesterol:PEG (53:45:2) and compound 9 ("22B") (drug:lipid ratio 0.1) dosed at 3 mg / kg demonstrate better platelet protection compared to liposomes containing DSPC:cholesterol:PEG (57:38:5.6) and compound 9 ("22B") (drug:lipid ratio 0.1) dosed at 3 mg / kg.

[0233] FIG. 7F shows that compound 7 was well tolerated at the doses tested.

[0234] FIG. 7G shows that liposomal formulations containing Compound 2 encapsulated in the internal medium of DSPC-containing liposomes demonstrate good platelet protection.

[0235] Example 11. 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.

[0236] 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.

[0237] 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.

[0238] FIG. 8 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. FIG. 8 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 FIG. 8, 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.

Claims

**Claim 1** A liposome composition comprising one or more liposomes, wherein each of said one or more liposomes comprises: (a) a lipid bilayer comprising a first lipid and a first sterol; (b) an internal medium comprising a first loading aid and a first medium; and an additional medium; and (c) a Bcl inhibitor encapsulated in said internal medium of said liposome and said lipid bilayer contains no Bcl inhibitor associated therewith or substantially no Bcl inhibitor associated therewith, said liposome composition. **Claim 2** The liposome composition according to claim 1, wherein said first lipid is a polymer-conjugated lipid 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), and DSG-PEG2000. **Claim 3** The lipid bilayer further comprises a second lipid, wherein said second lipid is a phospholipid; or said second lipid is egg sphingomyelin, distearoylphosphatidylcholine (DSPC) or dihydrosphingomyelin (DHSM), the liposome composition according to claim 1. **Claim 4** The liposome composition according to claim 1, wherein said first medium is an aqueous medium; and said aqueous medium is an acidic aqueous medium. **Claim 5** The liposome composition according to claim 1, wherein said additional medium is an organic medium; or said additional medium is dimethyl sulfoxide (DMSO). **Claim 6** The liposome composition according to claim 1, wherein said first loading aid is an ionic loading aid selected from the group consisting of ammonium sulfate (AS), sucrose octasulfate ammonium (NH4SOS), sucrose octasulfate potassium (KSOS), sucrose octasulfate triethanolammonium (TEA(OH)SOS), sucrose octasulfate triethylammonium (TEASOS), ammonium citrate, and sodium citrate. **Claim 7** The internal medium further comprises a second loading aid; the second loading aid is an ionic loading aid selected from the group consisting of ammonium sulfate (AS), sucrose octasulfate ammonium (NH4SOS), sucrose octasulfate potassium (KSOS), sucrose octasulfate triethylammonium (TEASOS), and sodium citrate. The liposome composition according to claim 1.

8. The liposome composition according to claim 1, wherein the first sterol is cholesterol or β-sitosterol.

9. The Bcl inhibitor is selected from a Bcl-2 inhibitor, a Bcl-XL inhibitor, and a Bcl-2 / Bcl-XL dual inhibitor; the Bcl inhibitor is a compound of formula (I): 【Chemical 1】 Or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof (wherein: V is 【Chemical 2】 ; W is H or 【Chemical Formula 3】 ; X is 【Chemical Formula 4】 ; Y is -NO ２ or -SO ２ CF ３ and; Z is 【Chemical Formula 5】 selected from the group consisting of) ; or or The compound of formula (I) is as follows: [Chemical Formula 6] The liposome composition according to claim 1.

10. Further comprising an additional therapeutic agent; or an alkylating agent, 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 receptor tyrosine kinase inhibitor, 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 liposome composition according to claim 1.

11. The liposome composition according to claim 1, wherein the liposome has an average diameter of about 50 nm to about 250 nm.

12. The liposome composition according to claim 1, further comprising a dispersion medium in which the liposome is suspended.

13. The liposome composition according to claim 1, having a polydispersity index (PDI) of about 0.001 to about 0.

5.

14. The liposome is as follows: First, encapsulating the Bcl inhibitor in the internal compartment of the liposome; contacting the liposome with an aqueous acid; protonating the Bcl inhibitor associated with the lipid bilayer; and separating the protonated Bcl inhibitor from the liposome, The liposomal pharmaceutical composition according to claim 1, which is prepared by a method comprising the above steps.

15. The liposomal pharmaceutical composition according to claim 14, wherein the protonated Bcl inhibitor is separated from the liposome by ultrafiltration.

16. The liposomal pharmaceutical composition according to claim 1, wherein the composition does not burst-release the Bcl inhibitor into the plasma, and the burst release is defined as a decrease of more than 50% of the Bcl inhibitor in the plasma in the first hour.

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