Modifiers for nanoparticle incorporation

Abstract

Various formulations for combination therapy of liposomes and SR-BI inhibitors that result in enhanced liposome uptake by target cells are provided, exemplified by combination therapy with CPX-351 and BLT-1. Also provided are various formulations for combination therapy of liposomes and ENT inhibitors that result in enhanced uptake of liposome-encapsulated cytarabine by target cells. Also provided are methods for using the formulations in combination therapy. Also provided are methods for reducing cardiotoxicity in age-group patient populations receiving therapy with cytarabine and daunorubicin, and CPX-351.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority under 119(e) of U.S. Provisional Application No. 63 / 493,969, filed on 3 April 2023, the disclosure thereof is incorporated herein by reference in its entirety. [Background technology]

[0002] Acute myeloid leukemia (AML) is a myeloid hematological malignancy associated with a median age of 65 at diagnosis (Siegel et al. CA Cancer J Clin, 2018;68(1):7-30(2018)). According to the American Association for Cancer Research, an estimated 19,520 new cases of AML were diagnosed in 2018, and an estimated 10,670 people died from AML (Siegel et al. ibid.). Since the late 1970s, the standard treatment for AML induction therapy has been a combination chemotherapy regimen consisting 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 cytotoxic substances to the protocol, but the overall survival benefit of conventional induction chemotherapy regimens remains poor in elderly patients with AML (Roboz Hematology, Am Soc Hematol Educ Program 2011:43-50 (2011)). As the biological nature of AML is further understood, several targeted agents are being investigated as new therapeutic approaches, particularly for patients with low-risk characteristics (DiNardo et al., Expert Opin Pharmacother, 6(1):95-106 (2015)). An alternative approach is to optimize cytarabine and anthracycline therapy by utilizing the current understanding of the pharmacodynamics of conventional drugs. For this purpose, CPX-351 (trade name: Vyxeos®) is a highly liposomal formulation of cytarabine and daunorubicin encapsulated in a 5:1 molar ratio, which has been shown to exhibit maximum synergy and minimum antagonism in vitro (Tardi et al., Leuk Res., 33(1):129-39 (2009)). In other words, at some molar ratios of cytarabine:daunorubicin, the combination is additive; at other ratios, the combination is far more effective; and at some ratios, the combination is actually detrimental.Liposome encapsulation significantly increases the plasma half-lives of cytarabine and daunorubicin, leading to even higher drug accumulation in the bone marrow compared to free cytarabine and daunorubicin (Feldman et al., Leuk Res. 36(10):1283-9 (2012), Lim et al., Leuk Res. 34(9):1214-23 (2010)). Importantly, the liposomal integrity of CPX-351 in circulation minimizes the release of free drugs, ensuring simultaneous delivery of the two drugs in an optimal molar ratio. This simultaneous delivery strategy eliminates the problems of differential in vivo distribution and metabolism. When the two drugs are administered in a free (unencapsulated) state, it is impossible to maintain the optimal synergistic molar ratio at the intended site of action due to the manner in which the two drugs are cleared from the systemic circulation. Human clinical trials using CPX-351 for myeloid leukemia demonstrated superior antileukemic activity, improving the median overall survival in patients to 9.56 months compared to 5.95 months in patients treated with the "7+3" regimen (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). This led to FDA approval in 2017 for the treatment of newly diagnosed treatment-related acute myeloid leukemia (t-AML) or AML with myelodysplastic-associated changes (AML-MRC) (Nikanjam et al., Cancer Chemother Pharmacol, 81(1):171-8 (2018)).

[0003] Ongoing research into the treatment of leukemia continues to provide advancements in treatment options for this disease. [Overview of the project]

[0004] Currently, it has been discovered that when specific liposomes containing one or more therapeutic agents along with inhibitors of cellular pathways selected for administration to target cells requiring treatment are administered, the uptake of the liposomes by cells increases significantly compared to the uptake observed in the same cells without inhibitor administration, provided that each agent is administered at a therapeutically useful dose.

[0005] In exemplary embodiments, the inhibitor is an inhibitor of scavenger receptor BI (SR-BI). Incorporating such an inhibitor at a therapeutically useful dose significantly increases liposome uptake by SR-BI-expressing cells compared to uptake observed in the same cells without the inhibitor.

[0006] SR-BI, which is involved in HDL uptake into cells, has recently been recognized as mediating the uptake of specific liposomes in leukemia cells (Di et al., Drug Dev.Ind.Pharm.45(1):21-26(2019)). Therefore, the increase in liposome uptake when SR-BI is exposed to known SR-BI inhibitors is a surprising result, as those skilled in the art would expect that inhibiting SR-BI would decrease liposome uptake. Even more surprisingly, the inventors discovered that while administration of exemplary SR-BI inhibitors at initial doses decreased liposome uptake by SR-BI-expressing cells as expected, administration of the same inhibitors at a second and higher dose counterintuitively increased liposome uptake by cells.

[0007] In exemplary embodiments, the present disclosure provides a method for increasing the cellular uptake of liposomes encapsulating one or more therapeutic agents by target cells expressing SR-BI. The method comprises administering liposomes to target cells and also administering an amount (e.g., a “therapeutic effective dose”) of an SR-BI inhibitor to the target cells sufficient to increase the uptake of the liposomes by the cells.

[0008] In exemplary embodiments, target cells are diseased cells that underexpress or overexpress SR-BI compared to identical non-disease cells. The formulations of the present invention utilize this overexpression or underexpression to achieve enhanced liposome delivery to cells.

[0009] In various embodiments, a method is provided for treating a disease in a subject requiring treatment of the disease. The method includes administering to a subject a therapeutically effective amount of liposomes containing one or more therapeutic agents effective for treating the disease, improving the symptoms of the disease, etc., and an amount of an SR-BI inhibitor selected such that the cellular uptake by target cells of the subject is increased compared to the cellular uptake of the liposomes by the same target cells in the absence of the SR-BI inhibitor.

[0010] In various embodiments, methods are provided for treating cancer, such as leukemia, such as acute myeloid leukemia, in subjects requiring cancer treatment. The method comprises administering to a subject a therapeutically effective dose of liposomes encapsulating one or more cancer chemotherapeutic agents, and an amount of an SR-BI inhibitor selected such that cellular uptake by target cells of the subject is increased compared to cellular uptake of the liposomes by the same target cells in the absence of the SR-BI inhibitor. In exemplary embodiments, the SR-BI inhibitor is effective in increasing liposome uptake by target cells at high initial doses of the SR-BI inhibitor, but at lower doses of the SR-BI inhibitor, it has no effect on uptake or reverses the effect, reducing uptake.

[0011] Also provided is a method for altering the adverse event profile of one or more therapeutic agents, for example, a liposomal formulation of CPX-351, by enhancing the delivery of the formulation to target cells. Delivery is enhanced by administering an SR-BI inhibitor in an amount selected such that uptake in the target cells is increased compared to cellular uptake of the liposome by the same target cells in the absence of the SR-BI inhibitor, thereby reducing delivery to non-target cells. In exemplary embodiments, the enhanced delivery to target cells results in less off-target delivery than in the absence of the inhibitor. Exemplary alterations to the adverse event profile include a reduction or improvement of at least one undesirable adverse event of the therapeutic agent in the target to which the therapeutic agent is administered.

[0012] Exemplary methods of the present invention are useful for reducing cellular resistance to one or more liposome-encapsulated therapeutic agents, such as cytarabine, daunorubicin, and combinations thereof. The methods include administering an SR-BI inhibitor to drug-resistant target cells in an amount selected such that the cellular uptake of liposome-encapsulated therapeutic agents by the target cells is increased compared to the cellular uptake of liposome-encapsulated drugs by the same target cells in the absence of the SR-BI inhibitor, or administering an SR-BI inhibitor in an amount selected such that the cellular efflux of liposome-encapsulated therapeutic agents (or deencapsulated therapeutic agents) is less than the efflux of drugs by the same target cells in the absence of the SR-BI inhibitor.

[0013] In various embodiments, pharmaceutical formulations are provided for carrying out one or more of the above methods. The pharmaceutical formulation comprises liposomes encapsulating one or more therapeutic agents, and an SR-BI inhibitor selected such that cellular uptake of the liposomes by target cells is increased compared to cellular uptake of the liposomes by the same target cells in the absence of the SR-BI inhibitor. In exemplary embodiments, the presence of the inhibitor in the formulation reduces delivery to non-target cells.

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

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

[0016] In an exemplary embodiment, a combination at a therapeutically useful dosage of a liposome containing cytarabine and an inhibitor of the equilibrative nucleoside transporter (ENT) (e.g., ENT1) results in a significant increase in the uptake of cytarabine encapsulated in the liposome by cells expressing ENT (e.g., ENT1) as compared to the uptake of unencapsulated cytarabine in the presence of the ENT inhibitor in the same cells.

[0017] In an exemplary embodiment, the present disclosure provides a method of combination therapy for a proliferative disorder, such as cancer, such as leukemia, such as acute myeloid leukemia, the method comprising administering to a subject in need of treatment a therapeutically effective amount of a liposome containing cytarabine and an amount of an ENT inhibitor effective to enhance the uptake of cytarabine encapsulated in the liposome by target cells that exhibit a genotype and / or phenotype associated with the proliferative disease as compared to the uptake of unencapsulated cytarabine by the same cells in the presence of the ENT inhibitor.

[0018] In exemplary embodiments, the present disclosure provides a method for enhancing the delivery of cytarabine into the intracellular compartment of a target cell in the presence of an ENT inhibitor. The method comprises administering to a subject requiring administration a therapeutically effective dose of liposomes containing cytarabine and a therapeutically effective dose of an ENT inhibitor, wherein the uptake of cytarabine encapsulated in liposomes by the target cell is enhanced compared to the uptake of unencapsulated cytarabine by the same cell in the presence of the ENT inhibitor, thereby providing enhanced delivery of cytarabine into the intracellular compartment.

[0019] In exemplary embodiments, the present invention's method utilizing an ENT inhibitor, similar to SRBI, alters the adverse drug reaction profile of the therapeutic agent. Exemplary alterations to the adverse drug reaction profile include a reduction or improvement of at least one undesirable adverse reaction of the therapeutic agent to the target to which the therapeutic agent is administered. In exemplary embodiments, enhanced delivery to target cells results in less off-target delivery than in the absence of the inhibitor.

[0020] In a further exemplary embodiment, a method is provided for treating acute myeloid leukemia in subjects requiring treatment for acute myeloid leukemia, the subjects being at least about 60 years of age. The method comprises administering a therapeutically relevant dose of CPX-351 to the subjects. Administration of CPX-351 in this patient population correlates with lower cardiotoxicity than administration of standard 7+3 therapy.

[0021] Further embodiments, objectives, and examples are described in embodiments for carrying out the invention. [Brief explanation of the drawing]

[0022] [Figure 1] Lipid receptor expression in leukemia cell lines. (a) Scavenger receptor class B1 (SR-BI), (b) Low-density lipoprotein receptor (LDLR), and (c) Low-density lipoprotein receptor-associated protein 1 (LRP1). The mean ± SE of at least three independent experiments is shown for each value. *p=0.01. **p=0.014. ***p=0.011. [Figure 2](a) Uptake of CPX-351 into leukemia cell lines as evaluated by flow cytometry. (b) Uptake of CPX-351 in HL-60 / AD treated with MK571. (c) Growth inhibition curve in HL-60 / AD. HL-60 / AD treated with control and MK571 were incubated with various concentrations of CPX-351 for 72 hours, and then IC50 was determined using the XTT assay. (d) Uptake of CPX-351 in K562 / ADM treated with thaliquidal. (e) Growth inhibition curve in K562 / ADM. K562 / ADM treated with control and thaliquidal were incubated with various concentrations of CPX-351 for 72 hours, and then IC50 was determined using the XTT assay. The mean ± SE of at least three independent experiments is shown for each value. *p=0.0001. **p=0.0015. [Figure 3] Inhibition of the CPX-351 uptake pathway by the addition of chlorpromazine, 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), or a low dose of lipid transport inhibitor-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. ***p=0.0031. The mean ± SE of at least three independent experiments is shown for each value. [Figure 4] Enhancement of CPX-351 uptake by addition of high-dose lipid transport inhibitor-1 (HD-BLT-1, 40 μM) alone, or in combination with chlorpromazine, 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), or by incubation at 4°C. (a) HL-60: *p<0.001. **p=0.0066. (b) K562: *p<0.001. **p=0.0019. ***p=0.001. (c) THP-1: *p<0.001. **p=0.0055. Mean ± SE of at least three independent experiments are shown for each value. [Figure 5]Analysis of patient samples: (a) Expression of scavenger receptor class B1 (SR-BI). (b) Uptake of CPX-351 alone or in combination with high-dose lipid transport inhibitor-1 (HD-BLT-1, 40 μM). [Figure 6] Uptake of daunorubicin into leukemia cell lines, and inhibition of the uptake pathway by addition of chlorpromazine, 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), low-dose lipid transport inhibitor-1 (LD-BLT-1, 400 nM), high-dose lipid transport inhibitor-1 (HD-BLT-1, 40 μM), or incubation at 4°C. The mean ± SE of at least three independent experiments is shown for each value. Each group was compared using one-way ANOVA, except for incubation at 4°C. (a) HL-60: p=0.77. (b) K562: p=0.073. (c) THP-1: p=0.063. Abbreviations: ns, not significant. [Figure 7] Inhibition of DNA synthesis by CPX-351. ***P<0.001. [Modes for carrying out the invention]

[0023] I. Introduction This disclosure provides novel conjugate formulations of liposomes encapsulating one or more therapeutic agents and inhibitors of selected cellular pathways. In exemplary embodiments, the inhibitor is an inhibitor of scavenger receptor BI (SR-BI). Also provided are combination therapies with these formulations and methods of using the novel formulations and combination therapies to treat diseases. This disclosure also shows novel methods for determining whether therapy with liposome-encapsulated therapeutic agents, or combination therapy with these liposomes and inhibitors, is appropriate for specific subjects requiring treatment of a disease. Supporting these various embodiments is the discovery that incorporating exemplary inhibitors of SR-BI at selected doses significantly increases liposome uptake by cells expressing SR-BI compared to uptake observed in the same cells without the inhibitor.

[0024] Liposomes are closed vesicles having at least one lipid bilayer surrounding an aqueous core. The intracellular space and lipid layer(s) of a liposome can capture a wide variety of substances, including drugs, cosmetics, diagnostic reagents, genetic material, and bioactive compounds. Because non-toxic lipids form the basis of liposomes, they generally exhibit low toxicity. This low toxicity, combined with the liposome's ability to extend the plasma circulating lifespan of drugs, makes liposomes a particularly useful vehicle for delivering pharmaceutically active drugs. In many cases, drugs delivered by liposomes result in superior clinical efficacy along with reduced toxicity.

[0025] SR-BI is recognized as a receptor that mediates HDL uptake into cells and has recently been recognized as mediating liposome uptake in leukemia cells (Di et al., Drug Dev.Ind.Pharm.45(1):21-26(2019)). SR-BI is a member of the CD36 superfamily. Each member contains a large extracellular domain flanked by two transmembrane (ML279) domains with short amino-terminated and carboxyl-terminated intracellular tails. Members of the CD36 family maintain approximately 30% sequence identity. They can differ in intracellular localization and ligand selectivity. For example, CD36 / SCARB3 can bind to HDL but cannot efficiently uptake HDL cholesterol via selective lipid uptake.

[0026] Inhibitors of SR-BI are recognized compounds. See, for example, U.S. Patent No. 9,884,851 and Raldue et al. Tox. Letters 175(1-3):1-7(2007).

[0027] The diabetes drug glybride, an inhibitor of sulfonylurea receptors SUR1 and SUR2, was a relatively weak inhibitor of cholesterol efflux, but it was found to have activity against SR-BI. Researchers at Sankyo discovered that protected piperazine R-138329 and R-154716 increased HDL cholesterol in both mice and hamsters, presumably by inhibiting HDL uptake mediated by SR-BI. Recently, a p38MAP kinase inhibitor (ITX-5061) has been reported to have a similar in vivo effect and appears to be a moderate inhibitor of SR-BI-mediated cholesterol uptake. See, for example, U.S. Patent No. 9,884,851.

[0028] Dockendorff et al., in U.S. Patent No. 9,884,851, demonstrate a range of consistent SR-BI activity across a certain range of novel synthetic small molecules prepared as SR-BI inhibitors. This reference also provides sufficient guidance to those skilled in the art for assays to determine whether a compound functions as an SR-BI inhibitor, as well as for various properties of compounds active against SR-BI, general aspects of SR-BI inhibitor biology, and so on.

[0029] Inhibitors of SR-BI as a class are exemplified by lipid transport inhibitor-1 and related compounds. While studying the mechanism by which liposomes are taken up by cells, the inventors unexpectedly discovered that SR-BI inhibitors, exemplified by BLT-1, increase cellular liposome uptake at selected doses.

[0030] This disclosure also provides novel conjugate formulations of cytarabine-containing liposomes and ENT inhibitors. Also provided are combination therapies using these formulations and methods of using the novel formulations and combination therapies to treat diseases. Supporting these various embodiments is the finding that exemplary ENT inhibitors at selected doses combined with cytarabine-containing liposomes enhance the uptake of liposome-encapsulated cytarabine by target cells compared to combinations of unencapsulated cytarabine and ENT inhibitors.

[0031] ENTs are polytopic endometrial proteins that mediate the transport of physiological nucleosides across the cell membrane (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 rapadosin (Rehan et al., SLAS Discovery, 24(10):953-968 (2019)).

[0032] 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 analogue 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 exemplary embodiments, the ENT inhibitor comprises NMBPR.

[0033] It was previously found that CPX-351 uptake is independent of human equilibrium nucleoside transporter 1 (hENT1) expression (Anderson et al., Leukemia Research, 74, 121-129 (2018), which is incorporated in its entirety herein by reference); however, how the administration of liposomes containing cytarabine in combination with an ENT inhibitor affects the delivery of cytarabine to target cells was previously unclear.

[0034] Hereinafter, we will refer in detail to embodiments of exemplary embodiments of the present disclosure shown in the accompanying drawings. The same reference numerals will be used throughout the drawings and the following detailed description to refer to the same or similar parts. Those skilled in the art will understand that the following detailed description is illustrative and not intended to limit in any way. Other embodiments of the present disclosure will be readily apparent to those skilled in the art who benefit from the present disclosure.

[0035] For clarity, not all of the typical features of the embodiments described herein are shown or explained. It will be understood that in the development of such actual embodiments, numerous embodiment-specific decisions will be made to achieve the developer's specific goals, such as compliance with application-related and business-related constraints, and that these specific goals will differ from embodiment to embodiment and from developer to developer.

[0036] Many modifications and variations of the embodiments shown in this disclosure can be made without departing from the spirit and scope of the exemplary embodiments, as will be obvious to those skilled in the art. The specific exemplary embodiments described herein are provided merely as examples, and this disclosure is limited only by the terms of such claims attached, along with the entire scope of equivalents granted in the claims.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art.

[0038] II. Terminology A.Definition Unless otherwise defined, all terms, notations, and other scientific or technical terms used herein have the same meaning as commonly understood by those skilled in the art in the field relating to this disclosure. Where applicable, terms having a commonly understood meaning are defined herein for clarity and / or immediate reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from the commonly understood meaning in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly used by those skilled in the art using conventional methodologies. Where necessary, procedures involving the use of commercially available kits and reagents are generally carried out according to protocols and / or parameters defined by the manufacturer, unless otherwise stated. All patents, applications, published applications, and other publications referenced herein are incorporated by reference in their entirety. If any definition set forth in this section contradicts or otherwise conflicts with any definition set forth in any patent, application, published application, or other publication incorporated herein by reference, the definition set forth in this section shall prevail over the definition set forth in any patent, application, published application, or other publication incorporated herein by reference.

[0039] The articles "a" and "an" are used herein to refer to one or more (i.e., at least one) grammatical objects of the article.

[0040] The terms “approximately” or “about” mean a range of acceptable error for a particular value, as determined by how the value is measured or determined, for example, by the limits of the measuring system or the degree of precision required for a particular purpose. For example, “approximately” may mean within one standard deviation or within two standard deviations, according to practice in the art. Alternatively, “approximately” may mean a range of up to 20%, preferably up to 10%, and more preferably up to 5% of a given value. Unless otherwise indicated, where a particular value is described in this application and claims, the term “approximately” means that an acceptable range of error should be assumed for that particular value.

[0041] As used herein, the term "CPX-351" refers to a liposomal formulation of cytarabine and daunorubicin encapsulated in a 5:1 molar ratio. The liposomes are formulated with DSPC / DSPG / Chol in a ratio of approximately 7:2:1.

[0042] When the term is used herein, a liposome refers to a closed vesicle having at least one lipid bilayer surrounding an aqueous core. The liposome's internal space and lipid layer(s) can capture a wide variety of substances, including drugs, cosmetics, diagnostic reagents, genetic material, and bioactive compounds. Exemplary liposomes used in the compositions and methods provided herein contain about 20% or less cholesterol. In various embodiments, the liposomes further contain about 1% to about 20% DSPG. In various embodiments, the liposomes contain one or more additional lipids. See, for example, U.S. Patent No. 8,518,437, which is incorporated herein in whole by reference for all purposes. In exemplary embodiments, the liposomes are formulated from DSPC / DSPG / Chol in a ratio of about 7:about 2:about 1.

[0043] The terms “therapeutic agent” or “drug” as used herein refer to the chemical portion used for various therapeutic purposes, including pharmaceutical uses.

[0044] 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. Examples of mammals include mice, rats, dogs, cats, pigs, sheep, horses, cattle, and humans. In some embodiments, the subject is a human, for example, a human who is or will be the subject of treatment, observation, or experimentation.

[0045] The terms “therapeutic dose” or “effective dose” refer to the amount of a compound disclosed and / or described herein that, when administered to a subject requiring such treatment, is sufficient to have a therapeutic effect as defined herein. The therapeutic dose will vary, for example, depending on the subject and condition being treated, the subject’s weight and age, the severity of the condition, the specific compound, the dosing regimen to be followed, the timing of administration, and the mode of administration, all of which can be readily determined by those skilled in the art. The therapeutic dose may be determined experimentally, for example, by assaying the blood concentration of the chemical entity, or theoretically by calculating its bioavailability.

[0046] Patient doses for the administration of either or both liposomes and SR-BI inhibitors are typically in the range of about 1 mg / day to about 10,000 mg / day, more typically about 10 mg / day to about 1,000 mg / day, and most typically about 50 to about 500 mg / day, after adding suitable adjuvants to obtain tablets if desired. In terms of the patient's body, typical doses are in the range of about 0.01 to about 150 mg / kg / day, more typically about 0.1 to about 15 mg / kg / day, and most typically about 1 to about 10 mg / kg / day, which can be, for example, 5 mg / kg / day or 3 mg / kg / day. Adjusting the dose to obtain a desired ratio of SR-BI inhibitor to liposomes is within the scope of the skill of a clinician or researcher skilled in the art.

[0047] With respect to SR-BI inhibitors, an exemplary "therapeutic effective dose" is the amount that increases liposome uptake by the target cells to which the inhibitor is administered.

[0048] With respect to ENT inhibitors, the exemplary “therapeutic effective dose” is the amount that enhances the uptake of liposome-encapsulated cytarabine by target cells compared to the uptake of unencapsulated cytarabine in the presence of the ENT inhibitor. Patient doses for these drugs are similar to those for SR-BI inhibitors in exemplary embodiments.

[0049] As used herein, “target cells” refers to cells that are administered liposomes and inhibitors and take up the liposomes. In exemplary embodiments, SR-BI and / or ENT are administered. Exemplary target cells express SR-BI and / or ENT. Exemplary target cells are diseased cells exhibiting the genotype and / or phenotype of the disease. Exemplary diseased cells overexpress or underexpress SR-BI and / or ENT. Target cells may also be “normal” cells that do not exhibit the genotype or phenotype of the disease. In exemplary embodiments, the degree of cellular uptake of liposomes by target cells is enhanced by administration of an SR-BI inhibitor, and normal target cells are used as a reference point for comparison to determine whether cellular uptake is increased or decreased in diseased cells. Such comparisons are used to determine whether a subject is a suitable candidate for combination therapy with liposomes and an SR-BI and / or ENT inhibitor, and / or to determine appropriate dose levels of one or both of these agents for that candidate.

[0050] As used herein with respect to target cells to which an SR-BI inhibitor is administered, “enhanced uptake by target cells” means the effect on target cells resulting from the administration of a selected dose of the SR-BI inhibitor, in which more liposomes are taken up by the target cells than are observed in the same cells and liposomes in the absence of the SR-BI inhibitor.

[0051] As used herein with respect to target cells to which an ENT inhibitor is administered, “enhanced uptake by target cells” means the effect on target cells resulting from the administration of a selected dose of the ENT inhibitor, in which more liposome-encapsulated cytarabine is taken up by the target cells than is observed in the same cells and with unencapsulated cytarabine in the presence of the ENT inhibitor.

[0052] "Treatment" (and related terms, e.g., "to treat," "to be treated," "to treat") includes one or more of the following: prevention of disease or disability (i.e., preventing the development of clinical symptoms of disease or disability); suppression of disease or disability; delaying or inhibiting the development of clinical symptoms of disease or disability; and / or mitigation of disease or disability (i.e., reducing or regressing clinical symptoms). This term includes situations in which disease or disability is already experienced by the subject, as well as situations in which disease or disability is not currently experienced but is expected to occur. This term encompasses both the complete and partial mitigation or prevention of a condition or disability, as well as the complete or partial mitigation of clinical symptoms of disease or disability. Thus, the compounds described and / or disclosed herein may prevent the exacerbation of an existing disease or disability, assist in the management of a disease or disability, or mitigate or eliminate a disease or disability. When used in a preventative manner, the compounds disclosed and / or described herein may prevent the development of disease or disability, or reduce the severity of a disease or disability that is likely to develop.

[0053] "SR-BI" refers to scavenger receptor B, type 1, which is involved in the movement of lipids between high-density lipoprotein (HDL) and cells.

[0054] "SR-BI inhibitors" refer to any member of the class of drugs that function to inhibit SR-BI-mediated cholesterol uptake by cells to which the inhibitor is administered, as exemplified by BLT-1.

[0055] "BLT-1" refers to lipid transport inhibitor-1, which is a thiosemicarbazone copper chelating agent and an SR-BI inhibitor.

[0056] "ENT" refers to equilibrium nucleoside transporters such as ENT1, ENT2, ENT3, and ENT4, which are polytopic endometrial membrane proteins that mediate physiological nucleoside transport across the cell membrane.

[0057] "ENT inhibitors" refer to any member of the class of drugs that function to inhibit ENT-mediated nucleoside transport across the cell membrane of the cells to which the inhibitor is administered, as exemplified by NBMPR.

[0058] III. Embodiments A. Method Currently, it has been discovered that when liposomes containing one or more therapeutic agents are administered to target cells expressing the scavenger receptor BI (SR-BI) together with an SR-BI inhibitor, both at therapeutically useful doses, the uptake of the liposomes by the target cells is significantly increased compared to the uptake observed in the same target cells without the inhibitor. Therefore, an exemplary embodiment provides a method for increasing the cellular uptake of liposomes containing one or more therapeutic agents in target cells expressing SR-BI. The method comprises administering liposomes to cells and administering an amount of an SR-BI inhibitor sufficient to increase the uptake of the liposomes by the target cells (e.g., a "therapeutic dose").

[0059] Exemplary inhibitors of SR-BI, including BLT-1, are known in the art, as are methods for assaying their activity against cells expressing this target, and for preparing and investigating novel inhibitors. See, for example, U.S. Patent No. 9,994,851.

[0060] In exemplary embodiments, an SR-BI inhibitor is effective in increasing liposome uptake by target cells at a first dose of the inhibitor. The exemplary first dose is greater than a smaller second dose in which cells show no increased uptake, show less uptake than seen at the first dose, or show lower uptake than target cells not treated with the SR-BI inhibitor.

[0061] In exemplary embodiments, target cells are abnormal cells (e.g., lesions) that underexpress or overexpress SR-BI compared to identical normal cells. The formulations of the present invention utilize this overexpression or underexpression to achieve enhanced liposome delivery to cells.

[0062] In exemplary embodiments, the liposomes contain at least 1 mol% of distearoylphosphatidylglycerol (DSPG) or distearoylphosphatidylinositol (DSPI).

[0063] In exemplary embodiments, liposomes contain approximately 20 mol% or less of cholesterol.

[0064] In exemplary embodiments, the liposomes contain distearoylphosphatidylcholine (DSPC).

[0065] In exemplary embodiments, the liposomes contain DSPC, DSPG, and cholesterol. The exemplary ratio of DSPC / DSPG / cholesterol is approximately 7:2:1 molar ratio.

[0066] In various embodiments, a method is provided for treating a disease in subjects requiring treatment of the disease. The method includes administering to a subject a therapeutically effective amount of liposomes containing one or more therapeutic agents effective for treating the disease, improving the symptoms of the disease, etc., and an amount of an SR-BI inhibitor selected such that the uptake of the liposomes by target cells is increased compared to the cellular uptake of the liposomes by the same target cells in the absence of the SR-BI inhibitor.

[0067] In exemplary embodiments, the disease is a proliferative disorder, such as cancer. In various embodiments, the disease is a blood disorder, such as a hematopoietic proliferative disorder. In exemplary embodiments, the disease is a leukemia, such as acute myeloid leukemia (AML).

[0068] Also provided is a method for altering the adverse event profile of a liposomal formulation, such as CPX-351, in which one or more therapeutic agents are encapsulated in liposomes, by enhancing the delivery of the formulation to target cells. Liposome uptake by target cells is enhanced by administering an SR-BI inhibitor in addition to the liposomes. The inhibitor is administered in an amount selected to increase liposome uptake by the target cells compared to cellular uptake of the liposomes by the same target cells in the absence of the inhibitor. Exemplary alterations to the adverse event profile include reducing the incidence or severity of one or more adverse events associated with the administration of a therapeutically effective dose of liposomes. Exemplary alterations to the adverse event profile include reducing or improving at least one undesirable adverse event of the therapeutic agent in the target to which the therapeutic agent is administered.

[0069] An exemplary method of the present invention is useful for reducing cellular resistance to one or more liposome-encapsulated therapeutic agents, such as cytarabine, daunorubicin, and combinations thereof. The method comprises administering an SR-BI inhibitor to drug-resistant target cells in an amount selected such that the cellular uptake of liposome-encapsulated therapeutic agents by the target cells is increased compared to the cellular uptake of liposome-encapsulated drugs by the same target cells in the absence of the SR-BI inhibitor, or an amount selected such that the cellular efflux of liposome-encapsulated therapeutic agents (or deencapsulated therapeutic agents) is less than the efflux of drugs (or other drugs) by the same target cells in the absence of the SR-BI inhibitor.

[0070] In various embodiments, methods for combination therapy of proliferative disorders, such as cancer, such as leukemia, such as acute myeloid leukemia, are provided. The method comprises administering to a subject requiring administration a therapeutically effective amount of liposomes containing one or more therapeutic agents useful for treating proliferative disorders and a therapeutically effective amount of a BLT-1 inhibitor. The liposomes and inhibitors are administered substantially simultaneously or sequentially in either order (i.e., inhibitor followed by liposomes, or liposomes followed by inhibitors). In various embodiments, the liposomes and the SR-BI inhibitors are administered sequentially in separate pharmaceutical formulations or combined in a single pharmaceutical formulation and administered in this manner.

[0071] In exemplary embodiments, the method of the present disclosure utilizes liposomes encapsulating one or more cancer chemotherapeutic agents. In various embodiments, the chemotherapeutic agents are cytarabine and daunorubicin. In some embodiments, cytarabine and daunorubicin are encapsulated in liposomes in a 5:1 ratio, and the liposomes contain DSPC / DSPG / cholesterol in a ratio of approximately 7:2:1. An exemplary liposome is CPX-351.

[0072] In an exemplary embodiment, a method is provided for enhancing the delivery of CPX-351 to an intracellular compartment of a target cell. The method comprises administering a therapeutically effective dose of CPX-351 and a therapeutically effective dose of an SR-BI inhibitor (e.g., BLT-1) to a subject requiring administration. The therapeutic agents are administered sequentially in separate pharmaceutical formulations or combined in a single pharmaceutical formulation. The amount of BLT-1 administered is effective in enhancing the uptake of CPX-351 by cells, thereby providing enhanced delivery of CPX-351 to this compartment. In this embodiment, liposome delivery to the intracellular compartment of the target cell is enhanced compared to delivery to the same compartment of the same target cell in the absence of the inhibitor.

[0073] In exemplary embodiments, a method is provided for altering the side effect profile of at least one therapeutic agent encapsulated in liposomes. The method comprises administering a therapeutically effective amount of liposomes and a therapeutically effective amount of an SR-BI inhibitor to a subject requiring administration. The amount of SR-BI inhibitor administered is effective in enhancing the uptake of liposomes by target cells, thereby enhancing the delivery of liposomes to target cells and reducing the amount of liposomes delivered to non-target cells, thereby altering the side effect profile of at least one therapeutic agent.

[0074] In various embodiments, at least one therapeutic agent is selected from cytarabine, daunorubicin, and combinations thereof. In an exemplary embodiment, the inhibitor of SR-BI is BLT-1.

[0075] In certain embodiments, a method is provided for determining whether administration of liposomes encapsulating at least one therapeutic agent is an appropriate treatment for a selected subject requiring treatment with at least one therapeutic agent. The method includes determining whether the target cells of the subject have an SR-BI expression level higher or lower 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 liposome-mediated treatment.

[0076] In various embodiments, the above method identifies candidates for combination therapy with liposomes and an SR-BI inhibitor. In various embodiments, SR-BI expression in target cells is below a predetermined threshold. In various embodiments, the liposomes contain at least 1 mol% distearoylphosphatidylglycerol (DSPG) or distearoylphosphatidylinositol (DSPI). In various embodiments, the liposomes contain about 20 mol% or less of cholesterol. In various embodiments, the liposomes contain distearoylphosphatidylcholine (DSPC). In various embodiments, the liposomes contain DSPC, DSPG, and cholesterol. In various embodiments, the liposomes contain DSPC, DSPG, and cholesterol in a molar ratio of about 7:2:1. In various embodiments, the liposomes are CPX-351. In various embodiments, the SR-BI inhibitor is BLT-1.

[0077] In various embodiments, the method further includes determining an appropriate dosage level of liposomes based on the determination of the SR-BI expression level in the target cells of the subject to which the liposomes are administered. For example, subjects expressing high levels of SR-BI on target cells may require a lower dosage of liposomes than subjects with low SR-BI expression, based on the principle that liposome uptake is greater in those target cells expressing even more SR-BI. In various embodiments, the dosage level is evaluated considering the administration of combination therapy to the subject using liposomes and an SR-BI inhibitor.

[0078] In various embodiments, the liposome is CPX-351. In various embodiments, the SR-BI inhibitor is BLT-1.

[0079] In exemplary embodiments, a therapeutically useful dose of liposomes containing cytarabine combined with an inhibitor of an equilibrium nucleoside transporter (ENT) (e.g., ENT1) results in a significant increase in the uptake of liposome-encapsulated cytarabine by cells expressing ENT (e.g., ENT1) compared to the uptake of unencapsulated cytarabine observed in the same cells in the presence of the ENT inhibitor (e.g., a "therapeutic effective dose").

[0080] In various embodiments, this disclosure provides a method for combination therapy of a disease in subjects requiring combination therapy. The method comprises administering to a subject requiring administration a therapeutically effective amount of liposomes containing cytarabine and an amount of an ENT inhibitor effective in enhancing the uptake of liposome-encapsulated cytarabine by target cells exhibiting proliferative disorder-associated genotypes and / or phenotypes compared to the uptake of unencapsulated cytarabine by the same cells in the presence of an ENT inhibitor.

[0081] In exemplary embodiments, the disease is a proliferative disorder, such as cancer. In various embodiments, the disease is a blood disorder, such as a hematopoietic proliferative disorder. In exemplary embodiments, the disease is a leukemia, such as acute myeloid leukemia (AML).

[0082] In exemplary embodiments, the disclosure provides a method for enhancing the delivery of a therapeutic agent to the intracellular compartment of a target cell in the presence of an ENT inhibitor. The method comprises administering to a subject requiring administration a therapeutically effective amount of liposomes containing a therapeutically effective amount of the therapeutic agent and a therapeutically effective amount of an ENT inhibitor, wherein the uptake of the therapeutic agent by the target cell is enhanced compared to the uptake of an unencapsulated therapeutic agent by the same cell in the presence of the ENT inhibitor, thereby providing enhanced delivery of the therapeutic agent to the intracellular compartment.

[0083] In exemplary embodiments, the present disclosure provides a method for enhancing the delivery of cytarabine into the intracellular compartment of a target cell in the presence of an ENT inhibitor, the method comprising administering to a subject requiring administration a therapeutically effective dose of liposomes containing a therapeutically effective dose of cytarabine and a therapeutically effective dose of an ENT inhibitor, wherein the uptake of cytarabine encapsulated in liposomes by the target cell is enhanced compared to the uptake of unencapsulated cytarabine by the same cell in the presence of the ENT inhibitor, thereby providing enhanced delivery of cytarabine into the intracellular compartment.

[0084] In exemplary embodiments, the present invention's method utilizing an ENT inhibitor, similar to SRBI, alters the adverse drug reaction profile of the therapeutic agent. Exemplary alterations to the adverse drug reaction profile include a reduction or improvement of at least one undesirable adverse reaction of the therapeutic agent to the target to which the therapeutic agent is administered. In exemplary embodiments, enhanced delivery to target cells results in less off-target delivery than in the absence of the inhibitor.

[0085] In some embodiments, the liposomes further contain daunorubicin. In exemplary embodiments, the liposomes contain CPX-351.

[0086] In some embodiments, the target cells are blood cells. In some embodiments, the target cells are leukemia cells.

[0087] In exemplary embodiments, target cells are abnormal cells (e.g., lesions) that underexpress or overexpress ENT compared to identical normal cells. The formulations of the present invention utilize this overexpression or underexpression to achieve enhanced liposome delivery to cells.

[0088] In an exemplary embodiment, the ENT inhibitor is NBMPR.

[0089] In some embodiments, liposomes and ENT inhibitors are administered sequentially in separate pharmaceutical formulations.

[0090] In some embodiments, liposomes and ENT inhibitors are combined in a single pharmaceutical formulation and administered to the subject in this form.

[0091] In exemplary embodiments, the liposomes contain at least 1 mol% of distearoylphosphatidylglycerol (DSPG) or distearoylphosphatidylinositol (DSPI).

[0092] In exemplary embodiments, liposomes contain approximately 20 mol% or less of cholesterol.

[0093] In exemplary embodiments, the liposomes contain distearoylphosphatidylcholine (DSPC).

[0094] In exemplary embodiments, the liposomes contain DSPC, DSPG, and cholesterol. The exemplary ratio of DSPC / DSPG / cholesterol is approximately 7:2:1 molar ratio.

[0095] In an exemplary embodiment, a method is provided for treating acute myeloid leukemia in subjects requiring treatment for acute myeloid leukemia, the subjects being at least about 60 years of age. The method comprises administering therapeutically relevant doses of CPX-351 and an SR-BI inhibitor to the subjects. Administration of CPX-351 in this patient population correlates with lower cardiotoxicity than administration of standard 7+3 therapy. In the exemplary embodiment, the SR-BI inhibitor is BLT-1.

[0096] B. Composition This disclosure provides a novel compound formulation comprising liposomes encapsulating one or more therapeutic agents and inhibitors (e.g., inhibitors of SR-BI and / or ENT).

[0097] In one embodiment, a pharmaceutical formulation is provided comprising a therapeutically effective amount of liposomes containing at least one encapsulated therapeutic agent and an inhibitor (e.g., an inhibitor of SR-BI and / or ENT). The liposomes and the inhibitor are present in a pharmaceutically acceptable carrier, diluent, etc.

[0098] In various embodiments, the pharmaceutical formulation comprises liposomes encapsulating one or more therapeutic agents, and an SR-BI inhibitor selected such that cellular uptake of the liposomes by target cells is increased compared to cellular uptake of the liposomes by the same target cells in the absence of the SR-BI inhibitor. In exemplary embodiments, the presence of the inhibitor in the formulation reduces delivery to non-target cells.

[0099] In exemplary embodiments, the liposome comprises a first lipid and a first sterol.

[0100] 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) (e.g., DSG-PEG2000), 1,2-dimiristoyl-rac-glycero-3-methoxypoly(ethylene glycol) (e.g., DMG-PEG2000), 1,2-dipalmitoyl-rac-glycero-3-methoxypoly(ethylene glycol) (e.g., DPG-PEG2000), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) (e.g., DSPE-PEG2000). In some embodiments, the polymer-bound lipid is 1,2-distearoyl-rac-glycero-3-methoxypoly(ethylene glycol) or 1,2-dimyristoyl-rac-glycero-3-methoxypoly(ethylene glycol). In some embodiments, the polymer-bound lipid is 1,2-distearoyl-rac-glycero-3-methoxypoly(ethylene glycol). In some embodiments, the polymer-bound lipid is DSG-PEG2000. In some embodiments, the lipid bilayer further comprises a second lipid. In some embodiments, the second lipid is a phospholipid. In some embodiments, the phospholipid is selected from the group consisting of phosphatidylcholine, sphingolipids, and hydrogenated sphingolipids. In some embodiments, the second lipid is sphingomyelin. In some embodiments, the second lipid is distearoylphosphatidylcholine (DSPC), hydrogenated sphingomyelin (dihydrosphingomyelin), or egg sphingomyelin. In some embodiments, the second lipid is distearoylphosphatidylcholine (DSPC) or hydrogenated sphingomyelin (dihydrosphingomyelin).In some embodiments, the second lipid is hydrogenated soy phosphatidylcholine (HSPC). In some embodiments, the second lipid is tamagosphingomyelin.

[0101] In some embodiments, the first lipid is a first phospholipid. In some embodiments, the phospholipid is selected from the group consisting of phosphatidylcholine, sphingolipids, and hydrogenated sphingolipids. In some embodiments, the first lipid is distearoylphosphatidylcholine (DSPC). In some embodiments, the first lipid is sphingomyelin. In some embodiments, the first lipid is hydrogenated sphingomyelin (dihydrosphingomyelin). In some embodiments, the lipid bilayer further comprises a second lipid. In some embodiments, the second lipid is a second phospholipid. In some embodiments, the second phospholipid is selected from the group consisting of phosphatidylcholine, sphingolipids, and hydrogenated sphingolipids. In some embodiments, the second lipid is distearoylphosphatidylglycerol (DSPG). In some embodiments, the second lipid is distearoylphosphatidylinositol (DSPI). In some embodiments, the second lipid is sphingomyelin. In some embodiments, the second lipid is hydrogenated sphingomyelin (dihydrosphingomyelin).

[0102] In some embodiments, the first sterol is cholesterol, a cholesterol derivative, or a phytosterol (e.g., β-sitosterol). In some embodiments, the first sterol is cholesterol. In some embodiments, the first sterol is β-sitosterol.

[0103] In some embodiments, the lipid bilayer comprises dihydrosphingomyelin (DHSM), cholesterol, and DSG-PEG2000. In some embodiments, the lipid bilayer comprises DHSM / cholesterol / DSG-PEG2000 in a molar ratio of approximately 53:45:2. In some embodiments, the lipid bilayer comprises distearoylphosphatidylcholine (DSPC), cholesterol, and DSG-PEG2000. In some embodiments, the lipid bilayer comprises DSPC / cholesterol / DSG-PEG2000 in a molar ratio of approximately 57:38:5.6.

[0104] In some embodiments, liposomes contain approximately 20 mol% or less of cholesterol.

[0105] In some embodiments, the liposomes contain at least about 1 mol% DSPG or DSPI.

[0106] In some embodiments, the liposomes contain distearoylphosphatidylcholine, distearoylphosphatidylglycerol, and cholesterol. In some embodiments, the liposomes contain distearoylphosphatidylcholine, distearoylphosphatidylglycerol, and cholesterol in a molar ratio of about 7:2:1.

[0107] In some embodiments, liposomes have an average diameter between approximately 50 nm and approximately 250 nm. In some embodiments, liposomes have an average diameter between approximately 50 nm and approximately 150 nm. In some embodiments, liposomes have an average diameter between approximately 50 nm and approximately 120 nm. In some embodiments, liposomes have an average diameter between approximately 50 nm and approximately 100 nm. In some embodiments, liposomes have an average diameter of approximately 80 nm. In some embodiments, liposomes have an average diameter between approximately 80 nm and approximately 150 nm. In some embodiments, liposomes have an average diameter of approximately 120 nm.

[0108] In some embodiments, the liposome composition has a polydispersity index (PDI) between about 0.001 and about 0.5. In some embodiments, the liposome composition has a polydispersity index (PDI) between about 0.001 and about 0.3. In some embodiments, the liposome composition has a polydispersity index (PDI) between about 0.001 and about 0.1. In some embodiments, the liposome composition has a polydispersity index (PDI) between about 0.1 and about 0.3. In some embodiments, the liposome composition has a polydispersity index (PDI) between about 0.1 and about 0.5. In some embodiments, the liposome composition has a polydispersity index (PDI) between about 0.3 and about 0.5.

[0109] In some embodiments, the liposome composition further comprises a carrier medium. In some embodiments, one or more liposomes are suspended in the carrier 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 physiological saline. In some embodiments, the carrier medium further comprises a buffer. In some embodiments, the buffer is HEPES buffer. In some embodiments, the buffer is PBS buffer. In some embodiments, the buffer is Tris buffer. In some embodiments, the buffer is MES buffer.

[0110] The pharmaceutical composition comprising the delivery vehicle of the present invention is prepared according to standard techniques and may contain water, buffered water, 0.9% physiological saline, 0.3% glycine, 5% dextrose, etc., individually or in combination, along with glycoproteins to enhance stability, such as albumin, lipoprotein, or globulin. These compositions may be sterilized by conventional, well-known sterilization techniques. The resulting aqueous solution may be packaged for use, or it may be filtered under sterile conditions, freeze-dried, and the freeze-dried preparation may be combined with a sterile aqueous solution before administration. If it is necessary to approximate physiological conditions, the composition may contain pharmaceutically acceptable auxiliary substances, such as pH adjusters and buffers, isotonic agents, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, or calcium chloride. In addition, the delivery vehicle suspension may contain lipid protectants to protect lipids from free radical and lipid peroxidation damage during storage. Lipophilic free radical inactivators such as α-tocopherol and water-soluble iron-specific chelating agents such as ferrioxamine are preferred.

[0111] The concentration of the delivery vehicle in the pharmaceutical formulation can vary widely, from less than approximately 0.05% by weight to typically around 2-5% by weight, or at least 2-5% by weight, and up to approximately 10-30% by weight, and is selected mainly by factors such as liquid volume and viscosity, according to the specific administration method chosen. For example, the concentration may be increased to reduce the amount of additive solution associated with the treatment. Alternatively, a delivery vehicle consisting of irritating lipids may be diluted to a low concentration to reduce inflammation at the administration site. For diagnostic purposes, the amount of delivery vehicle administered will depend on the specific label used, the condition being diagnosed, and the clinician's judgment.

[0112] In exemplary embodiments, the pharmaceutical composition of the present invention is administered intravenously. The dosage of the delivery vehicle formulation will depend on the ratio of the drug to lipids, as well as the opinion of the prescribing physician based on the patient's age, weight, and condition.

[0113] If a single composition contains multiple activators, the procedure essentially follows the steps outlined above. When administering drugs with separate delivery vehicle compositions, the administrations should be timed to maintain the desired ratio. This is usually achieved by administering the compositions simultaneously in calculated proportions.

[0114] In some embodiments, the liposome composition further comprises a therapeutic agent outside of one or more liposomes. In exemplary embodiments, the inhibitor of SR-BI and / or ENT is located outside the liposomes.

[0115] Liposomes can be prepared as described in “Liposomes: Rational Design” (AS Janoff ed., Marcel Dekker, Inc., New York, NY) or by additional techniques known to those skilled in the art.

[0116] The compositions of the present invention may be administered to humans, as well as warm-blooded animals including livestock and / or bird species. In addition to pharmaceutical compositions, formulations suitable for veterinary use may be prepared and administered in a manner suitable for the subject. For the treatment of human diseases, a qualified physician will determine how the compositions of the present invention should be used in terms of dosage, schedule, and route of administration, using established protocols. If the drug encapsulated in the delivery vehicle composition of the present invention exhibits reduced toxicity to the healthy tissue of the subject, such use may utilize dose escalation.

[0117] Preferably, the pharmaceutical composition of the present invention is administered parenterally, i.e., intra-arterial, intravenous, intraperitoneal, subcutaneous, or intramuscularly. More preferably, the pharmaceutical composition is administered intravenously or intraperitoneally by bolus injection. See, for example, Rahman, et al., U.S. Patent No. 3,993,754; Sears, U.S. Patent No. 4,145,410; Papahadjopoulos, et al., U.S. Patent No. 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.

[0118] In exemplary embodiments, the formulation comprises a therapeutically effective dose of CPX-351 and a therapeutically effective dose of an inhibitor (e.g., an inhibitor of SR-BI and / or ENT).

[0119] In exemplary embodiments, the formulation comprises a lipid transport inhibitor-1 (BLT-1), which is an SR-BI inhibitor, and a liposome containing at least one encapsulated therapeutic agent.

[0120] In exemplary embodiments, the formulation includes a combination of CPX-351 and lipid transport inhibitor-1 in a pharmaceutically acceptable carrier, diluent, etc.

[0121] In various embodiments, the formulation comprises NBMPR and a liposome containing at least one encapsulated therapeutic agent. In an exemplary embodiment, the formulation comprises NBMPR and a liposome containing cytarabine.

[0122] In exemplary embodiments, the formulation includes a combination of CPX-351 and NBMPR in a pharmaceutically acceptable carrier, diluent, etc.

[0123] In an exemplary embodiment, a kit is provided comprising a first container containing a therapeutically effective amount of liposomes containing at least one therapeutic agent encapsulated therein, and a second container containing a therapeutically effective amount of an SR-BI inhibitor. The kit further includes instructions for a clinician to administer the liposomes and the SR-BI inhibitor to a subject requiring treatment with these agents. The instructions optionally detail a procedure for preparing therapeutically effective amounts of the liposomes and / or the SR-BI inhibitor before administering the combination to the subject.

[0124] In various embodiments, kits are provided. An exemplary kit comprises a container containing a therapeutically effective amount of liposomes containing at least one therapeutic agent encapsulated therein, and a second container containing a therapeutically effective amount of an ENT inhibitor, the kit further including instructions for a clinician to administer the liposomes and the ENT inhibitor. In an exemplary embodiment, a container is provided containing liposomes containing a therapeutically effective amount of cytarabine, and a second container containing a therapeutically effective amount of an ENT inhibitor, the kit further including instructions for a clinician to administer the liposomes and the ENT inhibitor. The instructions optionally detail a procedure for preparing therapeutically effective amounts of liposomes and / or ENT inhibitors before administering the combination to a subject.

[0125] The therapeutic agents in the composition may be formulated separately in individual compositions in which each therapeutic agent is stably associated with an appropriate delivery vehicle. These compositions can be administered to a subject separately. In exemplary embodiments, the pharmacokinetics of the compositions are adjusted so that the ratio of therapeutic agents administered is maintained at the therapeutic target. It is therefore useful to construct a kit containing, in separate containers, a first composition containing a delivery vehicle stably associated with at least one first therapeutic agent, and in a second container, a second composition containing a delivery vehicle stably associated with at least one second therapeutic agent. These containers can then be packaged into a kit. The kit also includes instructions regarding the mode of administration of the compositions to a subject, which include a description of at least the ratio of the amounts of each composition administered. Alternatively, or further, the kit is constructed so that the amounts of the compositions in each container are pre-measured so that the combination of the contents of one container and the contents of the other represents the correct ratio. Alternatively, or additionally, the containers may be marked with measuring scales that allow for the distribution of appropriate amounts according to visible markings. The container itself may be usable for administration; for example, a kit may contain appropriate amounts of each composition in separate syringes. A formulation containing a pre-formulated therapeutic agent in the correct proportions may also be packaged in this way so that the formulation is administered directly from a syringe pre-packaged in the kit.

[0126] This disclosure presents novel methods, compositions, and kits. Supporting these various embodiments is the discovery that a representative SR-BI inhibitor, lipid transport inhibitor-1, administered in conjunction with liposomes encapsulating one or more therapeutic agents, significantly increases the uptake of liposomes, and consequently the therapeutic agent(s), by cells expressing scavenger receptor BI (SR-BI), at a therapeutic effective dose (HD-BLT-1). This result is surprising, given that administration of low doses of BLT-1 (LD-BLT-1) induces the opposite effect, reducing cellular uptake of liposomes.

[0127] The compositions and methods of the present invention are exemplified by a combination of liposome CPX-351, liposomes encapsulating cytarabine and daunorubicin, and the SR-BI inhibitor BLT-1. However, as shown herein, the methods, compositions, and principles of this disclosure are not limited to these exemplary embodiments.

[0128] This disclosure also provides compositions and methods exemplified by the combination of liposome CPX-351, which is a liposome encapsulating cytarabine and daunorubicin, and the ENT inhibitor NBMPR. However, as shown herein, the methods, compositions, and principles of this disclosure are not limited to these exemplary embodiments.

[0129] This disclosure also provides a method for treating a selected patient population with AML with CPX-351, thereby achieving therapeutic efficacy with lower-than-expected cardiotoxicity. In exemplary embodiments, the patient population includes individuals aged 60 years or older.

[0130] The combination of cytarabine and daunorubicin is the standard induction chemotherapy in treated patients with AML. However, the clinical efficacy of this combination has been limited in the elderly population. Because the two drugs have different clearances and metabolisms, co-administration of free drugs results in changes in the molar ratio at the pharmacological site of action over time, leading to intracellular molar ratios that may be additive, synergistic, or antagonistic. CPX-351 is a liposomal formulation encapsulating cytarabine and daunorubicin in the maximum synergistic ratio (5:1 molar ratio), thereby significantly increasing the systemic half-life of the encapsulated drugs as well as increasing the accumulation of the two drugs in leukemia cells in the optimal ratio. CPX-351 has received FDA approval for the treatment of adults with newly diagnosed treatment-related AML and AML with myelodysplastic-associated changes.

[0131] In a further exemplary embodiment, a method is provided for treating acute myeloid leukemia in subjects requiring treatment for acute myeloid leukemia, the subjects being at least about 60 years of age. The method comprises administering a therapeutically relevant dose of CPX-351 to the subjects. In the exemplary embodiment, cardiotoxicity markers with CPX-351 are lower in patients in this age group than in patients in this age group receiving standard 7+3 therapy.

[0132] The following examples are provided to illustrate exemplary embodiments of the present invention and are not intended to define or limit its scope. [Examples]

[0133] Example 1 1.1: Materials and Methods Cell culture and reagents HL-60 human promyelocytic leukemia cells (ATCC, VA, USA: CCL-240) were cultured in RPMI-1640 medium containing 20% ​​fetal bovine serum (FBS, catalog number 173012; NICHIREI BIOSCIENCES INC, Tokyo, Japan) and maintained at 37°C in a 5% CO2-humidified atmosphere. K562 chronic myeloid leukemia cells (JCRB Cell Bank, Osaka, Japan: JCRB0019), K562 / ADM adriamycin-resistant derivative of the K562 cell line due to P-glycoprotein expression (JCRB Cell Bank: JCRB1002), THP-1 human acute monocytic leukemia cells (JCRB Cell Bank: JCRB0112.1), and HL-60 / AD cytarabine and daunorubicin-resistant derivatives of the HL-60 cell line due to the expression of multidrug resistance-associated proteins (MRPs) established in the inventors' laboratory. 25The cells were cultured in RPMI-1640 medium with 10% FBS and maintained at 37°C in a 5% CO2 humidified atmosphere. CPX-351 was kindly provided by Jazz Pharmaceuticals (Oxford, UK). Cytarabine and daunorubicin were purchased from Selleck Chemicals (Houston, TX, USA).

[0134] Handling of CPX-351 The CPX-351 liposome formulation for injection is supplied in a 50 mL vial and is a sterile, pyrogenic, purple, lyophilized product containing 100 units, each unit corresponding to 1.0 mg of cytarabine and 0.44 mg of daunorubicin (as a base). The product was reconstituted with 19 mL of ultrapure water and gently stirred at room temperature for 10 minutes. Working aliquots of the reconstituted product were frozen and stored at -20°C.

[0135] Protein Corona Analysis CPX-351 was mixed with FBS in a 1:1 volume ratio and incubated at room temperature for 90 minutes. After incubation, the sample was centrifuged at 14,000 rpm for 15 minutes, and then the pellet was resuspended in phosphate-buffered saline (PBS). This procedure was repeated three times to wash the sample and remove unbound proteins. 26Next, the protein pellet was subjected to trypsin digestion and guanidine conversion in solution using a trypsin digestion and guanidine conversion kit (Thermo Fisher Scientific, Bremen, Germany) according to the manufacturer's instructions. Analysis was performed by liquid chromatography-tandem mass spectrometry (LC-MS / MS) using 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 (UniProtKB2022_01 results). The absolute concentration of the protein was determined by utilizing the mean MS signal response of the two or three most appropriate ionized peptides for a given protein using a well-established label-free technique. (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).

[0136] Flow cytometry for cell membrane lipid receptor expression Lipid receptor expression in each leukemia cell line was measured using a BD FACS Canto II cytometer (BD Bioscience, Heidelberg, Germany) and analyzed with FlowJo software (BD Biosciences). Cells were resuspended at a density of 1 × 10⁶ cells / mL in 100 μL of PBS containing 0.5% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO, USA). For the analysis of scavenger receptor class B1 (SR-BI) and low-density lipoprotein receptor-related protein 1 (LRP1), APC-conjugated SR-BI antibody (Miltenyi Biotec, Bergisch Gladbach, Germany), APC-conjugated LRP1 antibody (Miltenyi Biotec), or human IgG1 isotype control antibody (Miltenyi Biotec) were added to each suspension and incubated in the dark at 2–8°C for 10 minutes according to the manufacturer's instructions. For the analysis of low-density lipoprotein receptor (LDLR), 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 at room temperature in the dark for 30 minutes according to the manufacturer's instructions. After incubation, cells were washed with PBS containing 0.5% BSA and analyzed by BD FACS Canto II. The percentage of protein-positive cells was determined by the median fluorescence intensity of gated leukemia cells, normalized by the corresponding isotype control, and expressed in arbitrary units (AU) ± standard error (SE).

[0137] Flow cytometry of cellular uptake of CPX-351 The uptake of CPX-351 into various leukemia cell lines was measured using flow cytometry with the fluorescence of free daunorubicin. Cells were resuspended in 1 mL of FBS at a density of 1 × 10⁶ cells / mL. CPX-351 was then added to these cells to a final concentration of 19.5 μM in daunorubicin equivalent. After incubation for 2 hours (37°C, 5% CO₂), the cells were washed with PBS and analyzed by BD FACS Canto II at excitation wavelength 480 nm and emission wavelength 590 nm. Intracellular daunorubicin content was determined by the median fluorescence intensity of gated leukemia cells, normalized by the corresponding negative control, and expressed as AU ± SE. 27,28 .

[0138] MRP inhibition and P-glycoprotein assay HL-60 / AD and K562 / ADM were resuspended in RPMI-1640 medium containing 10% FBS at a density of 1 × 10⁶ cells / mL. The MRP inhibitor MK-571 (50 μM, Cayman Chemical, Ann Arbor, MI, USA) was added to HL-60 / AD and incubated for 30 minutes (37°C, 5% CO₂). 29 The P-glycoprotein inhibitor thaliquidal (1 μM, Selleck Chemicals, Houston, TX, USA) was added to K562 / ADM and incubated for 10 minutes (37°C, 5% CO2). 30 Subsequently, CPX-351 was added to these cells to a final concentration of 3.9 μM in daunorubicin equivalent. After incubation for 4 hours (37°C, 5% CO2), the cells were washed with PBS and analyzed by BD FACS Canto II using the method described above.

[0139] CPX-351 uptake pathway inhibition assay Uptake of CPX-351 into leukemia cell lines is controlled by chlorpromazine (CME inhibitor; TCI, Tokyo, Japan) at 10 μg / mL. 31 25 μM 5-(N-ethyl-N-isopropyl)-amiloride (EIPA, a micropinocytosis inhibitor; MedChemExpress, Monmouth Junction, NJ, USA)32 , low-dose lipid transport inhibitor-1 (LD-BLT-1, 400 nM, a selective inhibitor of SR-BI; Merck, Darmstadt, Germany), and high-dose lipid transport inhibitor-1 (HD-BLT-1, 40 μM) 33~35 were inhibited by. These inhibitors were added to the cells alone or in combination and incubated for 1 hour (37 °C, 5% CO2). Then, CPX-351 was added to the cells, and after a 2-hour incubation (37 °C, 5% CO2), BD FACS Canto II analysis was performed using the method described above.

[0140] Analysis of patient specimens Peripheral blood or bone marrow specimens were obtained from newly diagnosed or relapsed / refractory AML patients. This study was conducted in accordance with the Helsinki Declaration, and informed consent was obtained from all patients. The protocol was approved by the Institutional Review Board of the University of Fukui School of Medical Sciences. Blood or bone marrow specimens were separated by Ficoll gradient.

[0141] Growth assay To evaluate the growth inhibitory effect on each cell line, the 3'-(1-[(phenylamino)-carbonyl-3,4-tetrazolium])-bis(4-methoxy-6-nitro)benzenesulfonic acid sodium hydrate (XTT) assay was performed with a slight modification using the manufacturer's protocol (Roche, Indianapolis, IN, USA). 36 . The 50% inhibitory concentration (IC50) values were determined from the growth inhibition curves generated for each treatment.

[0142] statistical analysis Data from at least three independent experiments were analyzed using GraphPad Prism (version 9.3.1). The Student's t-test was used to compare two groups. One-way analysis of variance was used to compare three or more groups. Statistical significance was determined using a two-sided P-value of < 0.05.

[0143] 1.2:Results Expression of CPX-351 protein corona and lipid receptor in leukemia cell lines Table 1 lists the abundant proteins constituting the CPX-351 protein corona, identified using the mean MS signal response of three best-in-class ionized peptides. Table 2 lists those identified using the mean MS signal response of two best-in-class ionized peptides. The CPX-351 liposome protein corona consists of apolipoprotein A-II (relative ratio: 47.34% ± SE: 6.7) and AI (5.25% ± 0.52), ligands for SR-BI. 37、38 , as well as apolipoprotein C-III (21.71% ± 1.17%) that binds to LDLR and LRP1. 39 It was mainly composed of apolipoproteins, including [specifically, apolipoproteins]. [Table 1] [Table 2]

[0144] SR-BI expression 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 showing significantly higher expression than HL-60 and K562 (p < 0.05 for both). SR-BI expression was significantly higher in HL-60 / AD (96.0 AU ± 15.3) than in parental HL-60 cells (p < 0.05), and similar in K562 / ADM (38.0 AU ± 1.7) and parental K562 cells (Figure 1A). LDLR was expressed in THP-1 (4.1 AU ± 0.06), but decreased in HL-60 (1.5 AU ± 0.08) and K562 (1.2 AU ± 0.03) (Figure 1B). LRP1 was expressed in THP-1 (1.7 AU ± 0.05), but not in HL-60 or K562 (Figure 1C). As a result, the inventors planned to analyze endocytosis in leukemia cell lines, focusing particularly on SR-BI, a receptor that binds to apolipoprotein A, a major component of protein coronavirus, and is expressed at various levels in all leukemia cell lines.

[0145] Cellular uptake of CPX-351 CPX-351 uptake 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 uptake being significantly higher than that of HL-60 and K562 (p < 0.01 for both). CPX-351 uptake 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 parental cells (Figure 2A). The IC50 of CPX-351 was significantly higher in both HL-60 / AD (7120 nM cytarabine equivalent, p<0.01) and K562 / ADM (512.7 nM cytarabine equivalent, p<0.01) compared to their respective parent cells (Table 3). When the MRP inhibitor MK-571 was added to HL-60 / AD, intracellular CPX-351 in HL-60 / AD significantly increased (9.1 AU ± 0.07) compared to the control (3.1 AU ± 0.2, p<0.01) (Figure 2B), and correspondingly, the IC50 in HL-60 / AD (478.3 nM cytarabine equivalent) decreased compared to the control (7120 nM, p<0.01) (Figure 2C). Similarly, when the P-glycoprotein inhibitor thaligidal was added to K562 / ADM, intracellular CPX-351 in K562 / ADM significantly increased (3.0 AU ± 0.1) compared to the control (1.5 AU ± 0.1, p < 0.01) (Figure 2D), and correspondingly, IC50 (142.2 nM in cytarabine equivalent) in K562 / ADM significantly decreased compared to the control (512.7 nM, p < 0.01) (Figure 2E). [Table 3]

[0146] Inhibition of the CPX-351 uptake pathway Treatment with chlorpromazine, a CME inhibitor, significantly reduced CPX-351 uptake in HL-60, K562, and THP-1 cells compared to control cells, with decreases of 18.5%, 50.6%, and 34.6%, respectively (p<0.01) (Figures 3A-C). Similarly, treatment with EIPA, a macropinocytosis inhibitor, significantly reduced uptake in HL-60, K562, and THP-1 cells compared to control cells, with decreases of 9.9%, 43.4%, and 30.9%, respectively (p<0.01) (Figures 3A-C). Furthermore, treatment with LD-BLT-1 significantly reduced uptake in HL-60, K562, and THP-1 cells by 17.0%, 27.7%, and 24.4%, respectively, compared to control cells (p<0.01) (Figures 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 uptake in HL-60, K562, and THP-1 by more than twofold compared to control cells, increasing by 334.6%, 249.3%, and 266.8%, respectively (p<0.01, for all) (Figures 4A-C). The enhancement of uptake by HD-BLT-1 was significantly inhibited in HL-60, K562, and THP-1 by adding EIPA to cell cultures containing HD-BLT-1, decreasing by 30.4%, 33.4%, and 23.9%, respectively (p<0.01, for all) (Figures 4A-C). When chlorpromazine was added to cell cultures containing HD-BLT-1, the enhancement of uptake by K562 was significantly inhibited (p<0.01), but uptake by HL-60 and THP-1 did not change significantly, decreasing by 23.7%, 1.4%, and 11.3%, respectively (Figures 4A-C). Uptake of free daunorubicin did not change significantly in HL-60, K562, or THP-1, regardless of whether these inhibitors were used alone or in combination (Figures 6A-C).When cell cultures containing HD-BLT-1 and CPX-351 were incubated at 4°C, all endocytosis pathways were inhibited, and no CPX-351 uptake was observed in HL-60 (1.4 AU ± 0.14), K562 (1.9 AU ± 0.24), and THP-1 (1.6 AU ± 0.07) cells (Figure 4A-C). However, uptake of the free drug daunorubicin was 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 CPX-351 uptake in resistant cells HL-60 / AD and K562 / ADM by more than twofold compared to control cells, increasing by 434.4% and 214.2%, respectively (p<0.01, respectively) (Figure 4D-E).

[0147] Expression of SR-BI and uptake of CPX-351 in patient samples Finally, the inventors investigated whether the enhancing effect of HD-BLT-1 could be detected in patient-derived leukemia cells. Leukemic blasts were collected from three AML patients. Sample 1 was from a 48-year-old male who experienced a relapse of FLT3 / ITD-positive AML with KMT2A rearrangement after receiving standard induction and palliative chemotherapy (French-American-British (FAB) classification: M4, World Health Organization (WHO) classification: AML with myelodysplastic-related changes). 40 Sample 2 was resistant to gilteritinib. Sample 2 was a 64-year-old male who experienced a relapse of AML with a combined karyotype (FAB:M0, WHO:AML, not specified) after receiving standard induction and palliative chemotherapy, and was resistant to azacitidine. Sample 3 was a 72-year-old female with newly diagnosed FLT3 / ITD-positive AML (FAB:M5, WHO:AML with NPM1 mutation). SR-BI expression was highest in Sample 3, followed by Samples 1 and 2 (Figure 5A). Similarly, CPX-351 uptake was highest in Sample 3, followed by Samples 1 and 2. Addition of HD-BLT-1 significantly increased CPX-351 uptake by more than twofold in Samples 1 and 3, but did not change uptake in Sample 2 (Figure 5B).

[0148] 1.3: Discussion Protein corona analysis of CPX-351 revealed that apolipoproteins AI and A-II are its major components. Therefore, we focused particularly on SR-BI, the lipid receptor for apolipoproteins AI and A-II, to investigate endocytosis in leukemia cell lines. In various cell lines, HL-60, K562, and THP-1, the addition of chlorpromazine and EIPA significantly reduced CPX-351 uptake. Similarly, the addition of LD-BLT-1 significantly reduced CPX-351 uptake in these cell lines. These findings suggest that, in addition to CME and macropinocytosis, the SR-BI-mediated uptake pathway may be an important mechanism for the internal translocation of CPX-351 into leukemia cells. SR-BI expression in HL-60 / AD and K562 / ADM was at least as high as in their respective parent cell lines, but CPX-351 uptake was significantly lower. The addition of MK571 to HL-60 / AD and talikidal to K562 / ADM significantly increased CPX-351 uptake and significantly decreased IC50, suggesting that MRP and P-glycoprotein expression may be a resistance mechanism to CPX-351. Addition of HD-BLT-1 more than doubled CPX-351 uptake in all cell lines, including resistant cells. This was significantly suppressed by the addition of EIPA. In patient samples, SR-BI expression and CPX-351 uptake showed similar trends. Furthermore, in patient samples, the addition of HD-BLT-1 resulted in a significant increase in CPX-351 uptake, while samples with low SR-BI expression did not show a significant increase in CPX-351 uptake.

[0149] 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 various pathological conditions including melanoma, lymphoma, hepatocellular carcinoma, and atherosclerosis. 41 The apolipoprotein-rich properties of these nanoparticles may allow them to be recognized by cells that overexpress apolipoprotein receptors. Previous studies have demonstrated that apolipoproteins AI and A-II are important components of the protein corona surrounding liposomes. 26、42~44 This is consistent with the findings of this study. SR-BI is the primary receptor involved in the selective internal translocation of cholesteryl esters derived from high-density lipoprotein (HDL) molecules, which consist of apolipoproteins AI and A-II. 45,46 SR-BI has recently attracted attention for its role in the development of cancer. 47 High 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 hepatocytes. 45,47 High expression of SR-BI is associated with breast 48 ,lung 49 , renal cell carcinoma 50 , and neuroblastoma 51 It is associated with a poor prognosis in [the disease]. However, the expression and significance of SR-BI in leukemia have not yet been fully analyzed. In this study, all cell lines used expressed SR-BI. Recently, various nanomedicines carrying therapeutic agents that target cells highly expressing SR-BI have been tested. 52~54 .

[0150] Interestingly, while LDL cholesterol is delivered to cells via LDLR binding and endocytosis, HDL particles bind to SR-BI on the cell surface, and the cholesteryl ester is selectively delivered into the cell interior without the entire lipoprotein particle being transported internally. 34,46SR-BI has been demonstrated to have the ability to form hydrophobic tunnels within the cell membrane, enabling the selective uptake of hydrophobic molecules that evade lysosome processing. BLT-1, a selective inhibitor of SR-BI, can bind to the cysteine ​​384 residue of SR-BI and inhibit the translocation of cholesteryl esters. 33 BLT-1 is highly specific to the SR-BI pathway and does not interfere with receptor-mediated endocytosis or other forms of intracellular vesicular transport. 33 In addition to inhibiting cholesteryl ester uptake via SR-BI, BLT-1 has the unique characteristic of increasing the binding affinity of SR-BI to HDL in a dose-dependent manner, resulting in enhanced HDL binding. 33,34 .

[0151] This study showed that CPX-351 uptake decreased upon the addition of LD-BLT-1, suggesting that the components of CPX-351 (cytarabine and daunorubicin) may be taken up by leukemia cells via a non-endocytotic mechanism mediated by SR-BI. However, CPX-351 uptake significantly increased upon the addition of HD-BLT-1, which was suppressed by the addition of EIPA. In each cell line, CPX-351 uptake tended to decrease when combined with chlorpromazine compared with EIPA when combined with EIPA. However, when CPX-351 was combined with HD-BLT-1, uptake decreased when EIPA was added, rather than chlorpromazine. The addition of HD-BLT-1 also increased CPX-351 uptake in patient samples, but not in samples with low SR-BI expression. These findings suggest that HD-BLT-1 enhances the binding of CPX-351 to SR-BI, potentially activating other endocytotic pathways such as macropinocytosis in addition to CME, leading to increased CPX-351 uptake in leukemia cells. Macropinocytosis is uniquely expressed by some cancer cells compared to healthy cells of the same type, and has recently attracted considerable attention as a potential method of selective drug delivery to cancer cells. 12,17For example, cancer cells with mutated KRAS activate macropinocytosis to actively take up nutrients such as glucose, lipids, and albumin, meeting their energy requirements for survival and proliferation. 55 HDL particles reconstituted with apolipoprotein E3 have been found to activate macropinocytosis and are efficiently taken up by glioblastoma cells. 56 .

[0152] When using nanoparticles in vivo, it is important to consider factors that may affect protein corona formation, such as higher serum concentrations, blood flow, and serum complexity. 19 This study analyzed the CPX-351 uptake pathways via CME, macropinocytosis, and SR-BI (non-endocytotic pathway). Other endocytotic pathways may also be involved in nanoparticle uptake. 10,57 .

[0153] In conclusion, the non-endocytotic pathway mediated by SR-BI, which binds to apolipoproteins AI and A-II, the main components of the CPX-351 protein corona, is a crucial nanoparticle uptake pathway in leukemia cells. Enhancement of the binding between CPX-351 and SR-BI via the addition of HD-BLT-1 may increase uptake through the activation of macropinocytosis. SR-BI may serve as a potential biomarker for CPX-351 therapy, and the combination of HD-BLT-1 and nanoparticle formulations may enhance the therapeutic effect in treatment.

[0154] Example 2 Drug sensitivity to cytarabine (ara-C) in the presence or absence of the nucleoside transporter inhibitor NBMPR. HL-60 cells were pre-incubated for 30 minutes with or without 1 μM NBMPR, and then treated with ara-C for a further 72 hours. Subsequently, cell proliferation inhibition (IC) was assessed using the XTT assay. 50The following was determined: NBMPR and S-(4-nitrobenzyl)-6-thioinosine are potent inhibitors of cell surface nucleoside transporters. The proliferation inhibitory effect of ara-C was reduced in the presence of NBMPR. [Table 4]

[0155] Drug sensitivity of daunorubicin (DNR) in the presence or absence of the nucleoside transporter inhibitor NBMPR. HL-60 cells were pre-incubated for 30 minutes with or without 1 μM NBMPR, and then treated with daunorubicin (DNR) for a further 72 hours. Subsequently, cell proliferation inhibition (IC) was performed using the XTT assay. 50 The following was determined: DNR inhibited HL-60 cell proliferation to the same extent, regardless of the presence of NBMPR. [Table 5]

[0156] Drug susceptibility of CPX-351 in the presence or absence of the nucleoside transporter inhibitor NBMPR. HL-60 cells were pre-incubated for 30 minutes with or without 1 μM NBMPR, and then treated with CPX-351 for a further 72 hours. Subsequently, cell proliferation inhibition (IC) was assessed using the XTT assay. 50 The presence of NBMPR was determined. As shown above, the uptake of ara-C into cells is inhibited when NBMPR is present. However, as shown in Table 6, CPX-351 inhibited the proliferation of HL-60 cells to the same extent regardless of the presence of NBMPR. [Table 6]

[0157] Example 3 Inhibition of DNA synthesis by CPX-351 HL-60 cells were pre-incubated with 1 μM CPX-351 at 37°C for 2 hours, and then incubated with 3H-thymidine at 37°C for a further 4 hours. DNA was then extracted from the cells, and its radioactivity was measured using a scintillation counter. DNA synthesis, calculated from the radioactivity of thymidine uptake into the cells' DNA, was compared between untreated cells and CPX-351-treated cells (Figure 7).

[0158] Example 4 background: CPX-351, a two-drug liposome containing daunorubicin and cytarabine in a 1:5 molar ratio, is approved in the United States and Europe for newly diagnosed treatment-related acute myeloid leukemia (AML) or AML with myelodysplastic changes. In a Phase 3 clinical trial (NCT01696084), CPX-351 demonstrated improved overall survival and a similar safety profile compared to 7+3, however, the safety of CPX-351 related to cardiac function was not fully accounted for and is an important consideration due to the known risk of anthracycline-derived cardiotoxicity. Here, we evaluate the impact of CPX-351 compared to 7+3 chemotherapy on cardiac complications in elderly patients with previously untreated high-risk AML, using data from the Phase 3 trial.

[0159] method: In the Phase 3 trial, patients (60-75 years old; AML according to WHO 2008 criteria) received CPX-351 (100 units / m² on days 1, 3, and 5). 2 , and in the second induction, on days 1 and 3) or 7+3 (cytarabine 100 mg / m²) 2 / day for 7 days [5 days for the second induction] and daunorubicin 60mg / m² 2Patients were randomized to receive the drug on days 1-3 (days 1-2 for the second induction). Cardiac dysfunction was assessed by cardiac adverse events (AEs) and echocardiographic findings, including left ventricular ejection fraction (LVEF) and total ventricular long axis deformation (GLS), at baseline and at two follow-up visits (Follow-up 1: 30-45 days after the last induction therapy, or before intensification or salvage therapy; Follow-up 2: 150 or 45 days (±10 days) after the last treatment).

[0160] result: The study population consisted of 118 patients (CPX-351 group: n=63, 7+3 group: n=55) with normal baseline LVEF (LVEF > 53%) and who underwent at least one ECHO measurement (LVEF or GLS) after baseline. The mean cumulative dose of daunorubicin during the study was lower in patients treated with CPX-351 compared to 7+3 (570 vs. 378 mg, respectively). The CPX-351 treatment group had a lower proportion of patients with LVEF < 53% (11% vs. 26%) or GLS ≤ 18% (25% vs. 38%) at follow-up 1 or 2 compared to the 7+3 therapy group. In follow-up 1 or 2, the CPX-351 group showed a lower frequency of clinically significant changes in LVEF (absolute change from baseline >10% and LVEF <53%) or changes in GLS (relative change from baseline >12% and GLS <18%) compared to the 7+3 group (8% vs. 16% and 21% vs. 44%, respectively). The incidence of all cardiac events was similar in both CPX-351-treated and 7+3-treated patients (38% vs. 40%). Tachycardia was more frequent in CPX-351-treated patients than in 7+3-treated patients (19% vs. 9%), and atrial fibrillation was more frequent in 7+3-treated patients than in CPX-351-treated patients (13% vs. 3%).

[0161] Conclusion: In addition to previously reported survival and safety data for CPX-351, this trial also demonstrated that CPX-351 has lower cardiotoxicity compared to the standard 7+3 therapy in elderly (60-75 year old) AML patients.

[0162] The present invention has been described with reference to various exemplary embodiments and examples. As will be apparent to those skilled in the art, other embodiments and variations of the present invention may be devised by those skilled in the art without departing from the true spirit and scope of the invention. The appended claims should be construed to include all such embodiments and equivalent variations.

[0163] Any patents, patent applications, and publications disclosed herein are incorporated herein by reference in their entirety.

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Claims

1. A pharmaceutical preparation comprising (a) a liposome containing one or more therapeutically effective amounts of a therapeutic agent, and (b) an inhibitor of SR-BI.

2. A kit comprising a first container containing liposomes encapsulating one or more therapeutically effective doses of therapeutic agents, and a second container containing a therapeutically effective dose of an SR-BI inhibitor, the kit further comprising instructions for a clinician to administer the liposomes and the SR-BI inhibitor.

3. The kit according to claim 2, wherein the instruction manual details a procedure for preparing a therapeutically effective amount of the pharmaceutical formulation containing the liposomes and / or the pharmaceutical formulation containing the SR-BI inhibitor before administering the liposomes and the SR-BI inhibitor to a subject.

4. A method for combination therapy of a proliferative disorder, e.g., cancer, e.g., leukemia, e.g., acute myeloid leukemia, the method comprising administering to a subject requiring administration a therapeutically effective amount of liposomes and an amount of an SR-BI inhibitor effective in enhancing the uptake of the liposomes by target cells exhibiting a genotype and / or phenotype associated with the proliferative disorder compared to the uptake of the liposomes by the same cells in the absence of the SR-BI+ inhibitor.

5. A method for enhancing the delivery of liposomes to an intracellular compartment of a target cell, the method comprising administering a therapeutically effective amount of the liposomes and a therapeutically effective amount of an SR-BI inhibitor to a subject requiring administration, wherein the amount of the administered SR-BI inhibitor is effective in enhancing the uptake of the liposomes by the target cell, thereby providing enhanced delivery of the liposomes to the intracellular compartment.

6. A method for altering the adverse effect profile of liposomes, the method comprising administering to a subject requiring administration a therapeutically effective amount of the liposomes and an amount of an SR-BI inhibitor effective in enhancing the uptake of the liposomes by target cells, and reducing the amount of liposomes delivered to non-target cells, thereby altering the adverse effect profile of the liposomes.

7. A method for reducing resistance to one or more therapeutic agents in target cells that exhibit resistance to one or more therapeutic agents, the method comprising administering to a subject requiring administration a therapeutically effective dose of one or more therapeutic agents encapsulated in liposomes, and an amount of SR-BI inhibitor effective for enhancing intracellular delivery of the one or more therapeutic agents to the target cells, reducing their efflux from the target cells, and a combination thereof, thereby reducing resistance in the target cells.

8. A method for enhancing the delivery of one or more therapeutic agents to an intracellular compartment of a target cell, the method comprising administering one or more therapeutic agents encapsulated in liposomes, wherein the liposomes are formulated with components selected for their ability to interact with scavenger receptor BI, thereby enhancing the delivery of the first and second therapeutic agents 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 liposomes contain distearoylphosphatidylcholine, distearoylphosphatidylglycerol, and cholesterol in a molar ratio of about 7:2:

1.

11. A method for determining whether administration of liposomes to a selected subject is an appropriate treatment for the subject, the method comprising determining whether the expression level of SR-BI in the target cells of the subject is higher than a pre-selected SR-BI expression level threshold; if the SR-BI expression level of the subject is above the threshold, the method for identifying the subject as a candidate for liposome-based treatment.

12. The method according to claim 11, further comprising determining an appropriate dosage level for a drug selected from the liposomes, the SR-BI inhibitor, and combinations thereof, based on determining the expression level of SR-BI in the selected target cells.

13. The method according to any one of the prior claims, wherein the one or more therapeutic agents are selected from cytarabine, daunorubicin, CPX-351, and combinations thereof.

14. The method according to any one of the prior claims, wherein the inhibitor of SR-BI is BLT-1.

15. The method according to any one of the prior claims, wherein the inhibitor of SR-BI is BLT-1.

16. The method according to any one of the prior claims, wherein the target cells are blood cells.

17. The method according to any one of the prior claims, wherein the target cells are leukemia cells.

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

19. The method according to any of the prior claims, wherein the liposomes and the SR-BI inhibitor are combined into a single pharmaceutical formulation and administered to the subject in this form.

20. A method for treating acute myeloid leukemia in a subject requiring treatment for acute myeloid leukemia, wherein the subject is a member of a population at least about 60 years of age, and the method comprises administering a dose of CPX-351 relevant to the treatment to the subject.

21. The method according to claim 20, wherein the marker of cardiotoxicity due to administration of CPX-351 is even lower in one or more members of the population compared to the same marker or cardiotoxicity in one or more members of the population receiving standard 7+3 therapy.

22. The method according to claim 8, wherein the liposomes contain cholesterol at a concentration of about 20 mol% or less.

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

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

25. A pharmaceutical preparation comprising (a) liposomes containing a therapeutically effective amount of cytarabine and (b) an ENT inhibitor.

26. A kit comprising a first container containing liposomes comprising a therapeutically effective amount of cytarabine, and a second container comprising a therapeutically effective amount of an ENT inhibitor, wherein the kit further comprises instructions for a clinician to administer the liposomes and the ENT inhibitor.

27. The kit according to claim 26, wherein the instruction manual details a procedure for preparing a therapeutically effective amount of a pharmaceutical formulation containing the liposomes and / or a pharmaceutical formulation containing the ENT inhibitor before administering the liposomes and the ENT inhibitor to the subject.

28. A method for combination therapy of proliferative disorders, e.g., cancer, e.g., leukemia, e.g., acute myeloid leukemia, the method comprising administering to a subject requiring administration a liposome containing a therapeutically effective amount of cytarabine and an amount of an ENT inhibitor effective in enhancing the uptake of liposome-encapsulated cytarabine by target cells exhibiting a genotype and / or phenotype associated with the proliferative disorder compared to the uptake of unencapsulated cytarabine by the same cells in the presence of an ENT inhibitor.

29. A method for enhancing the delivery of cytarabine to an intracellular compartment of a target cell in the presence of an ENT inhibitor, the method comprising administering to a subject requiring administration a therapeutically effective amount of liposomes containing cytarabine and a therapeutically effective amount of an ENT inhibitor, wherein the uptake of cytarabine encapsulated in liposomes by the target cell is enhanced compared to the uptake of unencapsulated cytarabine by the same cell in the presence of the ENT inhibitor, thereby providing enhanced delivery of cytarabine to the intracellular compartment.

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

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

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

33. The method according to any one of claims 25 to 32, wherein the target cells are blood cells.

34. The method according to any one of claims 25 to 33, wherein the target cells are leukemia cells.

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

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