RU2786077C2 - Analyses and methods for selection of treatment scheme for individual with leukemia - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to the field of medicine; it can be used for the treatment of hemoblastosis. A method for the treatment of hemoblastosis in an individual includes the administration of effective amount of a liposomal composition containing cytarabine and daunorubicin incapsulated together in a molar ratio of 5:1 (CPX-351), and FLT-3 inhibitor selected from a group consisting of from quizartinib, midostaurin, tandutinib, sorafenib, sunitinib, lestaurtinib, crenolanib, gilteritinib, AST-487, dovitinib and linifanib. In this case, the specified liposomal composition and FLT-3 are administered simultaneously, or the specified liposomal composition is administered before the treatment with FLT-3 inhibitor.

EFFECT: invention provides for an increase in the efficiency of treatment of hemoblastosis due to a synergetic effect of a combination of CPX-351 and FLT-3 inhibitor.

4 cl, 11 dwg, 5 tbl, 5 ex

Description

Cross-reference to related applications

[0001] This application claims priority based on U.S. Provisional Application No. 62/254109, filed Nov. 11, 2015. The contents of this application are incorporated herein by reference.

Technical field

[0002] The present invention relates to the field of diagnosis and treatment of malignant neoplasms. More specifically, it relates to the identification of individuals who will benefit from treatment using a synergistic liposome combination of daunorubicin and cytarabine.

Prior Art

[0003] CPX-351 is a nanoscale (100 nm diameter) low cholesterol liposomal formulation containing cytarabine and daunorubicin co-encapsulated in a 5:1 molar ratio (US Pat. Nos. 8,022,279 and 8,431,806) shown to be optimally synergistic and vivo, and in vivo. Several preclinical studies have shown significant improvements in efficacy over the conventional free drug combination, and more importantly, CPX-351 provides improved complete remission and survival rates compared to standard of care in clinical studies, one of which was in elderly patients. with newly diagnosed acute myelogenous leukemia (AML) and the other in adult patients with first relapse of AML. The current treatment for AML is the "standard of care", also referred to as the "7+3" treatment.

[0004] The present invention is focused on identifying patient populations that benefit from treatment with CPX-351, as opposed to the current "standard of care," and on assays that take into account the heterogeneity of malignancies, t. they occur in different patients.

[0005] The efficacy observed in clinical studies using CPX-351 can be attributed to 1) increased concentrations of cytarabine : daunorubicin maintained in the bloodstream in a synergistic ratio for long periods of time (thus avoiding antagonistic ratios), 2) increased accumulation and persistence of CPX-351 in the bone marrow, and 3) selective accumulation and cytotoxicity of intact CPX-351 liposomes in leukemic cells compared to normal cells in the bone marrow. CPX-351 has been shown to be very effective in rapidly clearing leukemic cells from the bloodstream and bone marrow in a large proportion of patients at high risk of AML, including those who did not respond to the "standard of care" 7+3 cytarabine:daunorubicin immediately prior to CPX-351 therapy. and in adult patients with advanced acute lymphocytic leukemia (ALL) and myelodysplastic syndrome (MDS).

[0006] Rapid ex vivo cytotoxicity assays using freshly isolated leukemia cells from patients with hematological malignancies can sometimes be useful in determining the spectrum of activity of therapeutic agents against hematological malignancies. In this regard, the authors of the present invention developed a method for collecting and purifying circulating blast cells from freshly obtained blood samples from patients with a wide range of hemoblastoses, including acute myelogenous leukemia (AML), ALL, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), myeloproliferative neoplasia (MPN) and lymphoma to assess antineoplastic activity in selected patients. Applicants have also determined the antiproliferative/cytotoxic activity profile for many new research agents, classified according to the type of hemoblastosis, as well as specific phenotypic profiles of subtypes of patients with such malignancies. In such assays, 50% and 90% growth inhibition concentrations (IC ₅₀ and IC ₉₀ , respectively) are used to predict whether treatment sensitivity reflects drug concentrations that are appropriate and what proportion of said patient class is sensitive enough to allow testing in a clinical trial setting. Thus, the correlation between drug susceptibility and subpopulation phenotypes allows the identification of biomarkers that can guide patient selection.

[0007] However, historically, ex vivo evaluation of nanoparticulate drug formulations (e.g., liposomes and nanoparticles) for cytotoxicity against malignant cells has generally not been performed due to the fact that such formulations are typically prepared so as to act as an in situ drug infusion reservoir, whereby the agents accumulate preferentially at the growth sites of the malignant neoplasm and, once there, they slowly release the free drugs, which are then taken up by the malignant cells. Because ex vivo drug release rates are typically much lower than those observed in vivo, the cytotoxic activities of encapsulated anticancer drugs are often of the order of magnitude lower ex vivo than those observed with the corresponding free drugs, and ex vivo testing is less reliable in predicting in vivo success .

[0008] Clearly, in the case of CPX-351, Applicants have shown that human leukemic cells take up cytarabine and daunorubicin in liposome-encapsulated form ex vivo and in vivo in an energy-dependent manner. Once taken up by vacuoles in the cytoplasm, liposomes generate a bioavailable drug, resulting in cytolytic activity. This not only delivers a synergistic 5:1 molar ratio of cytarabine:daunorubicin, but also results in an ex vivo cytotoxic activity of CPX-351 (taking into account IC ₅₀ values) comparable to, and in some cases greater than, that of the free drugs as presented in the present description.

[0009] In pilot studies of ex vivo cytotoxicity of CPX-351 against fresh leukemic blasts from patients with various leukemic conditions, IC ₅₀ values of as little as 50 nM cytarabine:10 nM daunorubicin were obtained under incubation conditions in which drug release from liposomes in the medium was undetectable ( Tyner, J., et al., Blood (2010) 116: Abstract 2886). This confirmed that the direct antileukemic activity of intact CPX-351 liposomes previously documented in leukemic cell lines was significant in blasts recently obtained from patients. This also makes it possible to separate the influence of genotypic/phenotypic cellular traits on the sensitivity of blasts to CPX-351, regardless of RK.

[0010] Thus, in contrast to the properties of nanoparticle compositions, as a rule, the study of cytotoxicity of CPX-351 ex vivo against fresh samples from patients and/or uptake of CPX-351 with fresh samples from patients provides an opportunity to predict the antineoplastic activity of CPX-351 in individual patients and types of populations.

[0011] The accepted standard of care for leukemia has remained constant for over 40 years and uses anthracycline and cytarabine sequentially in their free forms as "7+3" therapy. Although this treatment is "standard", some patients experience side effects and/or a poor prognosis. CPX-351 may represent a positive alternative for these patients. Thus, there is a need to stratify patients with hematological malignancies so that they can be administered CPX-351 as an appropriate therapy. Because CPX-351 and/or conventional treatment regimens may not be effective for every individual, there is a need to identify diagnostic tests, agents and/or markers that can facilitate the selection of an appropriate treatment regimen for an individual with hematopoietic malignancies.

Description of the invention

[0012] In one aspect, the present invention is based on the evaluation of ex vivo cytotoxicity of CPX-351 and/or capture by a wide range of blast types recently obtained from patients with hematological malignancies, including cells from major groups of hemoblastoses such as AML and CLL, as well as other subtypes. diseases. The results correlate with patient outcomes after in vivo treatment.

[0013] Hemoblastosis includes acute myelogenous leukemia (AML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), myeloproliferative neoplasia (MPN), and lymphomas.

[0014] Thus, in this aspect, the invention relates to a method for predicting the likelihood that administration of CPX-351 to a human will be effective in treating hemoblastosis in an individual, the method comprising

exposing malignant cells of said individual to CPX-351 in ex vivo cell culture; and

measuring the response of said cells to said treatment;

where an individual whose cells show a response to said treatment is identified as an individual for whom treatment with CPX-351 is likely to be effective. Response can be measured as cytotoxicity, for example, by determining the IC ₅₀ or IC ₉₀ or measuring the uptake of CPX-351.

[0015] Individuals thus identified are administered CPX-351 in an effective amount.

[0016] In a second aspect, the invention relates to the identification of individuals who will benefit most from replacing the more expensive treatment of CPX-351 with the standard of care, thus potentially leading to regulatory approval for the use of CPX-351 for these individuals. These individuals are identified using the specific genetic and phenotypic characteristics described below.

[0017] In specific embodiments, the identification method includes determining the presence or absence of a Fms-like receptor tyrosine kinase 3 (FLT-3) gene mutation in said individual, whereby the individual having a mutation in said FLT-3 gene is identified as an individual who will receive benefit from treatment with CPX-351, and in some embodiments, the method further comprises administering an effective amount of CPX-351 to said individual and/or determining the presence or absence of a nucleophosmin 1 (NPM-1) gene mutation in said individual, whereby the individual having the mutation in said NPM-1 gene is identified as an individual that would benefit from treatment with CPX-351, and in some embodiments, the method further comprises administering an effective amount of CPX-351 to said individual and/or determining the presence or absence of a mutation in the CCAAT enhancer binding protein alpha gene (SEBPα) in the specified individual, through wherein an individual having a mutation in said CEBPα gene is identified as an individual who will benefit from treatment with CPX-351, and in some embodiments, the method further comprises administering an effective amount of CPX-351 to said individual.

[0018] In the case of FLT-3 mutations, the mutation may be an activating mutation of this gene, in particular, damage to FLT3-IDT.

[0019] A single biomarker or combination of biomarkers, such as those described above, that may indicate that a patient (e.g., with leukemia or at risk of leukemia) for a treatment regimen including CPX-351 includes, as non-limiting examples, at least one or more FLT-3 gene mutations, including FLT3-ITD damage or FLT3-TKD damage, and any combination of mutations thereof, or combination with other genetic markers such as mutations in the NPM-1 gene and/or the CEBPα gene. If approved, CPX-351 may be used in the absence or presence of other anticancer treatment (eg, radiation therapy, surgery) for hematopoietic malignancy in individuals selected for carrying at least one or more of the biomarkers provided herein. In alternative embodiments, if approved, CPX-351 can be used as a bone marrow conditioning agent for the treatment of such cancer in individuals selected for carrying at least one or more of the biomarkers provided herein.

[0020] In addition to the FLT-3, NPM-1 and CEBPα markers, other karyotypes may contain one or two CPX-351 responsive alleles according to the European LeukemiaNet (ELN) guidelines described in Example 1 below.

[0021] Thus, the invention also relates to a method for identifying an individual having a cancer who would benefit from treatment with CPX-351, wherein the method comprises determining the genotype of the individual according to the ELN system to classify the individual as having favorable risk, intermediate-I, intermediate-II or adverse risk. Individuals with an intermediate-II or adverse risk are identified as those likely to benefit from treatment with CPX-351 and will be treated in this way. The ELN system is based on a 7+3 response to standard treatment, and patients at unfavorable risk respond poorly to it.

[0022] It should be understood that the ELN classification can be combined with the genetic markers mentioned above to determine individuals who will benefit from treatment.

[0023] Thus, in general, the present invention in the above aspects relates to assays, methods, systems and kits for selecting a treatment regimen for an individual with a malignant neoplasm of the hematopoietic system (for example, leukemia) or a risk of developing a malignant neoplasm of the hematopoietic system (for example, leukemia), treating an individual with a hematopoietic malignancy (eg, leukemia), and/or improving the efficacy of a treatment regimen recommended or used in an individual with a hematopoietic malignancy (eg, leukemia) or at risk of developing a hematopoietic malignancy (eg, leukemia ).

[0024] A test sample for use in the assays, methods, systems, or kits provided herein can be obtained from a biological sample of an individual, such as a sample of bone marrow, or blood, or plasma, or serum of an individual.

[0025] Depending on the choice of at least one biomarker provided herein, a test sample may be subjected to one or more assays, e.g., including but not limited to genotyping assays, expression assays (e.g., protein and/or transcript levels ), other assays that can determine the genotype, or any combination thereof. Many such assays are known in the art and many are commercially available, such as microarrays (eg ^Affymetrix® ) and sequencing (eg Illumina).

[0026] Hemoblastosis includes acute myelogenous leukemia (AML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), myeloproliferative neoplasia (MPN), and lymphomas.

[0027] In another aspect, the invention relates to a method for improving the efficacy of treatment of CRX-351 in a hemoblastotic individual having an activating FLT-3 gene mutation, the method comprising administering an effective amount of an FLT-3 inhibitor in combination with CRX-351. The combination can be administered simultaneously, or in the same composition, or CPX-351 is administered first. Brief description of the drawings

[0028] Figure 1A is a diagram showing the ex vivo sensitivity of AML patient cells to CPX-351. IC ₅₀ values correlate with more specific AML risk subtypes annotated in the diagram. The IC ₅₀ values of CPX-351 are given for patients in each of these risk groups.

[0029] Figure 1B shows the correlation of risk categories with ex vivo response in terms of IC ₅₀ . As can be seen, although the majority of individuals in the intermediate-I, intermediate-II and adverse risk groups showed favorable IC ₅₀ values; individuals in the favorable risk group predominantly did not show this. In each case, some abnormal values were observed indicating the importance of individual responses.

[0030] Figure 1C shows the correlation between clinical response to a standard 7+3 treatment regimen and ex vivo IC ₅₀ response. There was little difference between groups with an ex vivo IC ₅₀ in the low range with respect to their response to standard treatment of 7+3.

[0031] Figures 2A-2C respectively show the ex vivo sensitivities of ALL, MDS/MPN and CLL patient cells to CPX-351. Leukemia cells were analyzed from peripheral blood or bone marrow samples from 127 patients with ALL, MDS/MPN or CLL. The IC ₅₀ values for these cases are given along with the specific diagnoses.

[0032] Figure 3A is a graph showing that FLT3-ITD AML cells show increased sensitivity to CPX-351-induced cytotoxicity. FLT3-ITD was positive in 14 patients and negative in 28. Figures 3B and 3C, respectively, show the results for individuals with NPM-1 and CEBPα mutants. Insertions in NPM-1 were identified in 13 patients and were not observed in 29; insertions/deletions of CEBPα were observed in 4 patients and were not detected in 34. Values are mean±SEM, with p-values shown in each chart.

[0033] Figures 4A-4C show a flow cytometric analysis of CPX-351 drug uptake and results showing a correlation between drug uptake and cytotoxicity.

[0034] Figures 5A-5C show uptake of intact CPX-351 by leukemia cells in the bone marrow compared to normal bone marrow cells; figures 5A and 5B, respectively, represent the capture of cytarabine and daunorubicin from CPX-351, and figure 5C represents the capture of lipid.

[0035] Figure 6 shows the response rates among patients with mutant FLT-3, NPM-1, and CEBPα treated with CPX-351 in clinical trials compared with standard treatment 7+3.

[0036] Figures 7A-7C compare survival rates of patients treated with CPX-351 compared to patients treated with standard 7+3 treatment with FLT-3, NPM-1, and CEBPα mutations, respectively.

[0037] Figures 8A and 8B compare IC ₅₀ values (Figure 8A) and normalized CPX-351 uptake measurements (Figure 8B) for various cell lines, including lines with and without FLT-3 mutations.

[0038] Figures 9A-9D compare uptake of CPX-351 with and without quisartinib pretreatment for two different cell lines with FLT-3 mutations.

[0039] Figures 10A-10C show the results of determining the synergistic interaction between CPX-351 and quisartinib or midostaurin. Figure 10A shows a sample of the charts that will be generated for various concentrations of these drugs as shown. Figure 10B depicts synergy according to the Additivity Bliss Algorithm (EOBA) (Berenbaum, M. C, Adv. Cancer Res. (1981) 35: 269-335). Figure 1C shows viability results for individual cell lines and various combination protocols, along with results from the EOBA assay.

[0040] Figures 11A-11D show synergy results using the Chow-Talalay algorithm (Chou and Talalay, Adv. Enzyme Reg. (1984) 22: 27-55). Figure 11A shows the position of the multiwell plate for various combinations of concentrations of drugs subjected to analysis. Figure 11B is an example of a chart useful for plotting the results for each combination. Figures 11C and 11D show the results for the combinations tested.

Methods for carrying out the invention

[0041] Because malignant neoplasms of the hematopoietic system are heterogeneous, i.e. not all patients with AML or other hematological malignancies have the same prognosis or underlying genotype, it is important to test individual human samples to determine a favorable treatment for a particular individual.

[0042] As noted above, in contrast to previous experience with liposome and nanoparticulate drug formulations, CPX-351 is successfully taken up by leukemia cells (predominantly normal bone marrow cells) along with encapsulated cytarabine and daunorubicin at the administered ratio. (Lim, W. S., et al., Leuk. Res. (2010) 34: 1214-1223.) This allows ex vivo testing of individual hemoblastoses for susceptibility to CRX-351 treatment. The assay may include the ability of cells to capture significant amounts of CPX-351 and/or cytotoxicity of CPX-351 to patient samples. This is important because of the heterogeneity in the response of individual patients to treatment in general and the advantage of early detection of whether treatment with CRX-351 would be beneficial for a particular individual. Typically, such ex vivo testing is limited to free drugs, as opposed to drugs delivered in nanoparticulate formulations, as these formulations are designed to accumulate in the bloodstream and release the contained therapeutic agents in such a way that only the agents themselves enter the malignant cells.

[0043] Thus, one aspect of the invention relates to ex vivo assays to determine whether a particular patient will successfully respond to CRX-351. Thus, malignant cells are obtained from a patient with hemoblastosis for ex vivo testing by bringing the cells into contact with CPX-351 and determining cytotoxicity, for example, determining the IC ₅₀ or IC ₉₀ against these cells and/or measuring the uptake of the liposome system by any conventional methods . As shown below, CPX-351 is delivered to leukemic cells in an intact form, thus making this ex vivo test reliable. Ex vivo cytotoxicity of CPX-351 against fresh blasts from AML patients and blasts from patients with acute lymphocytic leukemia (ALL), lymphoma, and myeloproliferative neoplasia correlates with genotypic and phenotypic profiles, as well as clinical outcomes for patients from whom blast samples were obtained.

[0044] It has also been found that mutations in specific genes, in particular FLT-3, NPM-1 and CEBPα, increase the susceptibility of hemoblastosis to CPX-351 if these mutations are observed in an individual, and, in particular, compared with standard treatment 7+3. Thus, these mutations can be used as cancer markers that will be successfully treated in vivo with CPX-351 as opposed to the standard 7+3 treatment regimen. The presence of these mutations can be measured directly by gene analysis or evaluation of markers indicative of the mutation using subsequent expression products. For example, FLT-3 activating mutations can be determined by increased FLT-3 activity in an individual or in malignant cells. Suitable substrates for this determination include blood, plasma, serum and saliva.

[0045] For example, in some embodiments, a suitable assay may include subjecting a test sample from a person diagnosed with leukemia or at risk of developing leukemia to at least one genotyping assay adapted to determine the genotypes of FLT-3 gene mutations (e.g., FLT3-ITD) and optionally using a treatment regimen comprising administering to a human an effective amount of CPX-351.

[0046] Similar outcomes were observed for NPM-1 gene mutations, CEBPα gene mutations, and in individuals falling into the intermediate-II and adverse risk categories of the ELN system.

[0047] As noted above, combinations of mutations determined by any suitable method can be used to improve the analysis. In particular, it is known that specific karyotypes, in combination with the mutational status of these genes, can be used to group patients according to their response to standard 7+3 treatment. This classification is described in Rollig, C, et al., J. Clin. oncol. (2011) 29:1-7 and published online as 10.1200/JCO.210.32.8500 May 31, 2011. This classification system is called the European LeukemiaNet (ELN) system, in which individuals with AML are classified as having a favorable risk, intermediate-I, intermediate-II and unfavorable risk. Individuals classified as having an intermediate-II or adverse risk, by definition, do not respond well to the standard 7+3 treatment regimen. In contrast, Applicants have found that these patients respond to CRX-351 in clinical studies. Thus, the identification of a particular patient's malignant cells as having an intermediate-II or adverse risk clearly indicates the desirability of treating the patient with CPX-351. The results presented by the applicants are unexpected in the light of experience with standard treatment.

[0048] In all of the above cases, after identification of a suitable individual, as appropriate, should be the introduction of CPX-351 in accordance with the approved protocol of these studies. As a result, CPX-351 can be administered in an effective amount to reduce at least one symptom associated with hemoblastosis. As noted above, hemoblastosis can be one of a number of such malignancies, including acute myelogenous leukemia (AML), ALL, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), myeloproliferative neoplasias (MPN) and lymphomas.

[0049] An effective amount of CPX-351 may be administered to a selected individual via a suitable route of administration, such as I.V. administration, in accordance with the CPX-351 schedule. Treatment protocols using CPX-351 are described in US Pat. No. 8,092,828, incorporated herein by reference.

[0050] Another aspect of the invention relates to the design of an improved system for the treatment of hematological malignancies in individuals with FLT-3 mutations, such as ITD, using the combination of CPX-351 with an FLT-3 inhibitor. FLT-3 inhibitors are available in the art and include quisartinib, midostaurin, tandutinib, sorafenib, sunitinib, lestaurtinib, crenolanib, gilteritinib, AST-487, dovitinib, and linifanib. However, it has surprisingly been found that the timing of administration is important: the two drugs must be administered simultaneously, in some cases in the same formulation, or CPX-351 must be administered prior to the inhibitor, including administration 10-24 hours before or within CPX-351 administration. . Long-term exposure to an FLT-3 inhibitor prior to CPX-351 administration has been shown to be counterproductive.

[0051] The present invention also relates to computer systems for use in any aspects of the assays and/or methods presented in the present description. For example, one embodiment provided herein relates to a computer system for obtaining data from at least one test sample obtained from at least one individual.

[0052] In some embodiments, the implementation of the determining module of the computer system can be configured to analyze at least one test sample to determine the presence or absence of at least two of the conditions presented above.

[0053] In some embodiments, the determination module may further comprise a comparison module adapted to compare data coming from the determination module with reference data stored on the storage device.

[0054] In some embodiments, the storage device can be further configured to store physical information about at least one individual, for example, containing indications of whether the tested individual has one or more detectable mutations in the FLT-3 gene, and/or the NPM- gene. 1 and/or CEBPα gene and/or ELN karyotypes.

[0055] The assays, methods, systems, and/or kits provided herein may be performed and/or used by more than one individual under the direction of a single authority authorizing and regulating these functions. Such individuals may charge a fee for the service provided to determine the presence or absence of at least one condition described herein in a human test sample, for example, to facilitate the selection of a treatment regimen for a person with hematological malignancies as part of a method for selecting a treatment regimen for a person. In one example, a suitable assay includes (a) obtaining a test sample from a person diagnosed with or at risk of developing AML; (b) subjecting the test sample to at least one biomarker assay (e.g., including but not limited to genotyping assays, expression assays (e.g., protein and/or transcript levels), other assays that can identify activated FLT-3 , or any combination thereof) to determine the parameters of at least one biomarker provided in the present description (for example, as non-limiting examples, FLT3-ITD); (c) determining, taking into account the parameters of the selected biomarkers, the presence of at least one specified condition; and (d) obtaining a result (eg, by way of non-limiting examples, a list of FLT3 gene mutations) indicating whether at least one of said conditions is determined in the test sample. If at least one condition is present, the manager may further select and implement a treatment regimen comprising administering an effective amount of CPX-351 to a human.

[0056] In some embodiments, one or more of the above method steps is performed by a non-human device.

[0057] The citation of publications or documents in this specification is neither an admission that any of them are relevant to the prior art, nor an admission as to their content or date of publication. All documents cited in the present description are, therefore, incorporated herein by reference.

EXAMPLES

[0058] The following examples are presented for illustrative purposes and not to limit the invention.

Preparation A

Composition of CPX-351 and a simple preparation

[0059] CPX-351 (cytarabine:daunorubicin liposomes for injection) was obtained as a sterile, pyrogen-free, purple, lyophilized product in 50 ml vials. Each vial contains 100 units, where 1 unit is equal to 1.0 mg of cytarabine and 0.44 mg of daunorubicin (as base). The material was reconstituted with 19 ml water for injection and gently inverted for 10 minutes at room temperature. Working aliquots of the reconstituted product were stored frozen for up to 12 months at -20°C.

[0060] For patient sample collection and preparation, peripheral blood (PB) or bone marrow (BM) samples were obtained prior to therapy from patients diagnosed with AML, ALL, MPN, or CLL. All samples were obtained with informed consent to a protocol approved by the Oregon Health & Science University Review Board. Blood or bone marrow samples were separated using a ficoll gradient followed by erythrocyte lysis using ammonium potassium chloride (ACK) buffer.

Example 1

Determination of cytotoxicity of CPX-351 ex vivo against leukemic cells of patients

[0061] Drug susceptibility testing was performed by standard, known methods. Briefly, mononuclear cells from samples from patients with myeloid leukemia were cultured in R10 (RPMI-1640 medium supplemented with 10% FBS (Atlanta Biologicals, Lawrenceville, GA), L-glutamine, penicillin/streptomycin (Invitrogen, Carlsbad, CA) and Fungizone ^® (Invitrogen)), supplemented with 10 ^-4 M 2-mercaptoethanol (Sigma). Cells from samples from patients with lymphoid leukemia were cultured in R20 (RPMI-1640 medium supplemented with 20% FBS (Atlanta Biologicals, Lawrenceville, GA), L-glutamine, penicillin/streptomycin (Invitrogen, Carlsbad, CA) and ^Fungizone® (Invitrogen) ) supplemented with 10 ^-4 M 2-mercaptoethanol (Sigma) and insulin-transferrin-sodium selenite (Invitrogen).

[0062] Cells were cultured in 96-well plates (50,000 cells per well) and exposed to stepwise concentrations of CPX-351 for 3 days, while the tetrazolium-based MTS assay (cell proliferation assay) was used to assess the relative numbers of viable cells in each well. ^96® AQueous One Solution, Promega). Viability of cells seeded in the absence of any drug was taken as 100% viability and each point of the CPX-351 dose-response curve was normalized to cell viability values for these drug-free conditions. Third order polynomial approximation was used to obtain IC ₅₀ values for each sample.

[0063] Because AML is currently a target indication for CPX-351 administration, showing promising signs of efficacy in multiple clinical trials of cohorts of AML patients with different clinical characteristics, AML blasts from peripheral blood or bone marrow samples from 53 AML patients were cultured at stepwise concentrations of CRX- 351 (10:2 µM; 1:0.2 µM; 0.1:0.02 µM; 0.01:0.002 µM) or no drugs for 3 days and relative viable cell counts were assessed using MTS analysis based on tetrazolium. The MTS values of cells cultured in the absence of drug were taken as 100% and the MTS values for each dose were normalized to the MTS values for these untreated cells. Third order polynomial approximation was used to calculate IC ₅₀ values for each sample. Complete demographic and clinical data were available for 42 of these cases and are summarized in Table 1. Complete cytogenetic data for assessing AML risk groups were available for 40 patients, with 3, 21, 12, and 4 patients falling into the favorable, intermediate-I groups. , intermediate-II and adverse risk, respectively, as classified using European LeukemiaNet (ELN) guidelines.

[0064] Favorable risk includes t(8;21) (q22;q22), inv(16) (p13.1q22) or t(16;16) (p13.1q22), mutant NPM1 with normal karyotype and mutant CEBPα with normal karyotype.

[0065] Intermediate-I risk includes mutant NPM1 with FLT3-ITD and normal karyotype, wild-type NPM1 with FLT3-ITD and normal karyotype, wild-type NPM1 without FLT3-ITD and normal karyotype.

[0066] Intermediate-II risk includes t(9;11) (p22;q23) and any cytogenetic parameters not classified as favorable or unfavorable.

[0067] Adverse risk includes inv(3) (q21q26.2) or t(3;3) (q21q26.2), t(6;9) (p23;q34), t(v;11) (v;q23 ), monosomy 5 or del(5q), monosomy 7, 17p anomaly, and complex karyotype (≥3 anomalies).

[0068] Most samples were obtained from patients with newly diagnosed de novo AML (34/42; 81%) and most patients were treated using a 7+3 treatment regimen after sample collection (32/42; 76%). Thirty-five (35) of the AML patients in the study were treated with a 7+3 treatment regimen after sample collection. Twenty-four (24) of these patients achieved an initial complete response, while 11 patients experienced disease progression. The IC ₅₀ values of CPX-351 for patients who have observed a complete response or progression of the disease are presented. The proportions of major demographic features as well as clinical parameters such as leukocyte count at baseline and at genetic-cytogenetic risk stratification were typical of the population of AML patients.

[0069] As evidenced by the data presented in Figure 1A and Table 2, primary blasts from AML patients were generally susceptible to ex vivo cytotoxicity of CPX-351. IC ₅₀ values ranged from 0.035:0.007 µM to 9.77:1.95 µM. All but one sample (98%) observed an IC ₅₀ below 1/10 of the human plasma concentration of CPX-351 72 hours after administration (60:12 µM cytarabine:daunorubicin), suggesting that a clinical response could potentially be achieved in the treatment of these patients with CPX-351.

[0070] As shown in Figure 1B, a potent antiproliferative response to ex vivo treatment with CPX-351 (low IC ₅₀ ) was observed in cells having intermediate-II or unfavorable risk cytogenetic abnormalities generally associated with poorer prognosis and resistance to conventional forms of chemotherapy.

[0071] As indicated in Table 2, a total of 17 samples were obtained from patients in the intermediate-II or adverse cytogenetic risk categories. As shown in Figure 1B, the overall ex vivo response to CRX-351 in this subgroup of patients is similar to that of patients at intermediate-II and favorable risk, with no significant differences in the ex vivo response to CRX-351 in the four risk groups. These ex vivo responses correlated well with observed clinical activity of CPX-351, with complete responses observed among a wide, diverse subset of AML patients, regardless of conventional risk stratification.

[0072] Clinical response to conventional treatment with 7+3 cytarabine:daunorubicin was compared with ex vivo response to CRX-351 among 35 AML patients treated with the 7+3 treatment regimen after their resulting blasts were also evaluated. Of these 35 patients, 24 achieved a complete response (CR) and 11 experienced disease progression (PD), as shown in Figure 1C. Leukemia blasts from these patients showed similar sensitivity to ex vivo CPX-351 cytotoxicity, whether or not they initially responded to the 7+3 regimen.

[0073] Ex vivo susceptibility to CPX-351 was also characterized in different subtypes of hemoblastoses. One hundred twenty-seven (127) additional patient samples were used, including patients with ALL (38), MPN/MDS (18) and lymphoma (71). The cytotoxic activities of CPX-351, as measured by IC ₅₀ , were determined for each individual patient sample ex vivo and are shown in Figures 2A-2C. A wide range of IC ₅₀ values were observed in each diagnostic category (0.03/0.006 μM - 10/2 μM). The median IC ₅₀ for all 180 patient samples tested (including 53 AML cases) was 0.558:0.112 µM, and the vast majority of cases (153/180, 85%) observed IC ₅₀ values below 2.0:0.4 µM, which 30-fold lower than reported 60:12 µM plasma drug concentrations at 72 h observed in patients with leukemia (Gordon, M., et al., Proceedings of the AACR (Apr 2016) 57: Abstract #287). The observed low median IC ₅₀ of CPX-351 relative to circulating drug concentrations was generally indicative of high activity of CPX-351 in potential inhibition of leukemia cell proliferation or survival in a wide range of diagnoses.

Example 2

The effect of gene mutations

[0074] Higher CPX-351 activity is observed in blasts from AML patients with the FLT3-ITD phenotype: FMS-like receptor tyrosine kinase (FLT3) plays an important role in normal hematopoiesis and leukemogenesis and is expressed in most AML blasts. In 20-25% of patients with AML, a duplication of the internal tandem in the FLT3 perimembrane domain (FLT3-ITD) occurs in the FLT3 gene, and it is associated with a poor prognosis in patients. Among the 42 AML patients in the study of Example 1 with known FLT3-ITD status, 14 patients were identified as carriers of the FLT3-ITD mutation and the remaining 28 were FLT3-ITD negative. The results of the ex vivo cytotoxicity study indicated that FLT3-ITD carriage was unexpectedly associated with higher susceptibility to CPX-351 induced cytotoxicity (as shown in Figure 3A). In particular, FLT3-ITD leukemic blasts showed significantly lower IC ₅₀ values (0.29:0.058 µmol) compared to samples from FLT3-ITD-negative patients (IC ₅₀ =1.32:0.26 µmol ). This difference in response to CPX-351 cytotoxicity between FLT3-ITD positive and negative samples was statistically significant (p=0.047). It should also be noted that FLT3-ITD-positive patients had a significantly higher white blood cell count (WBC) with a mean result of 91,000/mm ³ compared to 29,000/mm ³ at diagnosis, p=0.0002.

[0075] Other common mutations, including those of nucleophosmin (NPM1) and CCAAT enhancer binding protein alpha (CEBPα), were also found in 13 and 4 patients, respectively. However, neither of these two common mutations significantly affected response to ex vivo CPX-351 treatment, although a trend towards higher sensitivity was observed in CEBPα positive cases (Figures 3B and 3C).

Example 3

Correlation between ex vivo susceptibility and uptake of CPX-351 in patient leukemic blasts

[0076] In preclinical animal models of leukemia, it was observed that after injection of CPX-351, leukemia cells in bone marrow grafts from these animals could easily take up cytarabine and daunorubicin, to a greater extent in intact liposomal form, with a synergistic drug ratio maintained, resulting in to increased and prolonged accumulation of drugs in leukemic cells compared with the introduction of a mixture of free drugs. The data below show an increased uptake of CPX-351 compared to free drugs. Thus, ex vivo uptake and cytotoxicity of CPX-351 can be used to predict the clinical success of CPX-351.

[0077] Vital frozen cells from patient samples previously screened for ex vivo CPX-351 susceptibility were exposed to the maximum test concentration of CPX-351 (10:2 μM cytarabine:daunorubicin) for 24 hours. Cells were then washed 3 times with PBS and analyzed for daunorubicin uptake by fluorescence using a BD FACSAria flow cytometer.

[0078] Flow cytometric analysis was performed on 12 patient samples as described above using intrinsic fluorescence of daunorubicin as a semiquantitative indicator of intracellularly bioavailable drug. Samples were obtained from 6 AML patients and 6 CLL patients and a wide range of IC ₅₀ values were observed in the initial ex vivo screening. Cells were exposed to stepwise concentrations of CPX-351 for 24 hours before being analyzed for daunorubicin uptake using a BD FACSAria flow cytometer. Live cells were identified in the FSC vs. scatterplot. SSC and calculated the total fluorescence intensity (figure 4A, left). The ratio of the mean fluorescence intensity of cells exposed to CPX-351 relative to untreated cells was used to derive the drug uptake score, with a score of 1 indicating no uptake and numbers above 1 indicating uptake of daunorubicin.

[0079] Statistical analysis: Student's t-test was used to compare CPX-351 activity in the mutant FLT3 (ITD), NPM1 and CEBPα and FLT3 (ITD), NPM1 and wild type CEBPα groups for independent samples with one-tailed p value. A p value <0.05 was considered significant. One-way ANOVA analysis was used to compare outcomes in different genetic-cytogenetic risk groups. Statistical analysis was performed using Prism software version 5.0a.

[0080] Compared to untreated cells, cells treated with CPX-351 showed a significant increase in the degree of intensity of intracellular fluorescence, indicating the presence of free daunorubicin, because. the fluorescence of daunorubicin encapsulated in CPX-351 is completely quenched. Differences in daunorubicin uptake between treated and untreated cell samples are presented as fold shift of mean fluorescence intensity (MFI) (Figure 4A, right). Figure 4B shows a comparison of uptake and cytotoxicity. When the CPX-351 IC ₅₀ values for these 12 samples were plotted against their respective MFI values, a strong correlation was observed between cell sensitivity to CPX-351 (IC ₅₀ ) and CPX-351 uptake efficiency (MFI) with a correlation coefficient of 0.703 (Figure 4C).

[0081] Selective uptake of bone marrow by leukemia cells, leaving CPX-351 intact, was demonstrated as follows: Bone marrow cells from the femur of CCRF-CEM (leukemia) transplanted mice, which were administered 1 dose of CPX-351, were harvested 18 hours after drug administration. facilities. Leukemic and normal bone marrow cells were separated using human CD45-specific magnetic particles and analyzed for uptake of CPX-351. Cytarabine and liposomal lipids were labeled with ³ H and ¹⁴ C, respectively, and quantified by liquid scintillation. Daunorubicin was analyzed by HPLC. The results are presented in figures 5A-5C. Each bar represents the mean±SE for 3 parallels with 10 femurs (5 mice) per parallel.

[0082] As shown in Figure 5C, leukemic cells took up approximately 225 pmol of liposomal labeled lipid per 10 ⁶ cells, but normal bone marrow only took up about 110 pmol/10 ⁶ cells. Drugs contained in liposomes were also preferentially taken up by leukemic cells, where cytarabine showed uptake of 24 pmol/10 ⁶ cells and daunorubicin approximately 16 pmol/10 ⁶ cells in the case of leukemic cells and, in each case, to a lesser extent in normal bone marrow. (approximately 3 pmol/10 ⁶ cells in the case of cytarabine and approximately 7 pmol/10 ⁶ cells in the case of daunorubicin). The molar ratio of the liposomal lipid to the drug contained therein is approximately 10:1, such that these results indicate that the drugs remained in the liposomes when taken up by these cells.

Example 4

CPX-351 Demonstrates Superior Antitumor Efficacy in FLT3-ITD+ AML Patients

[0083] In this example, a Phase 3 clinical study was performed where AML patients were screened for carriage of an activating mutation in the FLT-3 gene. These AML patients found to have an AML-ITD+ activating mutation were administered 3 doses of CPX-351 in a cycle of administration consisting of a first stage of administration on day 1, a second stage of administration on day 3, and a third stage of administration on day 5. Treatment protocols using CPX-351 are described in US Pat. No. 8,092,828. Patient response, including analysis of plasma and/or bone marrow samples, was measured and monitored, and response rates and survival were measured. Also included were subgroups of patients positive for NPM1 and/or CEBPα mutation. The original study, which focused on FLT-3 mutations, also included individuals with NPM-1 and CEBPα mutations. The patients participating in the study are presented in Table 3.

*hypomethylating agent

[0084] The responses of individuals with and without FLT-3 mutations are presented in Table 4.

[0085] Figure 6 presents the response rates as a comparison of treatment with CPX-351 and 7+3 in the case of carriers of these mutations. As shown in Figure 6, among carriers of the ITD or TKD mutation in FLT-3, a response rate of 68.2% was observed in the case of CPX-351 and only 25.0% in the case of 7+3. Among patients with the NPM-1 mutation, a response rate of 92.3% was observed in the case of CPX-351, but only 58.3% in the case of 7+3. In the case of individuals with CEBPα mutations, the proportion of responders in the case of CPX-351 was 33.3%, and in the case of 7+3-20.0%.

[0086] Survival rates for participants with and without FLT-3 mutations in this study are presented in Table 5.

[0087] Survival rates for various cohorts are also presented in Figures 7A-7C. In all cases, carriers of the mutations significantly improved survival; in particular, 25% of individuals with a CEBPα mutation experienced a survival of more than 27 months when treated with CPX-351, while none of the patients treated with 7+3 survived more than 3 months.

Example 5

Susceptibility of FLT-3 mutant cells to CPX-351 and combined treatment: CPX-351 + FLT-3 inhibitor

[0088] In this example, cell viability and intracellular uptake based on daunorubicin fluorescence as determined by flow cytometry was measured as described in Examples 1 and 3.

[0089] A. Susceptibility of AML cell lines (including MOLM-13 and MOLM-14 containing FLT-3-ITD and ME-1 (containing mutant, activated FLT-3)) to CPX-351 and uptake of CPX-351 was determined. these cells. Cells not containing the FLT-3 mutation were also compared, including U-937, HL-60, KG-1 and GDM-1. Cultures of these cells were treated with CPX-351 and analyzed for IC ₅₀ and uptake of CPX-351. Figure 8A presents the results regarding IC ₅₀ as a graph of percent cell viability versus CPX-351 concentration in nM. As shown in Figure 8A, FLT-3 mutant cells had lower IC ₅₀ than non-mutated FLT-3 cells. Figure 8B shows the capture results; CPX-351 concentration in nM is plotted against the mean fluorescence intensity (MFI) normalized to intrinsic fluorescence. Cells containing FLT-3 mutations were more efficient in capturing CPX-351 than cells without mutations.

[0090] B. To determine the effect of combination treatment with FLT-3 inhibitors, cells were exposed to CPX-351 with or without treatment with quisartinib or midostaurin (FLT-3 inhibitors). In one protocol, ME-1 and MOLM-14 cells were pretreated with 10 nM quisartinib for 0, 2, 8, 16, and 24 hours, followed by the addition of 100 μM CPX-351 for 2 hours. CPX-351 uptake was measured by daunorubicin fluorescence as determined by flow cytometry, normalized to the untreated control. The results are presented in figures 9A-9D. The x-axis of the graphs plots CPX-351 uptake in standard fluorescence units, and the y-axis plots normalized cell numbers. As shown in Figures 9A and 9B, all untreated cells showed no capture, while cells treated with CPX-351 were eventually sorted into two populations of cells, one of which captures CPX-351 more efficiently than the other. From figures 9A and 9B it is obvious that, since increased the pretreatment time, the ability of some cells to capture CPX-351 decreased. The results presented in figures 9C and 9D are combinations of different time points up to 24 hours.

[0091] C. To determine synergy, cell lines were plated in 384-well plates and exposed to individual increasing concentrations of CPX-351 and FLT-3 inhibitor. CPX-351 and FLT-3 inhibitors were added (a) simultaneously (C+Q or C+M), (b) with a 24 hour pretreatment with CPX-351 (C→Q or C→M), or (c) 24 - hour pre-treatment with FLT-3 inhibitor (Q→C or M→C). The EOBA algorithm was used to determine synergy for each drug combination.

[0092] Figure 10A is a schematic of the protocol along with numerical values for the corresponding nM concentrations of CPX-351 or FLT-3 inhibitor. Figure 10B presents the results in terms of antagonistic, additive or synergistic effect, which can be obtained by measuring viability at various concentrations of the two drugs used using the EOBA algorithm. Figure 1°C presents the results of viability experiments as data for various combinations of concentrations and various protocols described above, and the final determination of synergy based on the EOBA algorithm. The results for KG-1, ME-1, MOLM-13 and MOLM-14 under the above protocols are shown in Figure 10C. As indicated, specific combinations of CPX-351 and quisartinib or midostaurin showed high synergy if the administration of CPX-351 preceded the administration of the FLT-3 inhibitor, or if they were administered simultaneously.

[0093] D. An alternative method for determining synergy was also used, which compared various combinations of FLT-3 inhibitors and protocols using two cell lines. The experiment described in paragraph C was carried out, in which case the Chow-Talalai algorithm was used to analyze the results. As shown in Figure 11A, various combinations of CPX-351 and FLT-3 inhibitor were tested in wells represented by filled cells. Figure 11B is a sample isobologram plotting the result of the Chow-Talalai analysis, i.e. Additivity Index (CI) for each of the concentrations shown in Figure 11A for all three of the protocols described above. Figure 11C shows the results for GDM-1 cells and MOLM-14 cells for the three protocols and concentration levels shown in Figure 11A. Circles on each graph indicate combinations of concentrations for each of the cells presented in figure 11A. As can be seen, in the case of MOLM-14 cells, the administration of CPX-351 simultaneously with or before quisartinib or midostaurin led to the observation of synergy in a large number of wells, while in the case of administration of these drugs before CPX-351, more antagonistic results are observed. The results for GDM-1 cells were not significantly different. Figure 11D is a series of graphs showing the number of data points falling into different categories based on their Combination Additivity Index (CI) values for each of the protocols shown in Figure 11C.

[0094] E. In conclusion, cell lines containing FLT-3-ITD or an FLT-3 activating mutation were more sensitive to CPX-351 and showed increased uptake of CPX-351 compared to cell lines with other genetic abnormalities.

[0095] Pretreatment with quisartinib for 16 hours resulted in reduced daunorubicin fluorescence in approximately 50% of the total cell population, indicating that long-term prior exposure to an FLT-3 inhibitor may reduce uptake of CPX-351 in this subpopulation.

[0096] However, consistent synergy was observed when CPX-351 and FLT-3 inhibitors were administered simultaneously or when cells were exposed to CPX-351 24 hours prior to exposure to the FLT-3 inhibitor.

Claims (5)

1. A method of treating hemoblastosis in an individual with hemoblastosis, comprising administering an effective amount of a liposomal formulation containing cytarabine and daunorubicin co-encapsulated in a 5:1 molar ratio and an FLT-3 inhibitor selected from the group consisting of quisartinib, midostaurin, tandutinib, sorafenib , sunitinib, lestaurtinib, crenolanib, gilteritinib, AST-487, dovitinib, and linifanib; and where said liposome formulation and FLT-3 are administered simultaneously, or where said liposome formulation is administered prior to treatment with an FLT-3 inhibitor, or where said liposomal formulation and FLT-3 inhibitor are administered in the same composition. 2. The method of claim 1, wherein said individual has a mutation in the Fms-like receptor tyrosine kinase 3 (FLT-3) gene. 3. The method of claim 2, wherein the mutation is an activating mutation in the FLT-3 gene. 4. The method of claim 1 wherein the hemoblastosis is selected from the group consisting of acute myelogenous leukemia (AML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), myeloproliferative neoplasias (MPN) and lymphomas .

Images