Modulators of nanoparticle uptake

Abstract

Various formulations for combination therapy with a liposome and an inhibitor of SR-BI, leading to enhanced uptake of the liposome by a target cell are provided and exemplified by combination therapy with CPX-351 and BLT-1. Also provided are various formulations for combination therapy with a liposome and an ENT inhibitor leading to enhanced uptake of liposome-encapsulated cytarabine by a target cell. Also provided are methods of using the formulations in combination therapy. Also provided is a method of reducing cardiotoxicity in an age-stratified patient population undergoing therapy with cytarabine and daunorubicin, CPX-351.

Description

MODULATORS OF NANOPARTICLE UPTAKECROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application Serial No. 63 / 493,969 filed on April 3, 2023, the entire disclosure of which is incorporated herein by reference for all purposes.BACKGROUND OF THE INVENTION

[0002] Acute myeloid leukemia (AML) is a myeloid hematologic neoplasm associated with a median age of 65 years at the time of diagnosis (Siegel et al. CA Cancer J Clin, 2018;68(l):7- 30 (2018)). According to the American Cancer Society, an estimated 19,520 new cases will be diagnosed with acute myeloid leukemia (AML) and an estimated 10,670 deaths in AML in 2018 (Siegel et al. Id.). Since the late 1970s, the standard of care for AML induction has consisted of combination chemotherapy comprised of cytarabine (days 1-7) and daunorubicin (days 1-3), known as the “7+3” regimen (Ofran et al. Leukemia. 30(8): 1796 (2016)).Attempts have been made to add additional cytotoxics to the protocol, however, the overall survival benefit from convention induction chemotherapy regimens remains dismal in older patients with AML (Roboz Hematology, Am Soc Hematol Educ Program 2011 :43-50 (2011)). As the biology of AML has become better understood, several targeted agents are under investigation as new therapeutic approaches, particularly for patients with poor risk characteristics (DiNardo et al., Expert Opin Pharmacother , 6(l ):95-106 (2015)). An alternative approach is to utilize the current understanding of the pharmacodynamics of the conventional drugs to optimize the cytarabine and anthracycline treatments. To that end, CPX-351 (Trade name: Vyxeos®), is an advanced liposomal formulation of cytarabine and daunorubicin encapsulated at a 5: 1 molar ratio, which is found to be maximally synergistic and minimally antagonistic in vitro (Tardi et al., LeukRes., 33(1): 129-39 (2009)). That is, at some molar ratios of cytarabine: daunorubicin the combination is additive, at others it is far more effective and in some ratios the combination is actually worse. Liposomal encapsulation markedly increases the plasma half-life of cytarabine and daunorubicin and leads to greater drug accumulation within the bone marrow comparing with free cytarabine and daunorubicin (Feldman et al. LeukRes. 36(10): 1283-9 (2012); Lim et al., LeukRes. 34(9): 1214-23 (2010)). Importantly, the liposomal integrity of CPX-351 in circulation ensures that there is minimal free drug release, as well as simultaneous co-delivery of the two drugs at the optimal molarratio. This co-delivery strategy eliminates the problem of differential biodistribution and metabolism. The manner in which the two drugs are cleared from the systemic circulation makes maintaining an optimal synergistic molar ratio at the intended site of action impossible with co-administration of the free (unencapsulated) drugs. Human clinical trials using CPX- 351 against myeloid leukemia reveal superior antileukemia activity and improved median overall survival in patients to 9.56 months comparing with 5.95 months in patients receiving “7+3” regimens (Lancet et al.. Blood, 123(21):3239-46 (2014); Cortes et al., Cancer. 121(2):234-42 (2015); Lancet et al., American Society of Clinical Oncology, 2016), which lead to FDA approval for the treatment of newly diagnosed therapy -related acute myeloid leukemia (t-AML) or AML with myelodysplasia-related changes (AML-MRC) in 2017 (Nikanjam et al.. Cancer Chemother Pharmacol, 81(1); 171-8 (2018)).

[0003] Ongoing research into the treatment of leukemia continues to provide advances in treatment options for this disease.BRIEF SUMMARY OF THE INVENTION

[0004] It has now been discovered that certain liposomes encapsulating one or more therapeutic agents administered with an inhibitor of a selected cellular pathway to a subject in need thereof, each in a therapeutically useful dosage, results in a significant increase in uptake of the liposome by cells relative to the uptake seen in an identical cell without administration of the inhibitor.

[0005] In an exemplary embodiment, the inhibitor is an inhibitor of Scavenger Receptor BI (SR-BI). Incorporating such an inhibitor in a therapeutically useful dosage results in a significant increase in uptake of the liposome by cells expressing SR-BI relative to the uptake seen in an identical cell without administration of the inhibitor.

[0006] SR-BI, implicated in HDL uptake into cells, was recently recognized as mediating uptake of certain liposomes in leukemia cells (Di et al., Drug Dev. Ind. Pharm. 45(l):21-26 (2019)). Thus, an increase in liposome uptake upon contacting SR-BI with a known inhibitor of SR-BI is a surprising result as one of ordinary skill in the art would expect the uptake of the liposome to decrease upon inhibiting SR-BI. The result is further surprising in view of the discovery' by the inventors that administering an exemplary' inhibitor of SR-BI at a first dosage results in the anticipated decrease in uptake of the liposomes by cells expressing SR-BI, yet administering the same inhibitor at a second, higher, dosage results in a counterintuitive increase in uptake of the liposomes by the cell.

[0007] In an exemplary embodiment, the disclosure sets forth a method of increasing the cellular uptake of a liposome encapsulating one or more therapeutic agents by a target cell expressing SR-BI. The method includes administering the liposome to the target cell and also administering to the target cell an amount of the inhibitor of SR-BI sufficient to increase uptake by the cell of the liposome (e.g., '‘a therapeutically effective amount”).

[0008] In an exemplary embodiment, the target cell is diseased and either underexpresses or overexpresses SR-BI relative to an identical undiseased cell. The formulations of the instant invention leverage this over-, under-expression to effect enhanced delivery of the liposome to the cell.

[0009] In various embodiments, there is a provided a method of treating a disease in a subject in need of such treatment. The method includes administering to the subject a therapeutically effective amount of a liposome encapsulating one or more therapeutic agents effective in treating the disease, ameliorating the symptoms of the disease, etc., and an amount of an inhibitor of SR-BI selected such that the cellular uptake by a target cell of the subject is increased relative to the cellular uptake of the liposome by the same target cell in the absence of the inhibitor of SR-BI.

[0010] In various embodiments, there is provided a method of treating cancer in a subject in need of such treatment, e.g., leukemia, e.g., acute myeloid leukemia. The method includes administering to the subject a therapeutically effective dosage of a liposome encapsulating one or more cancer chemotherapeutic agents and an amount of a SR-BI inhibitor selected such that the cellular uptake by a target cell of the subject is increased relative to the cellular uptake of the liposome by the same target cell in the absence of the inhibitor of SR-BI. In an exemplary embodiment, the inhibitor of SR-BI is effective at increasing the uptake of the liposome by the target cell at a first higher dosage of the SR-BI inhibitor but either has no effect on uptake or reverses the effect and reduces the uptake at a lower dosage of the SR-BI inhibitor.

[0011] Also provided are methods of altering the side effect profile of a liposomal formulation of one or more therapeutic agent, e.g., CPX-351, by enhancing delivery of the formulation to a target cell. The delivery is enhanced by administering an inhibitor of SR-BIin an amount selected such that the uptake by a target cell of the subject is increased relative to the cellular uptake of the liposome by the same target cell in the absence of the inhibitor of SR-BI, thereby reducing delivery to non-target cells. In an exemplary embodiment, the enhanced delivery to the target cell leads to less off target delivery than when the inhibitor is not present. An exemplary alteration of the side effect profile includes a diminishment or amelioration of at least one undesirable side effect of the therapeutic agent on the subject to whom it is administered.

[0012] An exemplary7method of the invention is useful to reduce cellular resistance to a one or more liposome encapsulated therapeutic agents, e.g., cytarabine, daunorubicin and combinations thereof. The method comprises administering to a drug resistant target cell an inhibitor of SR-BI in an amount selected such that the cellular uptake of the liposome encapsulated therapeutic agent(s) by the target cell is increased relative to the cellular uptake of the liposome encapsulated agent(s) by the same target cell in the absence of the inhibitor of SR-BI, or the cellular efflux of the liposome encapsulated therapeutic agent(s) (or deencapsulated therapeutic agent(s)) is less than the efflux of the agent(s) relative to the same target cell in the absence of the inhibitor of SR-BI.

[0013] In various embodiments, there is provided a pharmaceutical formulation for carrying out one or more of the methods set forth above. The pharmaceutical formulation includes a liposome encapsulating one or more therapeutic agents and a SR-BI inhibitor selected such that the cellular uptake of the liposome by a target cell is increased relative to the cellular uptake of the liposome by the same target cell in the absence of the inhibitor of SR-BI. In an exemplary embodiment, the presence of the inhibitor in the formulation reduces delivery to non-target cells.

[0014] In an exemplary embodiment, the liposome includes one or more cancer chemotherapeutic agents. In various embodiments, the chemotherapeutic agents are cytarabine and daunorubicin. In some embodiments, the cytarabine and daunorubicin are encapsulated in the liposome in a constant 5: 1 ratio. An exemplary7liposome is CPX-351.

[0015] In various embodiments, there is provided a pharmaceutical formulation comprising therapeutically effective amounts of a liposome and an inhibitor of SR-BI in an amount selected such that the cellular uptake of the liposome by a cell of the subject is increased relative to the cellular uptake of the liposome by the same cell of the subject in the absence ofthe inhibitor of SR-BI. In various embodiments, there is provided a pharmaceutical formulation comprising therapeutically effective amounts of CPX-351 and an inhibitor of SR- BI in an amount selected such that the cellular uptake of the liposome by a cell of the subject is increased relative to the cellular uptake of the liposome by the same cell of the subject in the absence of the inhibitor of SR-BI. The CPX-351 and inhibitor of SR-BI are combined in a pharmaceutically acceptable carrier.

[0016] In an exemplary embodiment, a combination of a liposome comprising cytarabine and an equilibrative nucleoside transporter (ENT) (e.g., ENT1) inhibitor in therapeutically useful dosages results in a significant increase in uptake of liposome-encapsulated cytarabine bycells expressing ENT (e.g., ENT1) relative to the uptake of unencapsulated cytarabine in the presence of the ENT inhibitor seen in an identical cell.

[0017] In an exemplary embodiment, the disclosure sets forth a method of combination therapy of a proliferative disorder, e.g., cancer, e.g.. leukemia, e.g., acute myeloid leukemia, the method comprising administering to a subject in need thereof a therapeutically effective amount of a liposome comprising cytarabine and an amount of an ENT inhibitor effective to enhance uptake of liposome-encapsulated cytarabine by a target cell exhibiting the genotype and / or phenotype associated with the proliferative disease relative to the uptake of unencapsulated cytarabine in the presence of the ENT inhibitor by an identical cell.

[0018] In an exemplary- embodiment, the disclosure sets forth a method of enhancing delivery of cytarabine into an intracellular compartment of a target cell in the presence of an ENT inhibitor. The method comprises administering to a subject in need thereof a therapeutically effective amount of a liposome comprising cytarabine and a therapeutically effective amount of the ENT inhibitor, wherein uptake of liposome-encapsulated cytarabine by the target cell is enhanced relative to the uptake of unencapsulated cytarabine in the presence of the ENT inhibitor by an identical cell, thereby providing enhanced delivery of the cytarabine to the intracellular compartment.

[0019] In exemplary embodiments, as with the SRBI, the method of the invention utilizing the ENT inhibitor alters the side effect profile of the therapeutic agent. An exemplary alteration of the side effect profile includes a diminishment or amelioration of at least one undesirable side effect of the therapeutic agent on the subject to whom it is administered. Inan exemplary embodiment, the enhanced delivery to the target cell leads to less off target delivery than when the inhibitor is not present.

[0020] In a further exemplary embodiment, there is provided a method of treating acute myeloid leukemia in a subject in need of such treatment, wherein the subject is at least about 60 years old. The method comprises administering a therapeutically relevant dose of CPX- 351 to the subject. Administration of CPX-351 in this patient population is correlated with lower cardiotoxicity than administration of standard 7+3 therapy.

[0021] Further embodiments, objects and examples are set forth in the Detailed Description.BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIGURE 1. The expression of lipid receptors in leukemia cell lines, (a) scavenger receptor class B type 1 (SR-BI), (b) low-density lipoprotein receptor (LDLR), and (c) low- density lipoprotein receptor-related protein 1 (LRP1). The mean ± SE for at least 3 independent experiments is shown for each value. * p = 0.01. ** p = 0.014. *** p = 0.011.

[0023] FIGURE 2. (a) The uptake of CPX-351 into leukemia cell lines evaluated by flow cytometry, (b) The uptake of CPX-351 in HL-60 / AD treated with MK571. (c) The growth inhibition curve in HL-60 / AD. Control and HL-60 / AD treated with MK571 were incubated with various concentrations of CPX-351 for 72 h, and the ICso was then determined by using the XTT assay, (d) The uptake of CPX-351 in K562 / ADM treated with tariquidar. (e) The growth inhibition curve in K562 / ADM. Control and K562 / ADM treated with tariquidar were incubated with various concentrations of CPX-351 for 72 h, and the ICso was then determined by using the XTT assay. The mean ± SE for at least 3 independent experiments is shown for each value. * p < 0.001. ** p = 0.0015.

[0024] FIGURE 3. Inhibition of the CPX-351 uptake pathway by the addition of chlorpromazine, 5-(N-ethyl-N-isopropyl)-Amiloride (EIPA), or low-dose blocks lipid transport-1 (LD-BLT-1, 400 nM). (a) HL-60. * p = 0.0011. ** p = 0.0014. *** p = 0.004. (b) K562. *p < 0.001. ** p = 0.0018. (c) THP-1. * p < 0.001. ** p = 0.0036. *** / ? = 0.0031. The mean ± SE for at least 3 independent experiments is shown for each value.

[0025] FIGURE 4. Enhancement of the CPX-351 uptake by the addition of high-dose blocks lipid transport-1 (HD-BLT-1, 40 pM) alone or in combination with chlorpromazine, 5-(N-ethyl-N-isopropyl)-Amiloride (EIP A), or incubation at 4°C. (a) HL-60. * p < 0.001. ** / ? = 0.0066. (b) K562. * / ? < 0.001. ** / ? = 0.0019. *** / ? = 0.001. (c) THP-1. * / ? < 0.001. ** / ? = 0.0055. The mean ± SE for at least 3 independent experiments is shown for each value.

[0026] FIGURE 5. Patient specimen analysis, (a) the expression of scavenger receptor class B type 1 (SR-BI). (b) the uptake of CPX-351 alone or in combination with high-dose blocks lipid transport-1 (HD-BLT-1, 40 pM).

[0027] FIGURE 6. The uptake of daunorubicin into leukemia cell lines and inhibition of the uptake pathway by the addition of chlorpromazine, 5-(N-ethyl-N-isopropyl)-Amiloride (EIP A), low-dose blocks lipid transport- 1 (LD-BLT-1, 400 nM), high-dose blocks lipid transport- 1 (HD-BLT-1, 40 pM), or incubation at 4°C. The mean ± SE for at least 3 independent experiments is shown for each value. One-way ANOVA was used to compare each group except for incubation at 4°C. (a) HL-60. p = 0.77. (b) K562. p = 0.073. (c) THP-1. p = 0.063. Abbreviations: n.s., not significant.

[0028] FIGURE 7. Inhibition of DNA synthesis by CPX-351. ***P<0.001.DETAILED DESCRIPTION OF THE INVENTIONI. Introduction

[0029] The present disclosure provides new combination formulations of a liposome encapsulating one or more therapeutic agents, and an inhibitor of a selected cellular pathway. In an exemplary embodiment, the inhibitor is an inhibitor of Scavenger Receptor BI (SR-BI).Also provided are combination therapies with these formulations, and methods of using the new formulations and combination therapies to treat disease. The present disclosure also sets forth new methods of determining whether therapy with a liposome encapsulated therapeutic or a combination therapy with this liposome and inhibitor is appropriate for a particular subject in need of treatment for a disease. Supporting these various embodiments is the discovery that incorporating an exemplary inhibitor of SR-BI, at selected dosages, results in a significant increase in uptake of the liposome by cells expressing SR-BI relative to the uptake seen in an identical cell without administration of the inhibitor.

[0030] Liposomes are closed vesicles having at least one lipid bilayer surrounding an aqueous core. The intra-liposomal space and lipid layer(s) can entrap a wide variety ofsubstances including drugs, cosmetics, diagnostic reagents, genetic material and bioactive compounds. Since non-toxic lipids act as the basis for liposomes, they generally exhibit low toxicity. The low toxicity coupled with the ability of liposomes to increase the plasma circulation lifetime of agents gives rise to liposomes as vehicles particularly useful for delivering pharmaceutically active agents. In many cases, liposome-delivered drugs result in superior clinical efficacy paired with reduced toxicity.

[0031] SR-BI is recognized as a receptor mediating the uptake of HDL into cells and was recently recognized as mediating uptake of liposomes in leukemia cells (Di et al., Drug Dev. Ind. Pharm. 45(l):21-26 (2019)). SR-BI is a member of the CD36 superfamily. Each member contains a large extra cellular domain flanked by two membrane - spanning (ML279) domains with short amino and carboxy - terminal intracellular tails. CD36 family members maintain about 30 % sequence identity. They can differ in subcellular localization and ligand preference. For example. CD36 / SCARB3 can bind HDL but is incapable of efficient uptake of HDL cholesterol via selective lipid uptake.

[0032] Inhibitors of SR-BI are recognized compounds. See, e.g., U.S. Pat. No. 9,884,851; Raldue et al. Tox. Letters 175(1-3): 1-7 (2007).

[0033] The diabetic dmg glyburide, an inhibitor of the sulfonylurea receptors SURI and SUR2, was also found to have activity at SR-BI, though it was a relatively weak inhibitor of cholesterol efflux. Researchers at Sankyo discovered that the protected piperazines R-138329 and R- 154716 increased HDL-cholesterol in both mice and hamsters, presumably by inhibiting SR-BI-mediated uptake of HDL Recently, a p38 MAP kinase inhibitor (ITX-5061) was reported to have similar in vivo effects and appears to be a moderate inhibitor of SR-BI- mediated cholesterol uptake. See, e.g., U.S. Pat. No, 9,884,851.

[0034] Dockendorff et al. in U.S. Pat. No. 9,884,851 demonstrate various activities towards SR-BI consistent across a range of novel synthetic small molecules prepared as SR-BI inhibitors. This reference also provides ample guidance for one of skill in the art to determine whether a compound functions as a SR-BI inhibitor, as well as assays for various properties of compounds active towards SR-BI, general aspects of SR-BI inhibitor biology, etc.

[0035] Inhibitors of SR-BI as a class are exemplified by Block Lipid Transport- 1 and related compounds. In researching the mechanism by which liposomes are taken up by cells, theinventors unexpectedly discovered that inhibitors of SR-BI, as exemplified by BLT-1, at selected dosages increase the uptake of liposomes by cells.

[0036] The present disclosure also provides new combination formulations of a liposome comprising cytarabine and an ENT inhibitor. Also provided are combination therapies with these formulations, and methods of using the new formulations and combination therapies to treat disease. Supporting these various embodiments is the discovery that an exemplary ENT inhibitor, at selected dosages in combination with a liposome comprising cytarabine, enhances uptake of liposome-encapsulated cytarabine by target cells compared to the combination of unencapsulated cytarabine and an ENT inhibitor.

[0037] ENTs are polytopic integral membrane proteins that mediate physiologic nucleoside transport across cellular membranes (Boswell-Casteel and Hays, Nucleosides Nucleotides Nucleic Acids , 36(1): 7-30 (2017)). ENT inhibitors include, but are not limited to, S-(4- nitrobenzyl)-6-thioinosine (NBMPR), dilazep, dipyridamole, and rapadocin (Rehan et al., SLAS Discovery, 24(10): 953-968 (2019)).

[0038] In some embodiments, the ENT inhibitor comprises a modified nucleoside. In some embodiments, the ENT inhibitor comprises adenosine. In some embodiments, the ENT inhibitor comprises a modified adenosine. In some embodiments, the ENT inhibitor comprises a derivative and / or analog of adenosine. In some embodiments, the ENT inhibitor comprises a nucleoside modified with a thionitrobenzyl moiety. In some embodiments, the ENT inhibitor comprises a purine moiety. In an exemplary embodiment, the ENT inhibitor comprises NMBPR.

[0039] It was previously found that CPX-351 uptake is not dependent on human equilibrative nucleoside transporter 1 (hENTl) expression (Anderson et al., Leukemia Research, 74. 121- 129 (2018), incorporated by reference herein in its entirety); however, it was previously unknown how administering a combination of a liposome comprising cytarabine and an ENT inhibitor would affect delivery' of cytarabine to target cells.

[0040] Reference will now be made in detail to implementation of exemplary' embodiments of the present disclosure as illustrated in the accompanying drawings. The same reference indicators will be used throughout the drawings and the following detailed description to refer to the same or like parts. Those of ordinary' skill in the art will understand that the following detailed description is illustrative only and is not intended to be in any waylimiting. Other embodiments of the present disclosure will readily suggest themselves to such skilled persons having benefit of this disclosure.

[0041] In the interest of clarity, not all of the routine features of the implementations described herein are shown and described. It will be appreciated that, in the development of any such actual implementation, numerous implementation-specific decisions are made in order to achieve the developer’s specific goals, such as compliance with application- and business-related constraints, and that these specific goals will vary from one implementation to another and from one developer to another.

[0042] Many modifications and variations of the exemplary embodiments set forth in this disclosure can be made without departing from the spirit and scope of the exemplary embodiments, as will be apparent to those skilled in the art. The specific exemplary embodiments described herein are offered by way of example only, and the disclosure is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

[0043] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.II. TermsA. Definitions

[0044] Unless defined otherwise, all terms of art, notations and other scientific terms or terminology used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which the present disclosure pertains. In some cases, terms with commonly understood meanings are defined herein for clarity’ and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer defined protocols and / or parameters 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 toor otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth in this section prevails over the definition that is incorporated herein by reference.

[0045] The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article.

[0046] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by how the value is measured or determined- e.g., the limitations of the measurement system, or the degree of precision required for a particular purpose. For example, "about” can mean within 1 or within 2 standard deviations, as per the practice in the art. Alternatively, "about" can mean a range of up to 20%, preferably up to 10%, and more preferably up to 5% of a given value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” means an acceptable error range for the particular value should be assumed.

[0047] The term “CPX-351” as used herein refers to a liposomal formulation of cytarabine and daunorubicin encapsulated at a 5: 1 molar ratio. The liposome is formulated with DSPC / DSPG / Chol at a about a 7:2: 1 ratio.

[0048] “Liposome”, as this term is used herein, refers to closed vesicles having at least one lipid bilayer surrounding an aqueous core. The intra-liposomal space and lipid layer(s) can entrap a wide variety of substances including drugs, cosmetics, diagnostic reagents, genetic material and bioactive compounds. An exemplary liposome of use in the compositions and methods provided herein includes no more than about 20% cholesterol. In various embodiments, the liposome further comprises from about 1% to about 20% DSPG. In various embodiments, the liposome comprises one or more additional lipid. See. e g., U.S. Pat. No, 8.518,437, incorporated herein by reference in its entirety for all purposes. In an exemplary embodiment, the liposome is formulated from DSPC / DSPG / Chol in a ratio of about 7 to about 2 to about 1.

[0049] The term "therapeutic agent" or "drug" as used herein refers to chemical moieties used in a variety of therapeutic, including pharmaceutical applications.

[0050] 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, for example a human that has been or will be the object of treatment, observation or experiment.

[0051] The term "therapeutically effective amount" or "effective amount" refers to that amount of a compound disclosed and / or described herein that is sufficient to affect treatment, as defined herein, when administered to a subject in need of such treatment. The therapeutically effective amount will vary depending upon, for example, the subject and disease condition being treated, the weight and age of the subject, the severity' of the disease condition, the particular compound, the dosing regimen to be followed, timing of administration, the manner of administration, all of which can readily be determined by one of ordinary skill in the art. The therapeutically effective amount may be ascertained experimentally, for example by assaying blood concentration of the chemical entity, or theoretically, by calculating bioavailability.

[0052] Patient doses for administration of either or both the liposome and the SR-BI inhibitor ty pically range from about 1 mg / day to about 10,000 mg / day, more ty pically from about 10 mg / day after adding suitable auxiliaries, if desired, to obtain tablets to about 1,000 mg / day, and most typically from about 50 to about 500 mg / day. Stated in terms of patient body, ty pical dosages can range from about 0.01 to about 150 mg / kg / day, more ty pically from about 0.1 to about 15 mg / kg / day, and most typically from about 1 to about 10 mg / kg / day, for example 5 mg / kg / day or 3 mg / kg / day. Adjusting the dosages to obtain a desired ratio of SR- BI inhibitor and liposome is within the skill of the ordinarily skilled clinician or researcher.

[0053] With respect to the inhibitor of SR-BI, an exemplary “therapeutically effective amount” is an amount that increases the uptake of the liposome by the target cell to w hich the liposome and inhibitor are administered.

[0054] With respect to the ENT inhibitor, an exemplary “therapeutically effect amount” is an amount that enhances uptake of liposome-encapsulated cytarabine by the target cell relative to the uptake of unencapsulated cytarabine in the presence of the ENT inhibitor. The patient doses for these agents, in an exemplary embodiment, are similar to those for the SR-BI inhibitor.

[0055] “Target cell”, as used herein, refers to a cell to which a liposome and an inhibitor are administered, and which uptakes the liposome. In exemplary embodiments, SR-BI and / orENT are administered. An exemplary' target cell expresses SR-BI and / or ENT. An exemplary target cell is a diseased cell exhibiting the genotype and / or phenotype of the disease. An exemplary diseased cell over- or under-expresses SR-BI and / or ENT. A target cell can also be a “normal” cell not exhibiting neither the genotype nor the phenotype of the disease. In exemplary7embodiments, in which the degree of cellular uptake of the liposome by a target cell is enhanced by administering an inhibitor of SR-BI, the normal target cell is used as reference point for comparison to determine if cellular uptake is greater or lesser in the diseased cell. Such comparisons are of use in determining whether a subject is an appropriate candidate for combination therapy with a liposome and an inhibitor of SR-BI and / or ENT, and / or an appropriate dosage level of either or both of these agents for the candidate.

[0056] “Enhance(d) uptake by' a target cell”, as used herein with reference to a target cell to which an inhibitor of SR-BI is administered, refers to an effect on a target cell from administering a selected dosage of an inhibitor of SR-BI in which more of a liposome is uptaken by the target cell than is observed with an identical cell and an identical liposome in the absence of administering the inhibitor of SR-BI.

[0057] “Enhance(d) uptake by a target cell”, as used herein with reference to a target cell to which an ENT inhibitor is administered, refers to an effect on a target cell from administering a selected dosage of an ENT inhibitor in which more liposome-encapsulated cytarabine is uptaken by' the target cell than is observed with an identical cell and unencapsulated cytarabine in the presence of the ENT inhibitor.

[0058] "Treatment" (and related terms, such as "treat", "treated", "treating") includes one or more of: preventing a disease or disorder (i.e., causing the clinical symptoms of the disease or disorder not to develop); inhibiting a disease or disorder; slowing or arresting the development of clinical symptoms of a disease or disorder; and / or relieving a disease or disorder (i.e.. causing relief from or regression of clinical symptoms). The term encompasses situations where the disease or disorder is already being experienced by a subject, as well as situations where the disease or disorder is not currently being experienced but is expected to arise. The term covers both complete and partial reduction or prevention of the condition or disorder, and complete or partial reduction of clinical symptoms of a disease or disorder. Thus, compounds described and / or disclosed herein may prevent an existing disease or disorder from worsening, assist in the management of the disease or disorder, or reduce or eliminate the disease or disorder. When used in a prophylactic manner, the compoundsdisclosed and / or described herein may prevent a disease or disorder from developing or lessen the extent of a disease or disorder that may develop.

[0059] “SR-BI” refers to Scavenger Receptor B, type 1, which mediates the transfer of lipids between high density lipoproteins (HDL) and cells.

[0060] ■‘Inhibitor of SR-BI” refers to any member of a class of agents that function to inhibit SR-BI mediated cholesterol uptake by a cell to which the inhibitor is administered, as exemplified by BLT-1.

[0061] '‘BLT-1” refers to Block Lipid Transport-1, a thiosemicarbazone copper chelator, which is a SR-BI inhibitor.

[0062] “ENT” refers to Equilibrative Nucleoside Transporter, such as ENTL ENT2, ENT3, and ENT4, which are polytopic integral membrane proteins that mediate physiologic nucleoside transport across cellular membranes.

[0063] “ENT inhibitor” refers to any member of a class of agents that function to inhibit ENT mediated nucleoside transport across a cell membrane of a cell to which the inhibitor is administered, as exemplified by NBMPR.III. EmbodimentsA. Methods

[0064] It has now been discovered that a liposome encapsulating one or more therapeutic agents administered with an inhibitor of Scavenger Receptor BI (SR-BI) to a target cell expressing SR-BI, both in a therapeutically useful dosage, results in a significant increase in uptake of the liposome by the target cell relative to the uptake seen in an identical target cell without administration of the inhibitor. Thus, in an exemplary embodiment, there is provided a method of increasing the cellular uptake of a liposome encapsulating one or more therapeutic agents in a target cell expressing SR-BI. The method includes administering theliposome to the cell and an amount of the inhibitor of SR-BI sufficient to increase uptake by the target cell of the liposome (e.g., "a therapeutically effective amounf ’).

[0065] Exemplary inhibitors of SR-BI, including BLT-1, are known in the art, as are methods for assaying their activity' towards cells expressing this target, and methods for preparing and testing new inhibitors. See, e.g., U.S. Pat. No. 9,994,851.

[0066] In an exemplary embodiment, the inhibitor of SR-BI is effective at increasing the uptake of the liposome by the target cell at a first dosage of the inhibitor. An exemplar}' first dosage is higher than a lower second dosage at which the cell shows no increase in uptake, an uptake less than that seen at the first dosage, or a lower degree of uptake than the target cell without administration of the inhibitor of SR-BI.

[0067] In an exemplary embodiment, the target cell is an abnormal cell (e.g., diseased) and underexpresses or overexpresses SR-BI relative to an identical normal cell. The formulations of the instant invention leverage this over-, under-expression to effect enhanced delivery of the liposome to the cell.

[0068] In an exemplary embodiment, the liposome comprises at least 1 mol % distearoyl phosphatidylglycerol (DSPG) or distearoyl phosphatidylinositol (DSPI).

[0069] In an exemplary embodiment, the liposome comprises about 20 mol % or less of cholesterol.

[0070] In an exemplary embodiment, the liposome comprises distearoylphosphatidykholine (DSPC).

[0071] In an exemplary embodiment, the liposome comprises DSPC, DSPG and Cholesterol. An exemplary ratio of DSPC / DSPG / Cholesterol is about a 7:2: 1 molar ratio.

[0072] In various embodiments, there is provided a method of treating a disease in a subject in need of such treatment. The method includes administering to the subject a therapeutically effective amount of a liposome encapsulating one or more therapeutic agents effective in treating the disease, ameliorating the symptoms of the disease, etc., and an amount of an inhibitor of SR-BI selected such that the uptake of the liposome by a target cell of the subjectis increased relative to the cellular uptake of the liposome by the same target cell in the absence of the inhibitor of SR-BI.

[0073] In an exemplary embodiment, the disease is a proliferative disease, e.g., cancer. In various embodiments, the disease is a hematological disease, e.g., a hematological proliferative disorder. In an exemplary embodiment, the disease is leukemia, e.g., acute myeloid leukemia (AML).

[0074] Also provided are methods of altering the side effect profile of a liposomal formulation in which one or more therapeutic agents is encapsulated in the liposome, e.g., CPX-351, by enhancing delivery of the formulation to the target cells. The uptake of the liposome by a target cell is enhanced by administering, in addition to the liposome, an inhibitor of SR-BI. The inhibitor is administered in an amount selected such that the uptake of the liposome by a target cell of the subject is increased relative to the cellular uptake of the liposome by the same target cell in the absence of the inhibitor. An exemplary alteration of the side effect profile includes reducing the incidence or severity of one or more adverse events associated with administration of a therapeutically effective amount of the liposome. An exemplary7alteration of the side effect profile includes a diminishment or amelioration of at least one undesirable side effect of the therapeutic agent on the subject to whom it is administered.

[0075] An exemplary' method of the invention is useful to reduce cellular resistance to one or more liposome encapsulated therapeutic agents, e.g., cytarabine, daunorubicin and combinations thereof. The method comprises administering to a drug resistant target cell an inhibitor of SR-BI in an amount selected such that the cellular uptake of the liposome encapsulated therapeutic agent(s) by the target cell is increased relative to the cellular uptake of the liposome encapsulated agent(s) by the same target cell in the absence of the inhibitor of SR-BI. or the cellular efflux of the liposome encapsulated therapeutic agent(s) (or deencapsulated therapeutic agent(s)) is less than the efflux of the agent(s) relative to the same target cell in the absence of the inhibitor of SR-BI.

[0076] In various embodiments, there is provided a method of combination therapy of a proliferative disorder, e g., cancer, e.g., leukemia, e.g., acute myeloid leukemia. The method comprises administering to a subject in need thereof a therapeutically effective amount of a liposome encapsulating one or more therapeutic agents useful in treating the proliferativedisease and a therapeutically effective amount of an inhibitor of BLT-1. The liposome and inhibitor are administered substantially simultaneously or sequentially in either order (i.e. , inhibitor followed by liposome, or liposome followed by inhibitor). In various embodiments, the liposome and the inhibitor of SR-BI are administered sequentially in separate pharmaceutical formulations or combined into a single pharmaceutical formulation and administered in this format.

[0077] In an exemplary embodiment, a method of this disclosure utilizes a liposome encapsulating one or more cancer chemotherapeutic agents. In various embodiments, the chemotherapeutic agents are cytarabine and daunorubicin. In some embodiments, the cytarabine and daunorubicin are encapsulated in the liposome in a 5: 1 ratio and the liposome comprises DSPC / DSPG / Cholesterol at about a 7:2: 1 ratio. An exemplary liposome is CPX- 351.

[0078] In an exemplary embodiment, there is provided a method of enhancing delivery of CPX-351 into an intracellular compartment of a target cell. The method comprises administering to a subject in need thereof a therapeutically effective amount of CPX-351 and a therapeutically effective amount of a SR-BI inhibitor (e.g., BLT-1). The therapeutic agents are administered sequentially in separate pharmaceutical formulations or combined into a single pharmaceutical formulation and administered in this format. The amount of BLT-1 administered is effective to enhance uptake of CPX-351 by the cell, providing enhanced delivery' of CPX-351 to this compartment. In this embodiment, the delivery of the liposome to the intracellular compartment of the target cell is enhanced relative to the delivery to the same compartment of the same target cell in the absence of the inhibitor.

[0079] In an exemplary' embodiment, there is provided a method of altering the side effect profile of at least one therapeutic agent encapsulated in a liposome. The method comprises administering to a subject in need thereof a therapeutically effective amount of the liposome and a therapeutically effective amount of SR-BI inhibitor. The amount of the SR-BI inhibitor administered is effective to enhance uptake of the liposome by a target cell, providing enhanced delivery of the liposome to the target cell, thereby decreasing the amount ofliposome delivered to a non-target cell and altering the side effect profile of the at least one therapeutic agent.

[0080] In various embodiments, the at least one therapeutic agent is selected from cytarabine, daunorubicin and a combination thereof. In an exemplary embodiment, the inhibitor of SR-BI is BLT-1.

[0081] In certain embodiments, there is provided a method of determining whether administering a liposome encapsulating at least one therapeutic agent is an appropriate therapy for a selected subject in need of treatment with the at least one therapeutic agent. The method comprises determining whether the target cell of the subject has an expression level of SR-BI greater or less than a pre-selected SR-BI expression level threshold; and if the SR- BI expression level is above this threshold identifying the subject as a candidate for therapy with the liposome.

[0082] In various embodiments, the method above identifies a candidate for combination therapy with the liposome and an inhibitor of SR-BI. In various embodiments, the expression of SR-BI in the target cell is below the predetermined threshold. In various embodiments, the liposome comprises at least 1 mol % distearoyl phosphatidylglycerol (DSPG) or distearoyl phosphatidylinositol (DSPI). In various embodiments, the liposome comprises about 20 mol % or less of cholesterol. In various embodiments, the liposome comprises distearoylphosphatidylcholine (DSPC). In various embodiments, the liposome comprises DSPC, DSPG and Cholesterol. In various embodiments, the liposome comprises DSPC. DSPG and Cholesterol at a molar ratio of about 7:2: 1. In various embodiments, the liposome is CPX-351. In various embodiments, the inhibitor of SR-BI is BLT-1.

[0083] In various embodiments, the method further comprises determining an appropriate dosage level for a liposome based on determining the expression level of SR-BI in a target cell of the subject to which the liposome is to be administered. For example, those subjects expressing a high level of SR-BI on the target cell may require a lower dosage of the liposome than those with a lower SR-BI expression on the principle that the cellular uptake of the liposome will be greater in those target cells expressing more SR-BI. In variousembodiments, the dosage level is assessed taking into account the administration of combination therapy for the subject using a liposome and a SR-BI inhibitor.

[0084] In various embodiments, the liposome is CPX-351. In various embodiments, the inhibitor of SR-BI is BLT-1.

[0085] In an exemplary embodiment, a combination of a liposome comprising cytarabine and an equilibrative nucleoside transporter (ENT) (e.g., ENT1) inhibitor in therapeutically useful dosages results in a significant increase in uptake of liposome-encapsulated cytarabine by cells expressing ENT (e.g., ENT1) relative to the uptake of unencapsulated cytarabine in the presence of the ENT inhibitor seen in an identical cell (e.g.. “a therapeutically effective amount”).

[0086] In various embodiments, the disclosure sets forth a method of combination therapy of a disease in a subject in need of such treatment. The method includes administering to a subject in need thereof a therapeutically effective amount of a liposome comprising cytarabine and an amount of an ENT inhibitor effective to enhance uptake of liposome- encapsulated cytarabine by a target cell exhibiting the genotype and / or phenotype associated with the proliferative disease relative to the uptake of unencapsulated cytarabine in the presence of the ENT inhibitor by an identical cell.

[0087] In an exemplary' embodiment, the disease is a proliferative disease, e.g., cancer. In various embodiments, the disease is a hematological disease, e.g., a hematological proliferative disorder. In an exemplary embodiment, the disease is leukemia, e.g., acute myeloid leukemia (AML).

[0088] In an exemplary embodiment, the disclosure sets forth a method of enhancing delivery of a therapeutic agent into an intracellular compartment of a target cell in the presence of an ENT inhibitor. The method comprises administering to a subject in need thereof a therapeutically effective amount of a liposome comprising the therapeutic agent and a therapeutically effective amount of the ENT inhibitor, wherein uptake of the therapeutic agent by the target cell is enhanced relative to the uptake of unencapsulated therapeutic agentin the presence of the ENT inhibitor by an identical cell, thereby providing enhanced deliver)' of the therapeutic agent to the intracellular compartment.

[0089] In an exemplary embodiment, the disclosure sets forth a method of enhancing deliver^' of cytarabine into an intracellular compartment of a target cell in the presence of an ENT inhibitor, the method comprising administering to a subject in need thereof a therapeutically effective amount of a liposome comprising cytarabine and a therapeutically effective amount of the ENT inhibitor, wherein uptake of liposome-encapsulated cytarabine by the target cell is enhanced relative to the uptake of unencapsulated cytarabine in the presence of the ENT inhibitor by an identical cell, thereby providing enhanced deliver)' of the cytarabine to the intracellular compartment.

[0090] In exemplar)' embodiments, as with the SRBI, the method of the invention utilizing the ENT inhibitor alters the side effect profile of the therapeutic agent. An exemplary alteration of the side effect profile includes a diminishment or amelioration of at least one undesirable side effect of the therapeutic agent on the subject to whom it is administered. In an exemplar ’ embodiment, the enhanced deliver ’ to the target cell leads to less off target deliver)' than when the inhibitor is not present.

[0091] In some embodiments, the liposome further comprises daunorubicin. In an exemplary embodiment, liposome comprises CPX-351.

[0092] In some embodiments, the target cell is a blood cell. In some embodiments, the target cell is a leukemia cell.

[0093] In an exemplar)' embodiment, the target cell is an abnormal cell (e.g., diseased) and underexpresses or overexpresses ENT relative to an identical normal cell. The formulationsof the instant invention leverage this over-, under-expression to effect enhanced delivery of the liposome to the cell.

[0094] In an exemplary embodiment, the ENT inhibitor is NBMPR.

[0095] In some embodiments, the liposome and the ENT inhibitor are administered sequentially in separate pharmaceutical formulations.

[0096] In some embodiments, the liposome and the ENT inhibitor are combined into a single pharmaceutical formulation and administered to the subject in this format.

[0097] In an exemplary embodiment, the liposome comprises at least 1 mol % distearoyl phosphatidylglycerol (DSPG) or distearoyl phosphatidylinositol (DSPI).

[0098] In an exemplary embodiment, the liposome comprises about 20 mol % or less of cholesterol.

[0099] In an exemplary embodiment, the liposome comprises distearoylphosphatidylcholine (DSPC).

[0100] In an exemplary' embodiment, the liposome comprises DSPC, DSPG and Cholesterol. An exemplary ratio of DSPC / DSPG / Cholesterol is about a 7:2: 1 molar ratio.

[0101] In an exemplary embodiment, there is provided a method of treating acute myeloid leukemia in a subject in need of such treatment, wherein the subject is at least about 60 years old. The method comprises administering a therapeutically relevant dose of CPX-351 to the subject and an inhibitor of SR-BI. Administration of CPX-351 in this patient population is correlated with lower cardiotoxicity than administration of standard 7+3 therapy. In an exemplary' embodiment, the SR-BI inhibitor is BLT-1.B. Compositions

[0102] The present disclosure provides new combination formulations of a liposome encapsulating one or more therapeutic agents and an inhibitor (e.g., an inhibitor of SR-BI and / or ENT).

[0103] In one embodiment, there is provided a pharmaceutical formulation comprising therapeutically7effective amounts of a liposome including at least one encapsulatedtherapeutic agent, and an inhibitor (e.g., an inhibitor of SR-BI and / or ENT). The liposome and inhibitor are in a pharmaceutically acceptable carrier, diluent, etc.

[0104] In various embodiments, the pharmaceutical formulation includes a liposome encapsulating one or more therapeutic agents and a SR-BI inhibitor selected such that the cellular uptake of the liposome by a target cell is increased relative to the cellular uptake of the liposome by the same target cell in the absence of the inhibitor of SR-BI. In an exemplary embodiment, the presence of the inhibitor in the formulation reduces delivery to non-target cells.

[0105] In an exemplary embodiment, the liposome comprises a first lipid and a first sterol.

[0106] In some embodiments, the first lipid is a polymer-conjugated lipid. In some embodiments, the polymer-conjugated lipid is a PEG-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 l,2-dimyristoyl-rac-glycero-3-methoxypoly(ethylene glycol). In some embodiments, the polymer-conjugated lipid is l,2-distearoyl-rac-glycero-3- methoxy poly (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 a phosphatidylcholine, a sphingolipid, and a hydrogenated sphingolipid. In some embodiments, the second lipid is sphingomyelin. In some embodiments, the second lipid is distearoyl phosphatidyl choline (DSPC). hydrogenated sphingomyelin (dihydrosphingomyelin), or egg sphingomyelin. In some embodiments, the second lipid is distearoyl phosphatidyl choline (DSPC) or hydrogenated sphingomyelin (dihydrosphingomyelin). In some embodiments, the second lipid is distearoyl phosphatidyl choline (DSPC). In some embodiments, the second lipid is hydrogenated sphingomyelin (dihydrosphingomyelin). In some embodiments, the second lipid ishydrogenated soybean phosphatidyl choline (HSPC). In some embodiments, the second lipid is egg sphingomyelin.

[0107] In some embodiments, the first lipid is a first phospholipid. In some embodiments, the phospholipid is selected from the group consisting of a phosphatidylcholine, a sphingolipid, and a hydrogenated sphingolipid. In some embodiments, the first lipid is distearoyl phosphatidyl choline (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 a phosphatidylcholine, a sphingolipid, and a hydrogenated sphingolipid. In some embodiments, the second lipid is distearoyl phosphatidyl glycerol (DSPG). In some embodiments, the second lipid is distearoyl phosphatidyl inositol (DSPI). In some embodiments, the second lipid is sphingomyelin. In some embodiments, the second lipid is hydrogenated sphingomyelin (dihydrosphingomyelin).

[0108] In some embodiments, the first sterol is cholesterol, a cholesterol derivative, or a phytosterol (such as beta-sitosterol). In some embodiments, the first sterol is cholesterol. In some embodiments, the first sterol is beta-sitosterol.

[0109] In some embodiments, the lipid bilayer comprises dihydrosphingomyelin (DHSM), cholesterol, and DSG-PEG2000. In some embodiments, the lipid bilayer comprises DHSM / cholesterol / DSG-PEG2000 at a molar ratio of about 53:45:2. In some embodiments, the lipid bilayer comprises distearoyl phosphatidyl choline (DSPC), cholesterol, and DSG- PEG2000. In some embodiments, the lipid bilayer comprises DSPC / cholesterol / DSG- PEG2000 at a molar ratio of about 57:38:5.6.

[0110] In some embodiments, the liposome comprises about 20 mol% or less of cholesterol.

[0111] In some embodiments, the liposome comprises at least about 1 mol % DSPG or DSPI.

[0112] In some embodiments, the liposome comprises distearoylphosphatidylcholine, distearoylphosphatidylglycerol, and cholesterol. In some exemplary embodiments, theliposome comprises distearoylphosphatidylcholine, distearoylphosphatidylglycerol, and cholesterol at about a 7:2: 1 molar ratio.

[0113] In some embodiments, the liposome has a mean diameter between about 50 nm and about 250 nm. In some embodiments, the liposome has a mean diameter between about 50 nm and about 150 nm. In some embodiments, the liposome has a mean diameter between about 50 nm and about 120 nm. In some embodiments, the liposome has a mean diameter between about 50 nm and about 100 nm. In some embodiments, the liposome has a mean diameter of about 80 nm. In some embodiments, the liposome has a mean diameter between about 80 nm and about 150 nm. In some embodiments, the liposome has a mean diameter of about 120 nm.

[0114] In some embodiments, the liposomal composition has a poly dispersity7index (PDI) between about 0.001 and about 0.5. In some embodiments, the liposomal composition has a poly dispersity index (PDI) between about 0.001 and about 0.3. In some embodiments, the liposomal composition has a poly dispersity index (PDI) between about 0.001 and about 0.1. In some embodiments, the liposomal composition has a poly dispersity index (PDI) between about 0.1 and about 0.3. In some embodiments, the liposomal composition has a poly dispersity index (PDI) between about 0.1 and about 0.5. In some embodiments, the liposomal composition has a poly dispersity index (PDI) between about 0.3 and about 0.5.

[0115] In some embodiments, the liposomal composition further comprises a carrier medium. In some embodiments, the one or more liposomes are suspended in a earner medium. In some embodiments, the carrier medium is a pharmaceutically acceptable solution. In some embodiments, the carrier medium is an aqueous dextrose solution. In some embodiments, the carrier medium is an aqueous sucrose solution. In some embodiments, the carrier medium is a saline solution. In some embodiments, the carrier 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 buff er. In some embodiments, the buffer is a MES buffer.

[0116] Pharmaceutical compositions comprising delivery vehicles of the invention are prepared according to standard techniques and may comprise water, buffered water, 0.9% saline, 0.3% glycine, 5% dextrose and the like, alone or in combination, including glycoproteins for enhanced stability, such as albumin, lipoprotein, globulin, and the like.These compositions may be sterilized by conventional, w ell-known sterilization techniques. The resulting aqueous solutions may be packaged for use or fdtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, and the like. Additionally, the delivery vehicle suspension may include lipid-protective agents which protect lipids against free-radical and lipid-peroxidative damages on storage. Lipophilic free-radical quenchers, such as alpha-tocopherol and water-soluble iron-specific chelators, such as ferrioxamine. are suitable.

[0117] The concentration of delivery vehicles in the pharmaceutical formulations can vary widely, such as from less than about 0.05%, usually at or at least about 2-5% to as much as 10 to 30% by weight and will be selected primarily by fluid volumes, viscosities, and the like, in accordance with the particular mode of administration selected. For example, the concentration may be increased to lower the fluid load associated with treatment. Alternatively, delivery vehicles composed of irritating lipids may be diluted to low concentrations to lessen inflammation at the site of administration. For diagnosis, the amount of delivery vehicles administered will depend upon the particular label used, the disease state being diagnosed and the judgment of the clinician.

[0118] In an exemplary embodiment, the pharmaceutical compositions of the present invention are administered intravenously. Dosage for the delivery vehicle formulations will depend on the ratio of drug to lipid and the administrating physician's opinion based on age, weight, and condition of the patient.

[0119] In the instance where a single composition containing more than one active agent is included, the above procedures are followed per se. Where the agents are administered in separate delivery' vehicle compositions, the administration should be timed in such a mannerthat the desired ratio is maintained. Typically, this is accomplished by simultaneously administering the compositions in the calculated proportions.

[0120] In some embodiments, the liposomal composition further comprises a therapeutic agent external to the one or more liposomes. In an exemplary embodiment, the inhibitor of SR-BI and / or ENT is external to the liposome.

[0121] Liposomes can be prepared as described in "Liposomes: Rational Design" (A.S. Janoff ed., Marcel Dekker, Inc., New York, NY), or by additional techniques known to those knowledgeable in the art.

[0122] Compositions of the present invention may be administered to warm-blooded animals, including humans as well as to domestic and / or avian species. In addition to pharmaceutical compositions, suitable formulations for veterinary use may be prepared and administered in a manner suitable to the subject. For treatment of human ailments, a qualified physician will determine how the compositions of the present invention should be utilized with respect to dose, schedule and route of administration using established protocols. Such applications may also utilize dose escalation should agents encapsulated in delivery' vehicle compositions of the present invention exhibit reduced toxicity to healthy tissues of the subject.

[0123] Preferably, the pharmaceutical compositions of the present invention are administered parenterally, i.e., intraarterially, intravenously, intraperitoneally, subcutaneously, or intramuscularly. More preferably, the pharmaceutical compositions are administered intravenously or intraperitoneally by a bolus injection. For example, see Rahman, et al., U.S. patent No. 3,993,754; Sears, U.S. patent No. 4,145,410; Papahadjopoulos, et al., U.S. patentNo. 4,235,871; Schneider, U.S. patent No. 4,224,179; Lenk, et al., U.S. patent No. 4,522,803; and Fountain, et al., U.S. patent No. 4.588,578.

[0124] In an exemplary embodiment, the formulation comprises a therapeutically effective amount of CPX-351 and a therapeutically effective amount of an inhibitor (e.g., an inhibitor of SR-BI and / or ENT).

[0125] In an exemplary embodiment, the formulation includes the SR-BI inhibitor Block Lipid Transport-1 (BLT-1) and a liposome containing at least one encapsulated therapeutic agent.

[0126] In an exemplary embodiment, the formulation comprises a combination of CPX-351 and Block Lipid Transport- 1 in a pharmaceutically acceptable carrier, diluent, etc.

[0127] In various embodiments, the formulation includes NBMPR and a liposome comprising at least one encapsulated therapeutic agent. In an exemplary embodiment, the formulation includes NBMPR and a liposome comprising cytarabine.

[0128] In an exemplary embodiment, the formulation comprises a combination of CPX-351 and NBMPR in a pharmaceutically acceptable carrier, diluent, etc.

[0129] In an exemplary embodiment, there is a provided a kit comprising a first vessel containing a therapeutically effective amount of a liposome comprising at least one therapeutic agent encapsulated therein, and a second vessel comprising a therapeutically effective amount of an inhibitor of SR-BI. The kit further comprises instructions for the clinician for administering the liposome and SR-BI inhibitor to a subject in need of treatment with these agents. The instructions optionally detail a procedure to prepare the liposome and / or inhibitor of SR-BI, in a therapeutically effective amount, prior to administering the combination to the subject.

[0130] In various embodiments, there is a provided a kit. An exemplary kit comprises a vessel comprising a therapeutically effective amount of a liposome comprising at least one therapeutic agent encapsulated therein, and a second vessel comprising a therapeutically effective amount of an ENT inhibitor, the kit further comprising instructions for the clinician for administering the liposome and the ENT inhibitor. In an exemplary embodiment, there is provided a vessel containing a therapeutically effective amount of a liposome comprisingcytarabine, and a second vessel comprising a therapeutically effective amount of an ENT inhibitor, the kit further comprising instructions for the clinician for administering the liposome and the ENT inhibitor. The instructions optionally detail a procedure to prepare the liposome and / or ENT inhibitor, in a therapeutically effective amount, prior to administering the combination to the subject.

[0131] The therapeutic agents in the compositions may be formulated separately in individual compositions wherein each therapeutic agent is stably associated with appropriate delivery vehicles. These compositions can be administered separately to subjects. In an exemplary' embodiment, the pharmacokinetics of the compositions are coordinated so that the ratio of therapeutic agents administered is maintained at the target for treatment. Thus, it is useful to construct kits which include, in separate containers, a first composition comprising delivery vehicles stably associated with at least a first therapeutic agent and, in a second container, a second composition comprising delivery vehicles stably associated with at least one second therapeutic agent. The containers can then be packaged into the kit. The kit also includes instructions as to the mode of administration of the compositions to a subject, at least including a description of the ratio of amounts of each composition to be administered. Alternatively, or in addition, the kit is constructed so that the amounts of compositions in each container is pre-measured so that the contents of one container in combination with the contents of the other represent the correct ratio. Alternatively, or in addition, the containers may be marked with a measuring scale permitting dispensation of appropriate amounts according to the scales visible. The containers may themselves be useable in administration; for example, the kit might contain the appropriate amounts of each composition in separate syringes. Formulations which comprise the pre-formulated correct ratio of therapeutic agents may also be packaged in this way so that the formulation is administered directly from a syringe prepackaged in the kit.

[0132] The present disclosure sets forth new methods, compositions and kits. Supporting these various embodiments is the discovery that an exemplary inhibitor of SR-BI, Block Lipid Transport-1, in a therapeutically effective dose (HD-BLT-1) administered in conjunction with a liposome encapsulating one or more therapeutic agents results in a significant increase in uptake of the liposome, and hence the therapeutic agent(s) by cells expressing Scavenger Receptor Bl (SR-BI). This result is surprising in view of the fact thatadministration of BLT-1 at lower dosages (LD-BLT-1) induces the opposite effect, decreasing cellular uptake of the liposome.

[0133] Compositions and methods of the invention are exemplified by the combination of the liposome CPX-351, a liposome encapsulating cytarabine and daunorubicin, and the SR-BI inhibitor BLT-1. As set forth herein, the methods, compositions and principles of the instant disclosure are, however, not limited to this exemplary embodiment.

[0134] The present disclosure also provides compositions and methods exemplified by the combination of the liposome CPX-351, a liposome encapsulating cytarabine and daunorubicin, and the ENT inhibitor NBMPR. As set forth herein, the methods, compositions and principles of the instant disclosure are, however, not limited to this exemplary embodiment.

[0135] The present disclosure also provides a method of treating a selected patient population with AML, with CPX-351, resulting in therapeutic efficacy with lower cardiotoxicity than would otherwise be expected. In an exemplary embodiment, the patient population includes subject over the age of 60.

[0136] Combination of cytarabine and daunorubicin is the standard induction chemotherapy in patients treated with AML. The clinical efficacy of the combination, how ever, has been limited in the older population. The clearance and metabolism of the two drugs are different, such that when the free drugs are co-administered, the molar ratio at the pharmacologic site of action will vary over time, resulting in intracellular molar ratios that may be additive, synergistic or antagonistic. CPX-351 is a liposomal formulation encapsulating cytarabine and daunorubicin at a maximally synergistic ratio (5: 1 molar ratio), which greatly increases the systemic half-life of the encapsulated drugs as well as the accumulation of the two drugs into leukemia cells at the optimal ratio. CPX-351 received FDA approval for the treatment of adults with newly diagnosed therapy -related AML and AML with myelodysplasia-related changes.

[0137] In a further exemplary embodiment, there is provided a method of treating acute myeloid leukemia in a subject in need of such treatment, wherein the subject is at least about 60 years old. The method comprises administering a therapeutically relevant dose of CPX- 351 to the subject. In an exemplary' embodiment, markers of cardiotoxicity due to CPX-351are lower in patients falling into this age group vs. these markers in patients in this age group to whom standard 7+3 therapy is administered.

[0138] The following Examples are offered to illustrate exemplar} embodiments of the invention and do not define or limit its scope.EXAMPLESEXAMPLE 11.1 Materials and Methods

[0139] Cell culture and reagents. HL-60 human promyelocytic leukemia cells (ATCC, VA, USA: CCL-240) was cultured in RPMI-1640 media with 20% fetal bovine serum (FBS, Cat# 173012; NICHIREI BIOSCIENCES INC, Tokyo, Japan) and maintained in a 5% CO2- humidified atmosphere at 37°C. K562 chronic myelogenous leukemia cells (JCRB Cell Bank, Osaka, Japan: JCRB0019), K562 / ADM adriamycin resistant derivative of K562 cell line due to expressing P-gly coprotein (JCRB Cell Bank: JCRB 1002), THP-1 human acute monocytic leukemia cells (JCRB Cell Bank: JCRB01 12.1), and HL-60 / AD cytarabine and daunorubicin resistant derivative of HL-60 cell line due to expressing multidrug resistance-associated protein (MRP) established in our laboratory25, were cultured in RPMI-1640 media with 10% FBS and maintained in a 5% CO2-humidified atmosphere at 37°C. CPX-351 was kindly provided by Jazz Pharmaceuticals (Oxford, UK). Cytarabine and daunorubicin were purchased from Selleck Chemicals (Houston, TX, USA).

[0140] CPX-351 handling. The formulation of CPX-351 liposome for injection was a sterile, pyrogen-free, purple, lyophilized product provided in 50 mL vials, each containing 100 units where 1 unit is equivalent to 1.0 mg of cytarabine and 0.44 mg of daunorubicin (as base). The product was reconstituted with 19 mL of ultrapure water and gently swirled for 10 minutes at room temperature. Working aliquots of the reconstituted product were stored frozen at -20°C.

[0141] Protein corona analysis. CPX-351 was mixed with FBS in a 1: 1 volume ratio and incubated at room temperature for 90 minutes. Following incubation, the samples were centrifuged for 15 minutes at 14,000 rpm, followed by resuspension of the pellet in phosphate-buffered saline (PBS). This procedure was repeated three times to wash the sample and remove unbound proteins.26 The protein pellets were then subjected to tryptic digestionusing an In-Solution Tryptic Digestion and Guanidination Kit (Thermo Fisher Scientific, Bremen, Germany) according to the manufacturer's instructions. Liquid chromatography - tandem mass spectrometry (LC-MS / MS) analysis was performed on an Orbitrap Elite hybrid ion trap-Orbitrap mass spectrometer (Thermo Fisher Scientific) equipped with a nanoelectrospray ion source. Raw data files were analyzed using Proteome Discover (version 1.4.0.288; Thermo Fisher Scientific) and searched against the UniProt database (UniProtKB 2022_01 results). A well-established label-free technique was used to determine the absolute concentration of proteins via the utilization of the average MS signal response of the most optimal two or three ionizing peptides to a given protein. (Silva JC, Gorenstein MV, Li GZ, Vissers JP, Geromanos SJ. Absolute quantification of proteins by LCMSE: a virtue of parallel MS acquisition. Mol Cell Proteomics. 2006:5(1): 144-156.).

[0142] Flow cytometry for cell membrane lipid receptor expression. Lipid receptor expression in each leukemia cell lines was measured using a BD FACS Canto II cytometer (BD Bioscience, Heidelberg. Germany) and analyzed with FlowJo software (BD Biosciences). Cells were re-suspended to a density of 1 x 106 cells / mL in 100 pL of PBS containing 0.5% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO, USA). For scavenger receptor class B type 1 (SR-BI) and low -density7lipoprotein receptor-related protein 1 (LRP1) analysis, APC-conjugated SR-BI antibody (Miltenyi Biotec, Bergisch Gladbach, Germany), APC-conjugated LRP1 antibody (Miltenyi Biotec), or human IgGl isotype control antibody (Miltenyi Biotec) was added to each suspension and incubated for 10 minutes in the dark at 2-8°C according to the manufacturer's instructions. For low-density7lipoprotein receptor (LDLR) analysis, PE-conjugated anti-LDLR antibody (C7 clone; Novus Biologicals, Littleton, CO. USA) or mouse IgG2b isotype control (Novus Biologicals) was added to each suspension and incubated for 30 minutes in the dark at room temperature according to the manufacturer's instructions. After incubation, cells w ere w ashed with PBS containing 0.5% BSA and analyzed by BD FACS Canto II. The protein-positive percentage was determined by the median fluorescence intensity of gated leukemic cells, normalized by the corresponding isotype controls, and expressed in arbitrary units (AU) ± standard error (SE).

[0143] Flow cytometry for cellular uptake of CPX-351. Uptake of CPX-351 into each leukemia cell lines w as measured using flow cytometry, utilizing the fluorescence of free daunorubicin. Cells were resuspended to a density7of 1 x 106 cells / mL in 1 mL of FBS alone.Then, CPX-351 was added to these cells to a final concentration of 19.5 pM in daunorubicin equivalents. After two hours of incubation (37 °C. 5% CO2), cells were washed with PBS and analyzed by BD FACS Canto II at an excitation wavelength of 480 nm and an emission wavelength of 590 nm. The intracellular daunorubicin content was determined by the median fluorescence intensity of gated leukemic cells, normalized by the corresponding negative controls, and expressed in AU ± SE.27,28

[0144] Inhibition of MRP and P-glycoprotein assay. HL-60 / AD and K562 / ADM were resuspended to a density7of 1 x 106 cells / mL in RPMI-1640 media with 10% FBS. MK-571 (50 pM, Cayman Chemical, Ann Arbor, MI, USA), an MRP inhibitor, was added to HL- 60 / AD and incubated for 30 minutes (37 °C, 5% CO2).29 Tariquidar (1 pM, Selleck Chemicals, Houston, TX, USA), a P-glycoprotein inhibitor, was added to K562 / ADM and incubated for 10 minutes (37 °C, 5% CO2).30 Then, CPX-351 was added to these cells to a final concentration of 3.9 pM in daunorubicin equivalents. After four hours of incubation (37 °C. 5% CO2), cells were washed with PBS and analyzed by BD FACS Canto II using the previously described method.

[0145] Inhibition of the CPX-351 uptake pathways assay. The uptake opf CPX-351 into the leukemia cell lines was inhibited with 10 pg / mL chlorpromazine (an inhibitor of CME; TCI, Tokyo, Japan),31 25 pM 5-(N-ethyl-N-isopropyl)-Amiloride (EIPA, an inhibitor of micropinocytosis; MedChemExpress, Monmouth Junction, NJ, USA), 32 low-dose blocks lipid transport-1 (LD-BLT-1, 400 nM, a selective inhibitor of SR-BI; Merck, Darmstadt, Germany), and high-dose blocks lipid transport-1 (HD-BLT-1, 40 pM).33-35 These inhibitors were added to cells alone or in combination and incubated for 1 hour (37 °C. 5% CO2). Thereafter, CPX-351 was added to the cells, and after two hours of incubation (37 °C, 5% CO2), BD FACS Canto II analysis was conducted using the previously described methods.

[0146] Patient specimen analysis. Peripheral blood or bone marrow specimens were obtained from patients with newly diagnosed or relapsed / refractory AML. This study was conducted in accordance with the Declaration of Helsinki, and informed consent w as obtained from all patients. The protocol was approved by the Institutional Review Board ofthe University of Fukui School of Medical Sciences. The blood or bone marrow specimens were separated on a Ficoll gradient.

[0147] Proliferation assay. To assess the growth-inhibitory effects on each cell lines, the sodium 3'-(l-[(phenylamino)-carbonyl-3,4-tetrazolium])-bis(4-methoxy-6-nitro) benzene sulfonic acid hydrate (XTT) assay was conducted using the manufacturer's protocols (Roche, Indianapolis, IN, USA) with slight modifications.36 The 50% inhibitory concentration (IC50) values were determined from the growth inhibition curves generated for each treatment.

[0148] Statistical analysis. Data from at least three independent experiments were analyzed using GraphPad Prism (Version 9.3.1). Student's t-test was used to compare the two groups. One-way ANOVA w as used to compare the three or more groups. Statistical significance was determined using tw o-sided P-values of < 0.05.1.2 Results

[0149] Protein corona of CPX-351 and lipid receptor expression in leukemia cell lines.The abundant proteins that comprise the protein corona of CPX-351 identified using the average MS signal response of the three best ionizing peptides are listed in Table 1. Those identified using the average MS signal response of the two best ionizing peptides are listed in Table 2. The protein corona of CPX-351 liposomes was primarily composed of apolipoproteins, including apolipoproteins A-II (relative ratio: 47.34% ± SE: 6.7) and A-I (5.25% ± 0.52), which are ligands for SR-BI,37’38and apolipoprotein C-III (21.71% ± 1.17), which binds to LDLR and LRP1.39Table 1 The abundant proteins identified in the protein corona of CPX-351 via the utilizationTable 2

[0150] Expression of SR-BI was detected in HL-60 (26.3 AU ± 3.8) . K562 (37.2 AU ± 3.4). and THP-1 (141.3 AU ± 24.7) (Figure 1A), with THP-1 having significantly higher expression than HL-60 and K562 (p<0.05, respectively). The expression of SR-BI was significantly higher in HL-60 / AD (96.0 AU ± 15.3) than in the parental HL-60 cells (p<0.05) and equivalent in K562 / ADM (38.0 AU ± 1.7) and the parental K.562 cells (Figure 1A). LDLR was expressed in THP-1 (4.1 AU ± 0.06) but was diminished in HL-60 (1.5 AU ± 0.08) and K562 (1.2 AU ± 0.03) (Figure IB). LRP1 was expressed in THP-1 (1.7 AU ± 0.05), but not in HL-60 or K562 (Figure 1C). As a result, we planned to analyze endocytosis in leukemia cell lines with a particular focus on SR-BI, a receptor that binds apolipoprotein A, a major component of the protein corona and is expressed at varying levels in all leukemia cell lines.

[0151] The cellular uptake of CPX-351. The uptake of CPX-351 was observed in HL-60 (10.1 AU ± 0.2), K562 (8.7 AU ± 0.2), and THP-1 (13.9 AU ± 0.6) (Figure 2A), with THP-1 having significantly higher uptake compared to HL-60 and K562 (p<0.01, respectively). The uptake of CPX-351 was significantly lower in HL-60 / AD (6.6 AU ± 0.4, p<0.01 ) and K562 / ADM (2.0 AU ± 0.03, p<0.01 ) compared to the parental cells (Figure 2A). The IC50 ofCPX-351 w as significantly higher in both HL-60 / AD (7120 nM in cytarabine equivalents, p<0.01) and K562 / ADM (512.7 nM in cytarabine equivalents, p<0.01) compared to their respective parental cells (Table 3). The addition of MK-571, an MRP inhibitor, to HL-60 / AD significantly increased the intracellular CPX-351 in HL-60 / AD (9.1 AU ± 0.07) compared to the control (3.1 AU ± 0.2, p<0.01) (Figure 2B), and correspondingly decreased the IC50 in HL-60 / AD (478.3 nM in cytarabine equivalents) compared to the control (7120 nM, p<0.01) (Figure 2C). Similarly, the addition of tariquidar, an P-gly coprotein inhibitor, to K562 / ADM significantly increased the Intracellular CPX-351 in K562 / ADM (3.0 AU ± 0.1) compared to the control (1.5 AU ± 0.1, p<0.01) (Figure 2D), and correspondingly decreased the IC50 in K562 / ADM (142.2 nM in cytarabine equivalents) compared to the control (512.7 nM, p<0.01) (Figure 2E).Table 3Ara-C 475.6 [318.6 - 717.0] 2167 [1050 - 4473] 57.47 [27.73 - 96.19] 13.62 [NA - 187.9] 3863 [2342 - 6450]DNR 18.52 [7.694 - 35.64] 170.3 [100.5 - 284.8] 15.13 [9.55 - 22.13] 3807 [2856 - 5070] 29.11 [15.28 - 52.38]* Ara-C equivalent. Abbreviations: 95% CI, 95% Confidence Interval; Ara-C, cytarabine; DNR, daunorubicin, IC50, median inhibition concentration

[0152] Inhibition of the CPX-351 uptake pathways. Treatment with chlorpromazine, an inhibitor of CME, significantly reduced the uptake of CPX-351 in HL-60, K562, and THP-1 compared to control cells, with reductions of 18.5%, 50.6%, and 34.6%, respectively (p<0.01, respectively) (Figure 3A-C). Similarly, EIPA, an inhibitor of macropinocytosis, treatment significantly reduced uptake in HL-60, K562, and THP-1 compared to control cells, with reductions of 9.9%, 43.4%, and 30.9%, respectively (p<0.01, respectively) (Figure 3A-C). Additionally, treatment with LD-BLT-1 also significantly reduced uptake in HL-60, K562, and THP-1 compared to control cells, with reductions of 17.0%, 27.7%, and 24.4%, respectively (p<0.01, respectively) (Figure 3A-C). In contrast to the decrease in CPX-351 uptake observed with LD-BLT-1 treatment, the addition of HD-BLT-1 significantly enhanced the uptake in HL-60, K562, and THP-1 by more than twofold compared to control cells, with increases of 334.6%, 249.3%, and 266.8%, respectively (p<0.01, respectively) (Figure 4A- C). The enhanced uptake by the addition of HD-BLT-1 was significantly inhibited by the addition of EIPA to the cell culture containing HD-BLT-1 in HL-60, K562, and THP-1, withreductions of 30.4%, 33.4%, and 29.3%. respectively (p<0.01, respectively) (Figure 4A-C). The addition of chlorpromazine to the cell culture containing HD-BLT-1 significantly inhibited the enhanced uptake in K.562 (p<0.01), but did not significantly alter uptake in HL- 60 and THP-1, with reductions of 23.7%, 1.4%, and 11.3%, respectively (Figure 4A-C). The uptake of free daunorubicin was not significantly altered in HL-60, K562, or THP-1, regardless of the presence or absence of these inhibitors, either alone or in combination (Figure 6A-C). When the cell culture containing HD-BLT-1 and CPX-351 was incubated at 4°C, which inhibit all endocytosis pathways, the uptake of CPX-351 was not observed in HL- 60 (1.4 AU ± 0.14), K562 (1.9 AU ± 0.24), and THP-1 (1.6 AU ± 0.07) (Figure 4A-C), while the uptake of free drug daunorubicin w as observed in these cells, 14.3 AU ± 1.4, 19.0 AU ± 3.4, and 22.5 AU ± 2.2, respectively (Figure 6A-C). The addition of HD-BLT-1 also significantly increased the uptake of CPX-351 in resistance cells, HL-60 / AD and K562 / ADM, by more than twofold compared to control cells, with increases of 434.4% and 214.2%, respectively (p<0.01, respectively) (Figure 4D-E).

[0153] SR-BI expression and CPX-351 uptake in patient specimens. Finally, we investigated whether the potentiating effect of HD-BLT-1 could also be detected in leukemia cells derived from patients. We collected leukemia blast cells from three patients with AML. Sample 1 was a 48-y ear-old man who experienced relapse of FLT3 / ITD-positive AML with KMT2A rearrangement (The French-American-British (FAB) classification: M4, World Health Organization (WHO) classification: AML with myelodysplasia-related changes)40after receiving standard induction and consolidation chemotherapy, and was resistant to gilteritinib. Sample 2 was a 64-year-old man who experienced relapse of AML with complex karyotype (FAB: M0, WHO: AML, not otherwise specified) after receiving standard induction and consolidation chemotherapy, and was resistant to azacitidine. Sample 3 w as a 72-year-old woman with newly diagnosed FLT3 / ITD-positive AML (FAB: M5, WHO: AML with NPM1 mutation). The expression of SR-BI was highest in sample 3. followed by sample 1 and sample 2 (Figure 5A). Similarly, the uptake of CPX-351 was highest in sample 3, followed by sample 1 and sample 2. The addition of HD-BLT-1 significantly increased the uptake of CPX-351 by more than twofold in sample 1 and sample 3, but did not alter uptake in sample 2 (Figure 5B).1.3 Discussion

[0154] Protein corona analysis of CPX-351 revealed that apolipoproteins, apolipoprotein A-I and A-II, are its primary component. Therefore, we examined endocytosis in leukemia cell lines with a particular focus on SR-BI, a lipid receptor for apolipoprotein A-I and A-II. In the various cell lines, HL-60, K562, and THP-1, the addition of chlorpromazine and EIPA significantly decreased CPX-351 uptake. Similarly, the addition of LD-BLT-1 significantly- decreased the uptake of CPX-351 in these cell lines. These findings suggest that, in addition to CME and macropinocytosis, the SR-BI-mediated uptake pathway may be a key mechanism by which CPX-351 is internalized in leukemia cells. SR-BI expression in HL- 60 / AD and K562 / ADM was at least as high as in their respective parental cell lines, but CPX- 351 uptake was significantly lower. The addition of MK571 to HL-60 / AD and tariquidar to K562 / ADM significantly increased CPX-351 uptake and significantly decreased IC50. suggesting that MRP and P -glycoprotein expression may be the resistance mechanism for CPX-351. When HD-BLT-1 was added, CPX-351 uptake increased more than twofold in all different cell lines, including resistant cells. This was significantly suppressed by the addition of EIPA. In patient samples, SR-BI expression and CPX-351 uptake tended to be similar. Additionally, in patient samples, the addition of HD-BLT-1 significantly increased CPX-351 uptake, while a sample with low SR-BI expression did not show significantly increased CPX- 351 uptake.

[0155] Various proteins, including human serum albumin, fibrinogen, apolipoproteins, transferrin, and complement proteins, have been identified as components of the protein corona surrounding nanoparticles.11Apolipoproteins have been shown to bind to lipoprotein receptors, such as SR-BI and LDLR, which are frequently observed in a variety of pathological conditions, including melanoma, lymphoma, hepatocellular carcinoma, and atherosclerosis.41This apolipoprotein-rich characteristic of nanoparticles may enable them to be recognized by cells that overexpress apolipoprotein receptors. Previous research has demonstrated that apolipoproteins A-I and A-II are key components of the protein corona surrounding liposomes,26,42'44which is consistent with the findings of this study. SR-BI is the primary receptor responsible for the selective internalization of cholesteryl esters from high- density lipoprotein (HDL) molecules, which consists of apolipoproteins A-I and A-II.45,46SR-BI has recently gained attention for its role in cancer development.47High expression of SR-BI has been observed in various types of cancer cells including choriocarcinoma cells, malignant epithelial cells, prostate cancer cells, breast cancer cells, and hepatoma cells.45,47High expression of SR-BI has been linked to poor prognosis in breast48, lung49, renal cellcancer50, and neuroblastoma.51However, the expression and significance of SR-BI in leukemia have not yet been fully analyzed. In this study, all the cell lines used expressed SR- BI. Recently, various nanomedicine carrying therapeutic agents targeting cancer cells with high expression of SR-BI have been tested.52'54

[0156] Interestingly, while LDL cholesterol is delivered to cells following binding and endocytosis via the LDLR, HDL particles bind to SR-BI on the cell surface, and cholestery l esters are selectively delivered to the interior of the cell without internalization of the entire lipoprotein particle.34,46SR-BI has been demonstrated to possess the ability to form a hydrophobic tunnel within cell membranes, allowing for the selective uptake of hydrophobic molecules which bypass lysosomal processing. BLT-1, a selective inhibitor of SR-BI, can bind to the cysteine 384 residue of SR-BI, inhibiting the transfer of cholestery l esters.33BLT- 1 is highly specific to the SR-BI pathway and does not interfere with receptor-mediated endocytosis or other forms of intracellular vesicular traffic.33In addition to inhibiting the uptake of cholesteryl esters via SR-BI, BLT-1 has the unique characteristic of increasing SR- BI's binding affinity7for HDL in a dose-dependent manner, resulting in enhanced HDL binding.33 34

[0157] In this study, the uptake of CPX-351 was reduced upon addition of LD-BLT-1, suggesting that the contents of CPX-351 (cytarabine and daunorubicin) may be taken up byleukemia cells through nonendocytic mechanisms mediated by SR-BI. However, the uptake of CPX-351 was significantly increased upon addition of HD-BLT-1, which was suppressed by the addition of EIPA. In each cell line, CPX-351 alone tended to decrease the uptake when combined with chlorpromazine, rather than EIPA. However, when CPX-351 was combined with HD-BLT-1, the addition of EIPA decreased the uptake, rather than chlorpromazine. The addition of HD-BLT-1 also increased CPX-351 uptake in patient samples, but not in a sample with low SR-BI expression. These findings suggest that HD-BLT-1 may enhance the binding of CPX-351 to SR-BI, activating other endocytic pathway s such as macropinocytosis in addition to CME, leading to an increased uptake of CPX-351 in leukemia cells. Macropinocytosis has recently received significant attention as a potential method of selective drug delivery7to cancer cells, as it is uniquely expressed by some cancer cells in comparison to healthy cells of the same ty pe.12,17For example, cancer cells with mutant KRAS activate macropinocytosis to actively take up nutrients such as glucose, lipids, and albumin to meet their energetic requirements for survival and proliferation.55HDL particlesreconstituted with apolipoprotein E3 have been found to activate macropinocytosis and are efficiently taken up by glioblastoma cells.56

[0158] When using nanoparticles in vivo, it is important to consider factors that may affect protein corona formation, such as higher serum concentration, blood flow, and higher serum complexity.19The present study analyzed the uptake pathways of CPX-351 through CME, macropinocytosis, and the nonendocytic pathway via SR-BI. Other endocytosis pathways may be active in nanoparticle uptake.10,57

[0159] In conclusion, a nonendocytic pathway mediated by SR-BI, which binds apolipoprotein A-I and All, a main component of the protein corona of CPX-351, is a significant uptake pathway for the nanoparticle in leukemia cells. The enhancement of the binding between CPX-351 and SR-BI through the addition of HD-BLT-1 may increase uptake through the activation of macropinocytosis. SR-BI can serve as a potential biomarker for CPX-351 therapy and that the combination of HD-BLT-1 and nanoparticle formulations can enhance therapeutic efficacy in treatment.EXAMPLE 2

[0160] Drug sensitivities of cytarabine (ara-C) in the presence or absence of a nucleoside transporter inhibitor NBMPR. HL-60 cells were pre-incubated for 30 min with or without 1 pM NBMPR, and then treated with ara-C for further 72 h. Subsequently, the cell growth inhibition (ICso) w as determined by using the XTT assay. NBMPR, S-(4-nitrobenzyl)-6- thioinosine, is a potent inhibitor of cell surface nucleoside transporters. Ara-C 's grow th inhibitory effect was reduced in the presence of NBMPR.Table 4. Drug sensitivities of ara-C in the presence or absence of a nucleoside transporter inhibitor NBMPR.

[0161] Drug sensitivities of daunorubicin (DNR) in the presence of or in the absence of a nucleoside transporter inhibitor NBMPR. HL-60 cells w ere pre-incubated for 30 min with or without 1 pM NBMPR, and then treated with daunorubicin (DNR) for further 72 h. Subsequently, the cell growth inhibition (IC50) was determined by using the XTT assay. DNR inhibited the growth of HL-60 cells to the same extent regardless of the presence of NBMPR.Table 5. Drug sensitivities of daunorubicin (DNR) in the presence of or in the absence of a nucleoside transporter inhibitor NBMPR.

[0162] Drug sensitivities o f CPX-351 in the presence or absence o f a nucleoside transporter inhibitor NBMPR. HL-60 cells were pre-incubated for 30 min with or without 1 pM NBMPR, and then treated with CPX-351 for further 72 h. Subsequently, the cell growth inhibition ( I C so ) was determined by using the XTT assay. As shown above, when NBMPR is present, ara-C uptake into the cells is inhibited. But. as shown in Table 6. CPX-351 inhibited the growth of HL-60 cells to the same extent regardless of the presence of NBMPR.Table 6. Drug sensitivities of CPX-351 in the presence or absence of a nucleoside transporter inhibitor NBMPR.EXAMPLE 3

[0163] Inhibition ofDNA synthesis by CPX-351. HL-60 cells were pre-incubated at 37 °C with 1 pM CPX-351 for 2 h, and then incubated at 37 °C with 3H-thymidine for further 4 h. Subsequently, DNA was extracted from the cells, and its radioactivity was measured by the scintillation counter. The DNA synthesis, which was calculated from the radioactivity of thymidine uptake into cellular DNA, was compared between untreated cells and CTP-351- treated cells (Figure 7).EXAMPLE 4

[0164] Background. CPX-351, a dual-drug liposomal encapsulation of daunorubicin and cytarabine in a synergistic 1 :5 molar ratio, is approved for newly diagnosed, therapy-related acute myeloid leukemia (AML) or AML with myelodysplasia-related changes in the United States and Europe. Whilst CPX-351 demonstrated improved overall survival and a similar safety profile vs 7+3 in the phase 3 clinical trial (NCT01696084). the safety of CXP-351 related to cardiac function has not been fully described, an important consideration, due to the known cardiot oxi city risk from anthracyclines. Here, we assess the impact of CPX-351 vs 7+3 chemotherapy on cardiac impairment in older patients with untreated high-risk AML, using data from the phase 3 trial.

[0165] Methods: In the phase 3 trial, patients (60-75 years old; AML according to WHO 2008 criteria) were randomized to receive CPX-351 (100 units / m2on days 1. 3 and 5; days 1 and 3 for second induction) or 7+3 (cytarabine 100 mg / m2 / day for 7 days [5 days for second induction] and daunorubicin 60 mg / m2on days 1-3 [days 1-2 for second induction]). Cardiac impairment was determined by cardiac adverse events (AEs) as well as echocardiogram (ECHO) measures including left ventricular ejection fraction (LVEF) and global longitudinal strain (GLS). which were assessed at baseline and two follow-up visits (follow-up 1: 30-45 days from last induction or prior to consolidation or salvage therapy; follow-up 2: Day 150 or 45 days (±10 days) from last treatment).

[0166] Results: The study population comprised 118 patients (CPX-351, n=63; 7+3, n=55) with normal baseline LVEF (LVEF >53%) and at least one post-baseline ECHO measure (LVEF or GLS). The mean cumulative dose of daunorubicin while on study was lower for patients receiving CPX-351 vs 7+3 (378 vs 570 mg. respectively). Lower proportions of CPX-351 vs 7+3-treated patients had LVEF <53% (11% vs 26%) or GLS <18% (25% vs 38%) at follow-up 1 or 2. A clinically significant change in LVEF (>10% absolute change from baseline and LVEF <53%) or GLS (>12% relative change from baseline and GLS <18%) was less common in patients treated with CPX-351 vs 7+3 (8% vs 16% and 21% vs 44%, respectively) at follow-up 1 or 2. The frequency of any cardiac disorder was similar in both CPX-351 and 7+3-treated patients (38% vs 40%). Tachycardia was most frequent in CPX-351 -treated patients than in 7+3-treated patients (19% vs 9%), while atrial fibrillation was most frequent in 7+3-treated patients than in CPX-351 -treated patients (13% vs 3%).

[0167] Conclusion: This study adds to the previously reported survival and safety data for CPX-351 to demonstrate that CPX-351 is also associated with less cardiotoxicity in older patients (60-75 years) with AML, compared with the standard of care 7+3 treatment.

[0168] The present invention has been illustrated by reference to various exemplary embodiments and examples. As will be apparent to those of skill in the art other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are to be construed to include all such embodiments and equivalent variations.

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Claims

WHAT IS CLAIMED IS:

1. A pharmaceutical formulation comprising: therapeutically effective amounts of (a) a liposome encapsulating one or more therapeutic agents; and (b) an inhibitor of SR-BI.

2. A kit comprising a first vessel containing a therapeutically effective amount of a liposome encapsulating one or more therapeutic agents, and a second vessel comprising a therapeutically effective amount of an inhibitor of SR-BI, the kit further comprising instructions for the clinician for administering the liposome and the inhibitor of SR-BI.

3. The kit according to claim 2, wherein the instructions detail a procedure to prepare a pharmaceutical formulation comprising the liposome and / or a pharmaceutical formulation comprising the inhibitor of SR-BI in a therapeutically effective amount, prior to administering the liposome and the inhibitor of SR-BI to the subject.

4. A method of combination therapy of a proliferative disorder, e.g., cancer, e.g., leukemia, e.g., acute myeloid leukemia, the method comprising administering to a subject in need thereof a therapeutically effective amount of a liposome and an amount of an inhibitor of SR-BI effective to enhance uptake of the liposome by a target cell exhibiting the genotype and / or phenotype associated with the proliferative disease relative to the uptake of the liposome by an identical cell absent the inhibitor of SR-BI+.

5. A method of enhancing delivery of a liposome into an intracellular compartment of a target cell, the method comprising administering to a subject in need thereof a therapeutically effective amount of the liposome and a therapeutically effective amount of an inhibitor of SR- BI, the amount of the inhibitor of SR-BI administered being effective to enhance uptake of the liposome by the target cell, thereby providing enhanced delivery of the liposome to the intracellular compartment.

6. A method of altering the side effect profile of a liposome, the method comprising administering to a subject in need thereof a therapeutically effective amount of the liposome and an amount of the inhibitor of SR-BI effective to enhance uptake of the liposome by a target cell, decreasing the amount of liposome delivered to a non-target cell, thereby altering the side effect profile of the liposome.

7. A method of reducing resistance to one or more therapeutic agents in a target cell displaying resistance to the one or more therapeutic agents, the method comprising administering to a subject in need thereof a therapeutically effective dosage of the one or more therapeutic agents encapsulated in a liposome, and an amount of an inhibitor of SR-BIeffective to enhance intracellular delivery to the target cell, reduce efflux from the target cell, and a combination thereof, of the one or more therapeutic agents, thereby reducing resistance in the target cell.

8. A method of enhancing delivery of one or more therapeutic agents to an intracellular compartment of a target cell, the method comprising administering to a subject the one or more therapeutic agent encapsulated in a liposome, wherein the liposome is formulated with components selected for their ability to interact with Scavenger Receptor BI, thereby enhancing the delivery of the first and second therapeutic agent to the intracellular compartment.

9. The method according to claim 8, wherein the liposome comprises distearoylphosphatidylcholine, distearoylphosphatidylglycerol, and cholesterol.

10. The method according to claim 9, wherein the liposome comprises distearoylphosphatidylcholine, distearoylphosphatidylglycerol, and cholesterol at about a 7:2: 1 molar ratio.

11. A method of determining whether administering a liposome to a selected subject is an appropriate therapy for the subject, the method comprising determining whether the expression level of SR-BI of a target cell of the subject is greater than a pre-selected SR-BI expression level threshold; and if the subject’s SR-BI expression level is above the threshold, identifying the subject as a candidate for therapy with the liposome.

12. The method of claim 11, further comprising determining an appropriate dosage level for an agent selected from the liposome, the SR-BI inhibitor, and a combination thereof based on determining the expression level of SR-BI in a target cell of the selected subject.

13. The method according to any preceding claim, wherein the one or more therapeutic agent is selected from cytarabine, daunorubicin, CPX-351 and a combination thereof.

14. The method according to any preceding claim, wherein the inhibitor of SR-BI is BLT- 1.

15. The method of any preceding claim, wherein the inhibitor of SR-BI is BLT-1.

16. The method of any preceding claim, wherein the target cell is a blood cell.

17. The method of any preceding claim, wherein the target cell is a leukemia cell.

18. The method according to any preceding claim, wherein the liposome and the inhibitor of SR-BI are administered sequentially in separate pharmaceutical formulations.

19. The method according to any of preceding claim, wherein the liposome and the inhibitor of SR-BI are combined into a single pharmaceutical formulation and administered to the subject in this format.

20. A method of treating acute myeloid leukemia in a subject in need of such treatment, wherein the subject is a member of a population aged at least about 60 years, the method comprising administering a therapeutically relevant dose of CPX-351 to the subject.

21. The method according to claim 20, wherein markers of cardiotoxicity due to CPX-351 administration are lower in one or more members of the population versus the same markers or cardiotoxicity in one or more members of this population to whom standard 7+3 therapy is administered.

22. The method according to claim 8, wherein the liposome comprises about 20 mol% or less of cholesterol.

23. The method according to claim 8, wherein the liposome comprises at least 45 mol% distearoylphosphatidylcholine (DSPC) or hydrogenated soybean phosphatidylcholine (HSPC).

24. The method according to claim 8, wherein the liposome comprises at least 1 mol% of distearoylphosphatidylglycerol or distearoylphosphatidylinositol.

25. A pharmaceutical formulation comprising therapeutically effective amounts of (a) a liposome comprising cytarabine; and (b) an ENT inhibitor.

26. A kit comprising a first vessel containing a therapeutically effective amount of a liposome comprising cytarabine, and a second vessel comprising a therapeutically effective amount of an ENT inhibitor, the kit further comprising instructions for the clinician for administering the liposome and the ENT inhibitor.

27. The kit according to claim 26, wherein the instructions detail a procedure to prepare a pharmaceutical formulation comprising the liposome and / or a pharmaceutical formulation comprising the ENT inhibitor in a therapeutically effective amount, prior to administering the liposome and the ENT inhibitor to the subject.

28. A method of combination therapy of a proliferative disorder, e.g., cancer, e.g., leukemia, e.g., acute myeloid leukemia, the method comprising administering to a subject in need thereof a therapeutically effective amount of a liposome comprising cytarabine and an amount of an ENT inhibitor effective to enhance uptake of liposome-encapsulated cytarabine by a target cell exhibiting the genotype and / or phenoty pe associated with the proliferative disease relative to the uptake of unencapsulated cytarabine in the presence of the ENT inhibitor by an identical cell.

29. A method of enhancing delivery of cytarabine into an intracellular compartment of a target cell in the presence of an ENT inhibitor, the method comprising administering to a subject in need thereof a therapeutically effective amount of a liposome comprising cytarabine and a therapeutically effective amount of the ENT inhibitor, wherein uptake of liposome-encapsulated cytarabine by the target cell is enhanced relative to the uptake of unencapsulated cytarabine in the presence of the ENT inhibitor by an identical cell, thereby providing enhanced delivery of the cytarabine to the intracellular compartment.

30. The method according to any one of claims 25-29, wherein the liposome further comprises daunorubicin.

31. The method according to any one of claims 25-30, wherein the liposome comprises CPX-351.

32. The method according to any one of claims 25-31, wherein the ENT inhibitor is NBMPR.

33. The method of any one of claims 25-32, wherein the target cell is a blood cell.

34. The method of any one of claims 25-33, wherein the target cell is a leukemia cell.

35. The method according to any one of claims 25-34, wherein the liposome and the ENT inhibitor are administered sequentially in separate pharmaceutical formulations.

36. The method according to any one of claims 25-35, wherein the liposome and the ENT inhibitor are combined into a single pharmaceutical formulation and administered to the subject in this format.

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