FIELD

[0001] The disclosure relates generally to the field of medicine and the treatment of diseases or disorders, and more particularly to the mobilization of stem cells and/or the treatment of a disease, disorder or condition involving CXCR4 receptor activity.

BACKGROUND

[0002] CXCR4 is a G protein-coupled chemokine (C-X-C motif) receptor spanning the cell membrane of white blood cells, hematopoietic stem cells, and cells of the brain and spinal cord. Its primary ligand is CXC12/SDF-1. This receptor is remarkably versatile, with roles as diverse as transducing biochemical signals by increasing intracellular calcium ion levels and enhancing MAPK1/MAPK3 activation, participating in the AKT signaling cascade, regulating cell migration, such as during wound healing, functioning as a receptor for extracellular ubiquitin; binding bacterial lipopolysaccharide (LPS) and mediating LPS-induced inflammatory responses, and playing a role in hematopoiesis and vascularization of the gastrointestinal tract.

[0003] One set of examples of disease processes in which CXCR4 receptor activity is involved is the group of diseases involving renal impairment. CXCR4 is significantly upregulated after renal injury and that sustained activation of CXCR4 expression contributes to an increased fibrotic response in the kidney. Removal of CXCR4 attenuates the fibrotic response.

[0004] Renal impairment manifests in various forms of kidney diseases and disorders and remains a significant health concern in the U.S. and throughout the world. Chronic kidney disease (/.e., CKD) refers to a group of kidney diseases and virtually all of these CKDs, lead to a progression of tissue injury culminating in kidney fibrosis.

[0005] Another set of examples in which CXCR4 plays a crucial role is cancer. CXCR4 facilitates cancer cell survival and may protect cells from cytotoxic therapy. Strategies have focused on the potential of CXCR4 inhibitors to enhance the cytotoxic effect of chemotherapy and immunotherapy. CXCR4 inhibition might also counteract immune evasion of tumor cells by altering the distribution of immune cells and/or activity in the tumor microenvironment. [0006] Despite considerable effort, cancer remains the third most common cause of death worldwide with 7.6 million deaths in any given year. Cancer deaths are estimated to increase to 13.1 million by 2030. Hematological malignancies remain a major form of cancer, and persist as health concerns associated with significant healthcare costs.

[0007] The thalassemias, including but not limited to alpha-thalassemia, beta thalassemia, and gamma-thalassemia, are a collection of genetically inherited diseases involving mutations to globin genes that compromise the ability of the encoded globin protein chains to form functional hemoglobin capable of efficiently transporting oxygen to tissues throughout the body. In its severest form, the disease can be lifethreatening.

[0008] Sickle cell disease is a genetically inherited missense mutation in the p-globin gene that, in homozygous individuals, results in sickled red blood cells that are deficient in oxygen transport, resulting in anemia. Moreover, the sickled red blood cells can interfere with blood flow leading to extremely painful, potentially life-threatening, crises.

[0009] Primary immunodeficiency diseases refer to a collection of diseases sharing the common characteristic of occurring in an immunodeficient subject. The diseases include major health concerns including autoimmune disorders exemplified by rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, neutropenia and WHIM syndrome (/.e., warts, hypogammaglobulinemia, infections, and myelokathexis). Collectively, these diseases constitute a significant health cost in reduced quality of life for many, and in terms of resources needed to diagnose and treat the diseases.

[0010] These and other diseases and disorders disclosed herein collectively constitute a significant health burden in terms of reductions in the quality of life, extending to life-threatening conditions, and result in a significant demand on the healthcare resources available in the U.S. and throughout the world. Faced with these challenges, biomedical research has delivered an increasingly detailed picture of the many biochemical pathways, including intersecting pathways, involved in these disease and disorder processes.

[0011] Balixafortide (POL6326, SPX6326) is a synthetic cyclic peptide consisting of 16 amino acids. It is a selective, potent competitive inhibitor of CXCR4.

[0012] Apparent from the foregoing is a continuing need for therapeutics to treat several diseases and disorders that currently burden mankind in terms of life- threatening challenges, quality-of-life issues, and significant health care costs. The development of therapeutics with more than one therapeutic effect, such as therapeutics effective and safe in treating multiple diseases or disorders, would provide significant benefits.

SUMMARY

[0013] The disclosure provides materials and methods for the mobilization of pluripotent stem cells and/or the treatment of a disease, disorder or condition involving CXCR4 receptor activity by administering high doses of the CXCR4 inhibitor balixafortide. Balixafortide is a therapeutic peptide having the amino acid sequence cyclo(-Tyr-His-Ala-Cys-Ser-Ala- ^D Pro-Dab-Arg-Tyr-Cys-Tyr-Gln-Lys- ^D Pro-Pro-) having a disulfide bond between Cys4 and Cysll (SEQ ID NO:1), or a pharmaceutically acceptable salt thereof. As noted in the associated sequence listing, ^D Pro is D-Proline and Dab, also known as Dbu, is (2S)-2,4-diaminobutyric acid. The disclosure is based on the surprising finding that high doses of the CXCR4 inhibitor balixafortide did not result in toxicity levels leading to unacceptable adverse events. As disclosed herein, relatively high doses of balixafortide of greater than 5.5 mg/kg were well-tolerated in animal models and in human trials. In particular, a 13-week repeated dose study in cynomolgus monkeys revealed no observed adverse effect level (NOAEL) for doses up to 45 mg/kg. Administration of the relatively high doses of balixafortide may yield greater therapeutic benefits while avoiding the unacceptable toxicity, thereby providing improved methods of treating diseases, disorders or conditions involving CXCR4 receptor activity.

[0014] In one aspect, the disclosure provides a composition comprising balixafortide for use in a method of treating a disease, disorder or condition involving CXCR4 receptor activity, wherein balixafortide is administered in a dose greater than 5.5 mg/kg.. Also provided is the use of a composition comprising balixafortide as described herein for the manufacture of a medicament for the treatment of a disease, disorder or condition involving CXCR4 receptor activity, wherein balixafortide is administered in a dose greater than 5.5 mg/kg. Also provided is the use of a composition comprising balixafortide as described herein for the treatment of a disease, disorder or condition involving CXCR4 receptor activity, wherein balixafortide is administered in a dose greater than 5.5 mg/kg. Also provided is a method for the treatment of a disease, disorder or condition involving CXCR4 receptor activity in a subject, comprising administering to said subject a dose greater than 5.5 mg/kg balixafortide of the pharmaceutical composition as described herein.

As used herein, a “disease, disorder or condition involving CXCR4 receptor activity” means a disease, disorder or condition in which CXCR4 receptor activity affects the magnitude, duration, or course of the disease, disorder or condition. In some embodiments, the disease or disorder is renal impairment, a primary immunodeficiency disease or disorder, a thalassemia, sickle cell disease, neutropenia, or cancer, preferably a thalassemia, sickle cell disease, or neutropenia, preferably a thalassemia or sickle cell disease. In some embodiments, balixafortide is administered in a dosage greater than 5.5 mg/kg, preferably greater than 5.5 to 60 mg/kg, preferably 6.6 to 50 mg/kg, preferably 9 to 20 mg/kg, preferably 11to16.5 mg/kg. In some embodiments, at least 6.05 mg/kg balixafortide is administered, preferably 6.6 mg/kg, preferably 6.9 mg/kg, preferably 7.5 mg/kg, preferably 9 mg/kg, preferably 11 mg/kg, preferably 13.5 mg/kg, preferably 16.5 mg/kg. In some embodiments, a plurality of doses of greater than 5.5 mg/kg balixafortide are administered, preferably doses greater than 5.5 mg/kg, preferably doses of at least 6.6 mg/kg, preferably doses of at least 6.9 mg/kg, preferably doses of at least 7.5 mg/kg, preferably doses of at least 9 mg/kg, preferably doses of at least 11 mg/kg, preferably doses of at least 13.5 mg/kg, preferably doses of at least 16.5 mg/kg. In some embodiments, balixafortide is administered in a dose or doses that maintains a balixafortide exposure of at least 75000 AUCo-24, preferably at least 90000 AUCo-24, preferably at leastl 50000 AUCo-24, preferably at least 200000 AUCO-24, preferably at least 250000 AUCo-24. In some embodiments, neither eribulin nor paclitaxel is administered.

[0015] In some embodiments, the administration of balixafortide induces a level of leukocytes in the blood of at least 20x10 ⁹ /L one day, two days three days, four days, five days and/or six days, after administration of balixafortide. In some embodiments, the renal impairment yields an estimated glomerular filtration rate of less than 90 mL/minute, preferably less than 60 mL/minute, preferably less than 30 mL/minute, preferably less than 15 mL/minute. In some embodiments, the renal impairment is chronic kidney disease, preferably wherein the chronic kidney disease has progressed to kidney fibrosis, acute kidney disease, renal artery disease, ureteral obstruction, or glomerulonephritis. In some embodiments, the cancer is a solid tumor, e.g. prostate cancer, or hematological malignancies such as leukemias and lymphomas. In some embodiments, the primary immunodeficiency disease or disorder is e.g. rheumatoid arthritis,. In some embodiments, the thalassemia is alpha-thalassemia or betathalassemia, optionally wherein a patient with beta-thalassemia has a damaged spleen, a dysfunctional spleen, or has had a splenectomy.

[0016] Another aspect of the disclosure is drawn to a composition comprising balixafortide for use in a method of mobilizing pluripotent stem cells in a human by administering greater than 5.5 mg/kg balixafortide, wherein the pluripotent stem cells exhibit a Sca-1 , c-kit, CD34, HLA, H-2K ^b , CD27, CD34, CD38, CD43, CD117, or CD150 pluripotent stem cell marker, preferably wherein the pluripotent stem cell marker is Sca-1 , c-kit, CD34, HLA, or H-2K ^b , optionally wherein the stem cells are hematopoietic stem cells or autologous stem cells. In some embodiments the use of a composition comprising balixafortide in a method of mobilizing pluripotent stem cells in a human by administering greater than 5.5 mg/kg balixafortide is provided, wherein the pluripotent stem cells exhibit a Sca-1 , c-kit, CD34, HLA, H-2K ^b , CD27, CD34, CD38, CD43, CD117, or CD150 pluripotent stem cell marker, preferably wherein the pluripotent stem cell marker is Sca-1 , c-kit, CD34, HLA, or H-2K ^b , optionally wherein the stem cells are hematopoietic stem cells or autologous stem cells. Also provided is the use of a composition comprising balixafortide as described herein for the manufacture of a medicament for mobilizing pluripotent stem cells in a human by administering greater than 5.5 mg/kg balixafortide, wherein the pluripotent stem cells exhibit a Sca-1 , c-kit, CD34, HLA, H-2K ^b , CD27, CD34, CD38, CD43, CD117, or CD150 pluripotent stem cell marker, preferably wherein the pluripotent stem cell marker is Sca-1 , c-kit, CD34, HLA, or H-2K ^b , optionally wherein the stem cells are hematopoietic stem cells or autologous stem cells. In some embodiments balixafortide is administered by contacting the pluripotent stem cells of the subject with greater than 5.5 mg/kg balixafortide, in vivo or ex vivo, wherein the pluripotent stem cells exhibit a Sca-1 , c-kit, CD34, HLA, H-2K ^b , CD27, CD34, CD38, CD43, CD117, or CD150 pluripotent stem cell marker, preferably wherein the pluripotent stem cell marker is Sca- 1 , c-kit, CD34, HLA, or H-2K ^b , optionally wherein the stem cells are hematopoietic stem cells or autologous stem cells.. In some embodiments, the stem cells are totipotent stem cells. In some embodiments, the stem cells exhibit a greater number of CD34+ stem cells than are induced by G-CSF alone. In some embodiments, the hematopoietic stem cells are more benign, more synchronized, or both, compared to hematopoietic stem cells induced by G-CSF. In some embodiments, the use further comprises modifying the mobilized stem cells using gene therapy. In some embodiments, the method is used for treating a primary immunodeficiency disease or disorder, , a thalassemia, sickle cell disease, cancer, or neutropenia, preferably wherein the mobilized pluripotent stem cells at least partially restore white blood cell count after chemotherapy when the disease is cancer, preferably wherein the hematopoietic stem cells are collected ex vivo prior to chemotherapy for introduction into the patient after chemotherapy when the disease is cancer, preferably wherein the hematopoietic stem cells are mobilized to reduce the risk of neutropenia during leukapheresis when the disease or disorder is neutropenia. In some embodiments, balixafortide is administered in a dosage greater than 5.5 mg/kg, preferably greater than 5.5 to 60 mg/kg, preferably 6.6 to 50 mg/kg, preferably 9 to 20 mg/kg, preferably 11 to 16.5 mg/kg. In some embodiments, at least 6.05 mg/kg balixafortide is administered, preferably 6.6 mg/kg, preferably 6.9 mg/kg, preferably 7.5 mg/kg, preferably 9 mg/kg, preferably 11 mg/kg, preferably 13.5 mg/kg, preferably 16.5 mg/kg. In some embodiments, a plurality of doses of greater than 5.5 mg/kg balixafortide are administered, preferably doses greater than 5.5 mg/kg, preferably doses of at least 6.6 mg/kg, preferably doses of at least 6.9 mg/kg, preferably doses of at least 7.5 mg/kg, preferably doses of at least 9 mg/kg, preferably doses of at least 11 mg/kg, preferably doses of at least 13.5 mg/kg, preferably doses of at least 16.5 mg/kg.

[0017] The disclosure also provides a method of mobilizing pluripotent stem cells, in particular, hematopoietic stem cells, in a subject, in particular a human subject, to treat a disease, disorder or condition involving CXCR4 receptor activity by administering greater than 5.5 mg/kg balixafortide to the subject. In some embodiments the use of a composition comprising balixafortide in a method of mobilizing pluripotent stem cells in particular, hematopoietic stem cells, in a subject, in particular a human subject, to treat a disease, disorder or condition involving CXCR4 receptor activity is provided, wherein greater than 5.5 mg/kg balixafortide is administered to the subject. Also provided is the use of a composition comprising balixafortide as described herein for the manufacture of a medicament for mobilizing pluripotent stem cells in particular, hematopoietic stem cells, in a subject, in particular a human subject, to treat a disease, disorder or condition involving CXCR4 receptor activity, wherein greater than 5.5 mg/kg balixafortide is administered to the subject .Balixafortide may be administered as the sole therapeutic, or may be administered with a secondary therapeutic such as G-CSF. In embodiments where a plurality of therapeutics are administered, the therapeutics may be administered together, separately but at the same time, or at offset times. In some embodiments, the disease or disorder is renal impairment, a primary immunodeficiency disease or disorder, a thalassemia, sickle cell disease, neutropenia, idiopathic pulmonary fibrosis (/.e., I PF), acute respiratory distress syndrome (/.e., ARDS), chronic obstructive pulmonary disease (/.e., COPD), or cancer (e.g., solid tumors like, e.g., prostate cancer, or hematological malignancies like, e.g., leukemias and lymphomas). In some embodiments, greater than 5.5 mg/kg balixafortide is administered. In some embodiments, the dosage of balixafortide is in the range of greater than 5.5 to 60 mg/kg, preferably 6.6 to 50 mg/kg, preferably 9 to 20 mg/kg, preferably 11 to 16.5 mg/kg. In some embodiments, at least 6.05 mg/kg, 6.6 mg/kg, or 6.9 mg/kg balixafortide is administered, which reflects a dosage increase of 10%, 20% or 25% relative to a 5.5 mg/kg dose. In some embodiments, at least 11 mg/kg or 16.5 mg/kg balixafortide is administered, which reflects a dosage increase of 100% or 200% relative to a 5.5mg/kg dose. In some embodiments, at least 6.05 mg/kg balixafortide is administered, preferably 6.6 mg/kg, preferably 6.9 mg/kg, preferably 7.5 mg/kg, preferably 9 mg/kg, preferably 11 mg/kg, preferably 13.5 mg/kg, preferably 16.5 mg/kg. In some embodiments, a plurality of doses of greater than 5.5 mg/kg balixafortide are administered, preferably doses greater than 5.5 mg/kg, preferably doses of at least 6.6 mg/kg, preferably doses of at least 6.9 mg/kg, preferably doses of at least 7.5 mg/kg, preferably doses of at least 9 mg/kg, preferably doses of at least 11 mg/kg, preferably doses of at least 13.5 mg/kg, preferably doses of at least 16.5 mg/kg.

[0018] In some embodiments, a plurality of doses of greater than 5.5 mg/kg balixafortide is administered daily. In some embodiments, balixafortide is administered in a dose or a plurality of doses that provides an exposure to the subject of at least 75000 units AUCo-24, including embodiments wherein balixafortide is administered in a dose or doses that provides an exposure to the human body of at least 90000 units AUCO-24, where the AUC units are hours x ng/mL therapeutic agent in the blood, i.e., AUCO-24 units represent the area under the plasma concentration curve from 0 to 24 hours of exposure. In some embodiments, balixafortide is administered in a dose or a plurality of doses that provides an exposure to the subject of at least 150000 units AUCO-24, including embodiments wherein balixafortide is administered in a dose or doses that provides an exposure to the subject of at least 200000 units AUCo-24 or 250000 units AUCo-24, where the AUC units are hours x ng/mL therapeutic agent in the blood, i.e., AUCo-24 units represent the area under the plasma concentration curve from 0 to 24 hours exposure. In some embodiments, balixafortide is administered in a dose or a plurality of doses that exceeds a threshold level of at least 150000 units AUCo-24 balixafortide over the first 24 hours following initial administration. In some embodiments, balixafortide is administered in a dose or a plurality of doses that exceeds a threshold level of at least 200000 units AUCo-24 balixafortide over the first 24 hours following initial administration. In some embodiments, balixafortide is administered in a dose or a plurality of doses that exceeds a threshold level of at least 250000 units AUCo-24 balixafortide over the first 24 hours following initial administration. In some embodiments, balixafortide is administered, but neither eribulin nor paclitaxel is administered.

[0019] In some embodiments, the administration of balixafortide induces a leukocyte level in the blood of at least 20x10 ⁹ /L, a lymphocyte level in the blood of at least 4x10 ⁹ /L, and a neutrophil level in the blood of at least 13x10 ⁹ /L, one day, two days, three days, four days, five days and/or six days, after administration of balixafortide. In some embodiments, the administration of balixafortide induced sustained leukocyte, lymphocyte and neutrophil levels in the blood of renal impaired patients compared to healthy volunteers. In some embodiments, the renal impairment yields an estimated glomerular filtration rate of less than 90 mL/minute, including embodiments yielding an estimated glomerular filtration rate of less than 60 mL/minute, less than 30 mL/minute, and less than 15 ml/minute. In some embodiments, the renal impairment is chronic kidney disease, acute kidney disease, renal artery disease, ureteral obstruction, or glomerulonephritis. In some embodiments, the chronic kidney disease has progressed to kidney fibrosis.

[0020] In some embodiments, the cancer is prostate cancer or hematological malignancies like, e.g., leukemias and lymphomas. In some embodiments, the cancer is prostate cancer, or hematological malignancies like, e.g., leukemias and lymphomas, wherein prostate cancer, or hematological malignancies like, e.g., leukemias and lymphomas have metastasized.

[0021] In some embodiments of this aspect of the disclosure, the primary immunodeficiency disease or disorder is rheumatoid arthritis. In some embodiments, the thalassemia is alpha-thalassemia or beta-thalassemia. In some embodiments, the thalassemia is beta thalassemia, including embodiments wherein the patient with betathalassemia has a damaged or dysfunction spleen or has had a splenectomy.

[0022] The disclosure will be better understood upon consideration of the following detailed description of the disclosure, including a consideration of the figures. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1. Graph showing the mean balixafortide plasma concentration as a function of time in human subjects having varying grades of renal impairment (normal renal function or mild, moderate or severe impairment). Results were graphed using a logarithmic scale. Blood samples were taken for measurement of plasma concentrations of balixafortide. These samples were collected immediately prior to dosing (pre-dose) and then at 0.5, 2, 2.5, 3, 4, 5, 7, 8, 10, 12, 20, 24, 38, 48, and 72 hours relative to infusion start. For pre-dose time points or those in the lag time between time zero and the first quantifiable amount, concentrations below the LLOQ (lower limit of quantitation) were calculated as zero. Balixafortide in plasma samples were analyzed with a G(C)LP validated analytical procedure. The results shown in the graph reveal a pharmacokinetic profile of healthy individuals (individuals with normal renal function) that was as expected. There was no significant difference during the first 6 hours post-start of balixafortide infusion. Terminal plasma concentration and AUCo-72h increased with renal dysfunction. Vertical bars indicate the standard deviation (/.e., SD).

[0024] Figure 2. White blood cell levels as a function of relative renal impairment. Data analysis was performed with GraphPadPrism Version 9.3.1. Statistical analysis was done with the mixed-effect analysis (Anova) using the Geisser-Greenhouse correction. Graph shows a box plot interleaved low-high with a line at mean value. In progressing from patients with mild to moderate to severe renal impairment, progressively higher counts of leukocytes, lymphocytes and neutrophils were measured (leukocytosis). These data indicate long pharmacological action. As Cmax is comparable in all groups it is not likely the driver of increased leukocytosis. Increased exposure over time (Area Under the Curve or AUC) triggered the increase in pharmacological effect. A doubling of balixafortide exposure, as measured by AUC, stimulated leukocyte mobilization by a factor of three. (A) Leukocyte levels (leukocyte profiles) by grade of renal impairment. (B) Lymphocyte levels (lymphocyte profiles) by grade of renal impairment. (C) Neutrophil levels (neutrophil profiles) by grade of renal impairment.

[0025] Figure 3. Graph of exposure to balixafortide measured as the area under the curve of blood concentration as a function of time (/.e., hours x ng/mL blood concentration) for the first 24 hours post-administration), or AUCo-24 (24 hours x ng/mL blood concentration) as a function of the estimated glomerular filtration rate (/.e., eGFR) for human subjects with normal kidney function, mild renal impairment, moderate renal impairment, and severe renal impairment. The graph shows increased exposure (AUC0-24) with increasing renal impairment as the elimination of balixafortide in the urine becomes increasingly compromised. Normal function is characterized by an eGFR of at least 90 mL/minute, mild renal impairment between 60-90 mL/minute, moderate renal impairment between 30-60 mL/minute, and severe renal impairment no greater than 30 mL/minute.

DETAILED DESCRIPTION

[0026] Disclosed herein are the results of experiments showing that high doses of CXCR4 inhibitor balixafortide (/.e., POL6326, SPX6326) surprisingly exhibit tolerable levels of toxicity such that high-dose administrations are suitable as efficacious for mobilization of stem cells, in particular pluripotent stem cells, and/or in the treatment of a disease, disorder or condition involving CXCR4 receptor activity. Balixafortide is a cyclic peptide having the following amino acid sequence: cyclo(-Tyr-His-Ala-Cys-Ser- Ala- ^D Pro-Dab-Arg-Tyr-Cys-Tyr-Gln-Lys- ^D Pro-Pro-) having a disulfide bond between Cys4 and Cys11 (SEQ ID NO:1), or a pharmaceutically acceptable salt thereof. The high-dose administrations of balixafortide (greater than 5.5 mg/kg) may be also useful in mobilizing stem cells that are more pluripotent than the stem cells induced by G-CSF in being more benign, more highly synchronized, or both, which provides an increased safety profile in inducing mobilization of stem cells and in treating a disease, disorder or condition involving CXCR4 receptor activity, such as renal impairment and cancer (e.g., solid tumors like, e.g., prostate cancer, or hematological malignancies like, e.g., leukemias and lymphomas). High-dose balixafortide administrations are also useful in treating primary immunodeficiency diseases and disorders, a thalassemia, sickle cell disease, and neutropenia.

[0027] Balixafortide may be administered as the sole therapeutic, or may be administered with a secondary therapeutic such as G-CSF. In embodiments where a plurality of therapeutics are administered, the therapeutics may be administered jointly, at the same time, or at offset times.

[0028] Balixafortide is effective as CXCR4 inhibitor and is formulated in pharmaceutical compositions for administration to subjects such as human patients suffering from a disease, disorder or condition involving CXCR4 receptor activity, including but not limited to renal impairment, cancer (e.g., solid tumors like, e.g., prostate cancer, or hematological malignancies like, e.g., leukemias and lymphomas), a primary immunodeficiency disease or disorder, inflammation, a thalassemia (e.g., alpha-thalassemia, beta-thalassemia, gamma-thalassemia), sickle cell disease, and neutropenia. Balixafortide is administered in a variety of doses that are therapeutically effective. In some embodiments, balixafortide is administered in a dosage of greater than 5.5 mg/kg, up to and including at least 45 mg/kg, providing the surprising advantage of delivering high exposures of the CXCR4 inhibitor with NOAEL at these dosages. In some embodiments, the dosage of balixafortide is greater than 5.5 mg/kg, such as a dosage of at least 6.05 mg/kg, which is 10% higher than a dose of 5.5 mg/kg. Embodiments are also provided in which the dosage of balixafortide is 6.6 mg/kg (20% higher than 5.5 mg/kg) or 6.9 mg/kg (25% higher than 5.5 mg/kg), or 11 mg/kg (100% higher than 5.5 mg/kg) or 16.5 mg/kg (200% higher than 5.5 mg/kg). In some embodiments, at least 6.05 mg/kg balixafortide is administered, preferably 6.6 mg/kg, preferably 6.9 mg/kg, preferably 7.5 mg/kg, preferably 9 mg/kg, preferably 11 mg/kg, preferably 13.5 mg/kg, preferably 16.5 mg/kg. In some embodiments, a plurality of doses of greater than 5.5 mg/kg balixafortide are administered, preferably doses greater than 5.5 mg/kg, preferably doses of at least 6.6 mg/kg, preferably doses of at least 6.9 mg/kg, preferably doses of at least 7.5 mg/kg, preferably doses of at least 9 mg/kg, preferably doses of at least 11 mg/kg, preferably doses of at least 13.5 mg/kg, preferably doses of at least 16.5 mg/kg.

[0029] In some embodiments, the dosage of the CXCR4 inhibitor balixafortide produces a specified exposure of the patient to the inhibitor. These dosages are conveniently defined in terms of the area under the curve (AUC) of blood concentration of the inhibitor over a defined period of time, such as the 24 hours following administration (i.e. , AUC0-24). Exemplary AUC0-24 dosages balixafortide are at least 75000 (h x ng/mL) or at least 90000 (h x ng/mL), as shown in Figure 3, which reflects the concentration of inhibitor in the blood (ng/mL) multiplied by the time of exposure in hours (h). Other exemplary AUC0-24 dosages of balixafortide are at least 150000 (h x ng/mL) or at least 200000 (h x ng/mL) or at least 250000 (h x ng/mL) which reflects the concentration of inhibitor in the blood (ng/mL) multiplied by the time of exposure in hours (h). In addition, suitable dosages of balixafortide are dosages that result in a blood concentration of the inhibitor that meets or exceeds a threshold level of at least 150000 units AUC0-24, at least 200000 units AUC0-24, or at least 250000 units AUC0-24 balixafortide over the first 24 hours following initial administration. [0030] Pharmaceutical compositions according to the disclosure that are suitable for infusion or injection can include balixafortide with or without a secondary therapeutic such as G-CSF, in combination with one or more pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or in combination with sterile powders that may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes that render the composition isotonic with the blood of the intended recipient or suspending or thickening agents known in the art.

[0031] Examples of suitable aqueous and non-aqueous carriers that may be employed in the pharmaceutical compositions provided herein include water for injection (e.g., sterile water for injection), bacteriostatic water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol such as liquid polyethylene glycol, and the like), sterile buffer (such as citrate buffer), and suitable mixtures thereof, vegetable oils, such as olive oil, injectable organic esters, such as ethyl oleate, and Cremophor EL™ (BASF, Parsippany, NJ). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability or infusibility exists. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0032] The composition should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. Prevention of such contamination can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[0033] Sterile injectable solutions can be prepared by incorporating the active compound(s) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those identified above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are freeze-drying (lyophilization), which yields a powder of the active compound(s) plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0034] Injectable depot forms can be made by forming microcapsule or nanocapsule matrices of a compound provided herein in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of therapeutic compound(s) to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot-injectable compositions are also prepared by entrapping the drug in liposomes, microemulsions or nanoemulsions, which are compatible with body tissue.

[0035] A compound as disclosed herein can be prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled- release composition, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such compositions can be prepared using standard techniques, or obtained commercially, e.g., from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to selected cells or tissues with monoclonal antibodies to cellular antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811, which is incorporated herein by reference in its entirety.

[0036] As used herein, "administration" and "administered by injection" refer to sterile injection using needle-and-syringe or an infusion apparatus and include, without limitation, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal or intrastemal injection or infusion.

[0037] In the following examples, experimental data is provided to establish that administrations of high doses of CXCR4 inhibitor balixafortide are efficacious and safe in mobilizing stem cells and treating diseases and disorders characterized by excess CXCR4 activity. EXAMPLES

Example 1

Balixafortide administration to renally impaired subjects

[0038] The experiments described in this Example establish that balixafortide at high doses of at least 5.5 mg/kg is both well-tolerated and safe for use as a therapeutic in renally impaired subjects. In support of that position, disclosed herein are the results of an open-label, non-randomized, monocenter, single-dose, Phase 1 study conducted to evaluate the pharmacokinetics (PK) and safety of balixafortide (/.e., POL6326) administered using a single two-hour intravenous infusion to subjects with renal impairment. The study design was to investigate the safety and tolerability of the single IV infusion of balixafortide to subjects with renal impairment.

[0039] The study involved an open-label, non-randomized, monocenter, single IV dose, The study included up to 40 males and females of at least 18 years of age who were divided into four groups - one group of subjects with normal renal function and three groups of subjects with stable renal impairment of varying relative severity (/.e., mild, moderate or severe) as assessed by estimated glomerular filtration rate (eGFR) using the Chronic Kidney Disease Epidemiology Collaboration (/.e., CKD-EPI) equation.

[0040] A single dose of 5.5 mg/kg balixafortide was administered to the subjects using a two-hour IV infusion. Plasma concentrations showed an almost linear increase until the end of infusion. Thereafter, concentrations showed a biphasic decline, an initial steep decline until approximately 5 h after start of infusion, followed by a slower decline in the terminal phase (see Figure 1). The Cmax and AUCo-~ (area under the curve of dose per unit time running from time zero to the end of the study) were derived from balixafortide plasma concentrations. All measured variables and derived PK parameters were listed individually and summarized by renal function group. AUCo-~ and Cmax of balixafortide were analyzed with descriptive statistics and graphical description. Analyses of Variance (ANOVA) were performed on the log-transformed values with 90% Cis for the group ratios of impaired/normal and regression models explaining the impact of eGFR value and creatinine clearance at screening on AUCo-~ and Cmax-

[0041] The pharmacokinetic results of the study (see Table 1) showed that with decreasing renal function, overall exposure of the patient or subject to balixafortide increased. AUCo-~ in participants with severe renal impairment (eGFR less than 30 mL/min) was more than twice as large as in participants with normal renal function (eGFR at least 90 mL/min). Maximum exposure (Cmax) was comparable over the four groups. Visual analysis of exposure characteristics AUC and Cmax versus total dose show no influence of total dose on Cmax, AUCo-~, AUCo-t, and AUCo-24h. In line with decreasing renal function, apparent terminal elimination half-life (ti/2) prolonged from 13 h to 32 h, while total clearance (CL/F) decreased from 5,373 mL/h to 1 ,900 mL/h, indicating that renal excretion is a major route of balixafortide elimination. As expected, renal clearance of balixafortide decreased with declining renal function. In participants with severe renal impairment, mean CLR was 249 mL/h compared to 1663 mL/h in the control group of participants with normal renal function (Table 2). With lower clearance rates, the fraction of the given dose excreted in urine (f _e ) also decreased from a mean of 25% in the control group to 9% and 6%, respectively, in participants with moderate or severe impairment (Table 2). In addition, in participants with severe renal impairment, non-renal clearance (CLNonR) was slightly reduced in comparison to participants with normal renal function (Table 2).

Table 1 : Descriptive statistics of balixafortide plasma PK characteristics

AUC = Area Under the Curve, GeoCV = Geometric coefficient of variation,

GeoMean = Geometric Mean, Cmax = observed maximum concentration, tmax = time of observed maximum concentration, ti/2 = apparent terminal elimination half-life,

CL/F = total clearance Table 2: Urine PK parameters of balixafortide

GeoCV = Geometric coefficient of variation, GeoMean = Geometric Mean, f _e = fraction excreted in urine, CLR = renal clearance, CLNOOR = non-renal clearance

[0042] While maximum exposure (Cmax) was only marginally affected, overall exposure of balixafortide increased with decreasing renal function. In moderate and severe renal impairment, overall exposure (AUCo-~) increased markedly (90% Confidence Interval (/.e., Cl) moderate: 126.29%, 226.38%; severe: 205.33%, 375.68%). The reduced renal clearance resulted in a prolongation of the half-life from about 13 h observed in participants with normal renal function to 27 h with moderately impaired renal function and 32 h with severely impaired renal function, respectively.

[0043] The increase in AUCo-~ in participants with severe renal impairment was slightly larger than estimated based on the population PK model, which predicted a smaller increase of AUC (1.8x) levels, if eGFR decreased from 90 to 30 mL/min/1.73 m ² . Participants with mild renal impairment only showed an increase in AUCo-~ of about 17%, which was not significantly higher than normal renal function (90% Cl: 87.67%, 157.15%).

[0044] The increases in exposure resulted from decreased renal excretion of balixafortide. Urine PK parameters were consistent with the observations in plasma. Mild renal impairment showed only a minor impact on balixafortide elimination in urine. The fractional elimination (/.e., f _e ) of 22% for balixafortide that was seen in participants with mild renal impairment was only slightly lower compared to participants with normal renal function (25%). The highest decrease in renal clearance was observed in the group with severe renal impairment (eGFR below 30 mL/min) and moderate renal impairment (eGFR 30 to 59 mL/min), where renal clearance was 248.52 mL/h and 484.84 mL/h, respectively, compared to 1662.62 mL/h in participants with normal renal function.

[0045] Regression analysis showed a clear association of increasing plasma exposure to balixafortide with decreasing renal function. In participants with eGFR below 60 mL/min, /.e., in the groups with moderate and severe renal impairment, exposure to balixafortide increased substantially (90% Cl moderate: 126.29%, 226.38%; severe: 205.33%, 375.68%) due to reduced renal clearance. The half-life in groups with moderate and severe renal impairment was prolonged to 27 h and 32 h, respectively, compared to 13 h observed in participants with normal renal function. Both variables for renal function applied in this study (eGFR and creatinine clearance at screening) may be considered to estimate exposure.

[0046] Infusion of 5.5 mg/kg balixafortide over 2 h was safe and well tolerated. Based on the observed safety and tolerability, no dose adjustment for participants with severe renal impairment (eGFR less than 30 mL/min) was required.

[0047] The safety results of this study are in line with the known pharmacological and safety profile of balixafortide. The majority of observed treatment-emergent adverse events (/.e., TEAEs) related to the treatment were leukocytosis (30 events in 30 subjects, 96.8%) and hypersensitivity (25 events in 24 subjects, 77.4%). The frequency of these events was similar across all groups.

[0048] Leukocytosis, and in particular neutrophil mobilization, is a pharmacological effect of balixafortide. In the study described in this Example, unexpectedly the administration of balixafortide shifted neutrophil levels in 93% (28/30 patients) of the patients from normal levels to high (/.e., at least 10 ⁹ neutrophils/L serum). On days 3 and 4, the percentage of patients having neutrophil levels shifting from the normal range to the high range was 43.5% (10/23 patients; day 2) and 22.7% (5/22 patients; day 4). The total number of patients varied by day post-administration due to patients missing scheduled appointments. Infusion-related-reaction-induced hypotension is described in the reference safety information of balixafortide. Except for leukocytosis and a few events of hypotension, a known side effect of balixafortide, no adverse effects on safety laboratory parameters, vital signs or ECG were observed.

[0049] Hypersensitivity reactions or infusion-related reactions (/.e., IRR) usually occur during balixafortide administration and quickly disappear after infusion stops. Thus, in case of emerging adverse events, the infusion can briefly be interrupted until signs disappear and then the infusion can be continued. To reduce the occurrence and/or severity of adverse hypersensitivity reactions, it is generally recommended that all subjects be pre-medicated with an antihistaminic drug, e.g., 10 mg loratadine, 2 h prior to receiving balixafortide. [0050] Renal impairment had no marked influence on the observed safety parameters, as the frequency and characteristics of TEAEs were similar in all four renal function groups. In some embodiments, subjects with reduced renal clearance, e.g., below 30 mL/min, should be closely monitored during drug administration. Notwithstanding any monitoring that may be implemented, the results of the study disclosed herein establish that there is no need for dose reductions in subjects with increasing renal impairment. More particularly, the results of this study showed higher systemic exposure with balixafortide and more pronounced elevations of white blood cells in general, including neutrophils, with increasing severity of renal impairment, but without a clear need for dosing reductions in subjects with renal clearance below 30 mL/min. The observed higher AUC exposure and concomitant stronger leukocyte mobilization indicate that higher doses in subjects with normal renal function might be beneficial for the desired pharmacological effect.

Example 2

Balixafortide administration to treat cancer

[0051] Balixafortide at dosages of greater than.5 mg/kg are useful in treating several disease or conditions beyond the renal impairment addressed in Example 1. As a CXCR4 inhibitor, balixafortide is expected to be an effective therapeutic in treating various cancers.

[0052] Several clinical studies have demonstrated the efficacy of balixafortide administered within a dosage range of 0.01-2.5 mg/kg administered by infusion over a period of 1-3 hours. Results showed that there were dose-proportional increases in Cmax and AUC. Exposure to balixafortide appeared to increase in a dose-proportional manner over the higher dose range (1-2.5 mg/kg). Decreasing the infusion time resulted in the predicted increase in Cmax levels with a change in total systemic exposure. Following a single IV administration, the average ti/2 of balixafortide in the lower dose range (0.01-0.6 mg/kg) in healthy subjects was 4.4 ± 1.4 h. In the higher dose range (1-2.5 mg/kg), the terminal ti/2 increased slightly to 6.7 ± 0.9 hours. These clinical trials examined the effect of balixafortide on various patient populations, including cancer patients (multiple myeloma patients) and patients with acute myocardial infarction. An additional study of patients with metastatic breast cancer investigated a balixafortide dose of 5.5 mg/kg. The balixafortide clearance rate in mBC patients was 4.4 L/h, lower than the CL of 6.5 L/h for healthy individuals. The distribution volume V _z was larger than for healthy patients (49 L versus 45 L) due to a larger volume of distribution of the central compartment, and a longer ti/2 than found in healthy subjects (16 h versus 6.6 h). The results of these studies led to the expectation that increasing the dosage of balixafortide beyond 5.5 mg/kg would be contra-indicated because of increasing toxicity evidenced by proliferating treatment-emergent adverse events (/.e., TEAEs).

[0053] In vitro, balixafortide was not cytotoxic to cells derived from the immortal human cell line HeLa and the monkey cell line Cos-2 (up to a concentration of 100 pM). Also, balixafortide did not elicit any hemolytic activity in human blood, as demonstrated up to a concentration of 100 pM. Further toxicology studies were conducted using mouse and monkey models, selected because of appropriate target expression and relevant pharmacology.

[0054] The mouse was considered a relevant rodent species because mice express the target for balixafortide, /.e., the CXCR4 receptor, and exhibit pharmacodynamic (/.e., PD) responses, such as mobilization of hematopoietic stem cells (/.e., HSCs) following administration of a CXCR4 inhibitor.

[0055] The monkey was selected as the non-rodent species because it was considered relevant to the assessment of safety for humans, as it expresses the CXCR4 receptor [1] and displays PD responses to CXCR4 antagonist administration [2], Also, higher exposures of balixafortide were better tolerated in the monkey than in, e.g., the dog. Consequently, monkey was chosen as the non-rodent species.

[0056] General in vivo toxicology studies lasted up to 13 weeks using CD-1 mice or using cynomolgus monkeys. Route of administrations chosen for the mouse and monkey were driven by technical constraints (mouse) and the intended human administration regimen(s). Several toxicology studies were conducted using the mouse model that showed that balixafortide was well-tolerated. A relevant example of such toxicological studies, including the dosages, duration, and observed results, is presented in Table 3. Table 3: In vivo toxicology study in mice

[0057] In vivo toxicology of balixafortide administration in monkeys was also assessed. In a corresponding study, varying doses of balixafortide were administered via a 2-hour infusion to cynomolgus monkeys once daily for three consecutive days during each week of the 13-week study. Dosages, duration and observed results for these in vivo studies of the effects of balixafortide on cynomolgus monkeys are presented in Table 4. Balixafortide was well-tolerated in all animals, with the exception of transient increases in penile sheath or vulva size at 200 mg/kg/day but only soon after dosing on Day 1. There were no balixafortide-related changes in body weight. Also, there were no balixafortide-related abnormalities at the functional observation battery, or electrocardiograph or ophthalmology examinations. In line with the CXCR4 antagonist mode of action, balixafortide produced dose-dependent increases in white blood cells, reaching statistical significance at 200 mg/kg/day in both sexes. The dose- dependent increases in white blood cells involved increases in mean neutrophil, eosinophil, basophil, lymphocyte and monocyte counts, as well as a dose-dependent increase in the number of CD34+ progenitor cells.

Table 4: In vivo toxicology study in monkeys

[0058] Cynomolgus monkeys were used to assess systemic exposure to balixafortide in a repeat-dose toxicity study. In this study, monkeys received repeat doses of balixafortide administered by IV over the course of 13 weeks. Dosing, PK characteristics, and safety margins assuming a human dosing of 5.5 mg/kg are collected in Table 5.

Table 5: Systemic exposure to balixafortide in repeat-dose toxicity studies and safety margins ^a Mean exposure through study (mean of both genders Day 1 and Day 14 or Day 28; 13-week study, mean of both genders Day 1 and Day 3 of Weeks 1 and 13). ^b Multiple of human exposure based on mean values for Cmax of 11700 ng/mL and AUC0-24 of 74300 ng*h/mL in breast cancer patients at 5.5 mg/kg.

[0059] The results obtained using the mouse and monkey animal models and in particular the determined NOAEL of 45 mg/kg/day led to the surprising realization that balixafortide at doses exceeding 5 mg/kg would be well-tolerated, creating the unexpected possibility of increasing the efficacy of balixafortide treatment of, e.g., cancer by increasing the balixafortide dosage beyond 5 mg/kg and in particular beyond 5.5 mg/kg. [0060] Supported by the experimental data disclosed in this Example and throughout the disclosure, provided is a method for treating cancer, such as solid tumors (e.g., prostate cancer) or hematological malignancies, such as leukemias and lymphomas, by administering a dose of greater than 5.5 mg/kg of balixafortide. In some embodiments, repeated doses of balixafortide are administered. In some embodiments, greater than 5.5 mg/kg balixafortide are administered to treat cancer such as solid tumors (e.g., prostate cancer) or hematological malignancies, such as leukemias and lymphomas. In some of these embodiments, the dose of balixafortide is significantly greater than 5.5 mg/kg, such as a dose that is greater than 5.5 mg/kg balixafortide by 10% (at least 6.05 mg/kg), 20% (at least 6.6 mg/kg), 25% (at least 6.9 mg/kg), 100% (at least 11 mg/kg), 200% (at least 16.5 mg/kg) or more.

Example 3

Treatment of diseases wherein CXCR4 receptor activity is involved

[0061] Treatment of renal impairment and cancer (e.g., solid tumors like, e.g., prostate cancer) or hematological malignancies, such as leukemias and lymphomas) have been described in detail in Examples 1 and 2, respectively. Treatment of other diseases wherein CXCR4 receptor activity is involved include a primary immunodeficiency disease or disorder, a thalassemia, sickle cell disease, and neutropenia. As disclosed herein, one or more therapeutically effective doses of a CXCR4 inhibitor such as balixafortide are administered to a patient suffering from one of these disorders by injection or infusion. Effective doses have been defined herein and include doses of greater than 5.5 mg/kg balixafortide, doses greater than 5.5 mg/kg balixafortide, such as doses of at least 6.05, 6.6 or 6.9 mg/kg, 11 mg/kg, or 16.5 mg/kg balixafortide, doses yielding an AUCo-24 of at least 75000 (h x ng/mL), at least 90000 (h x ng/mL), at least 150000 (h x ng/mL), or at least 250000 (h x ng/mL), or a dose of balixafortide yielding a blood concentration that meets or exceeds a threshold level of at least 150000 units AUCo-24, 200000 units AUCo-24, or 250000 units AUCo-24 balixafortide over the first 24 hours following initial administration.

[0062] Balixafortide may also be combined with secondary therapeutics such as G- CSF. In some of these embodiments, the administration of the CXCR4 inhibitor balixafortide provides a therapy in which the dosage of the secondary therapeutic is reduced. For example, G-CSF is contra-indicated in the treatment of beta-thalassemia because of toxic effects at therapeutic levels. Administration of the CXCR4 inhibitor balixafortide provides a therapy in which the G-CSF dosage is reduced to sub-toxic (/.e., lower than unacceptably toxic) levels (including not administering any G-CSF), resulting in an effective treatment for beta-thalassemia.

References

[0063] 2. Larochelle A, Krouse A, Metzger M, Orlic D, Donahue RE, Fricker S, et al. AMD3100 mobilizes hematopoietic stem cells with long-term repopulating capacity in nonhuman primates. Blood. 2006;107(9):3772-8.

[0064] 3. Karpova D, Ritchey J K, Holt M S, Abou-Ezzi G, Monllish D, Batoon L, Millard S, Spohn G, Wiercinska E, Chendamarai E, Yang W, Christ S, Gehrs L, Schuettpelz L G, Dembowsky K, Pettit A R, Rettig M P, Bonig H, and DiPersio J F. Continuous blockade of CXCR4 results in dramatic mobilization and expansion of hematopoietic stem and progenitor cells. Blood 2017; 129921): 2939-49.

[0065] The disclosure has presented embodiments to facilitate disclosure of the subject matter, but the only limitations to be placed upon the disclosure are the limitations found in the claims.

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