Identification and validation of fetal hemogobin-induction by idasanutlin for the treatment of sickle cell disease

Abstract

The present application relates to the use of idasanutlin, or a pharmaceutically acceptable salt, a solvate, an isomer, or a functional derivative thereof for the treatment of hemoglobinopathies, including sickle cell disease, thalassemia, sickle cell beta thalassemia (Hb S / β Th), and leukocytosis as well as myeloproliferative conditions, polycythemia, and acute and chronic hemolytic anemia. It was found that idasanutlin increases HbF levels in multipotent erythroleukemia, hematopoietic stem cells, and sickle cell disease cells to provide another therapy for treatment of sickle cell disease.

Description

IDENTIFICATION AND VALIDATION OF FETAL HEMOGOBIN-INDUCTION BY IDASANUTLIN FOR THE TREATMENT OF SICKLE CELL DISEASEFIELD

[0001] The present invention relates to induction of fetal hemoglobin for the treatment of sickle cell disease.BACKGROUND

[0002] Sickle cell disease affects more than 100,000 people in the United States and 8 million people worldwide. In Canada, almost 8,000 people are affected with sickle cell disease. Worldwide, at least 300,000 children affected with a severe hemoglobinopathy are born each year. The true prevalence of thalassemia in North America is not known; however, it appears to be increasing, especially with increased immigration from affected regions.

[0003] It has been identified that a birth incidence for sickle cell disease is about 1 in 2400 births in a specific population. The disease is characterized by sickling of red blood cells that subsequently causes anemia, necrosis, infections, and severe painful episodes known as sickle cell crisis when blood vessel blockages restrict blood flow to vital tissue and organs. Molecularly, sickle cell disease is caused by a mutation in hemoglobin, which carries oxygen in red blood cells, resulting in polymerization of protein fibers that distort the cell’s shape under hypoxic conditions. Normal adult hemoglobin (HbA) is composed of four proteins, two a-globin and two [3-globin subunits (a2|32). A mutation in [3-globin can lead to the formation of abnormal sickle hemoglobin (HbS). The expression of these genes is temporally regulated in which distinct globin genes are produced during different stages of development.

[0004] Individuals with sickle cell disease spend approximately $1 .7 million on disease-related medical expenses over their lifetime. Accordingly, this highlights the enormous economic burden affecting individuals living with sickle cell disease and support and validate the utility of the HbF-inducing agent idasanutlin. This agent that can decrease the health care utilization of resources due to sickle cell disease has the potential to generate $3 billion in revenue in North America every year.SUMMARY

[0005] It has now been found that the compounds which act as inhibitors of specific proteins that are involved in controlling cell proliferation may be used for the treatment of sickle cell disease by controlling proliferation of hematopoietic stem cells andthereby altering hematopoiesis to increase the number of fetal hemoglobin-expressing erythroid progenitors.

[0006] The present disclosure relates to a compound of formula: 4- ((2R,3S,4R,5S)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)-4-cyano-5- neopentylpyrrolidine-2-carboxamido)-3-methoxybenzoic acid, referred to as idasanutlin (RG7388).

[0007] The present disclosure provides a compound of formula (idasanutlin), or a pharmaceutically acceptable salt, or a solvate, or an isomer, or a functional derivative thereof.

[0008] The present disclosure also encompasses idasanutlin in a physiologically or pharmacologically acceptable buffer, carrier, and / or excipient and in combination with hydroxyurea or other cytostatic drugs as approved therapies for the treatment of sickle cell disease.

[0009] In some embodiments there is provided:

[0010] 1. A method of treating a hemoglobinopathy in a subject in need thereof, comprising administering to the human an effective amount of 4-((2R,3S,4R,5S)-3-(3- chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)-4-cyano-5-neopentylpyrrolidine-2- carboxamido)-3-methoxybenzoic acid (idasanutlin), or a pharmaceutically acceptable salt, or a solvate, or an isomer, or a functional derivative thereof, wherein the subject is a human.

[0011] 2. The method of embodimnt 1 , further comprising the administration of one or more of hydroxyurea, L-glutamine (Endari), crizanlizumab (Adakveo), or penicillin in combination to optimize safety, tolerability and effectiveness for the desired outcome to reduce sickling crisis..

[0012]

[0013] 3. The method of embodiment 1 or 2, wherein said hemoglobinopathy is sickle cell disease, thalassemia major, thalassemia intermedia, sickle cell beta thalassemia (Hb S / p Th), polycythemia, leukocytosis, myeloproliferative conditions or acute and chronic hemolytic anemia.

[0014] 4. The method of embodiment 1 , wherein said hemoglobinopathy is sickle cell disease, thalassemia major, thalassemia intermedia, thalassemia major, sickle cell beta thalassemia (Hb S / p Th), polycythemia, leukocytosis, myeloproliferative conditions or acute and chronic hemolytic anemia.

[0015]

[0016] 5. The method of any one of embodiments 1- 5, wherein the subject is an adult or a child.

[0017] 6. The method of any one of embodiments 1 -5 , wherein the idasanutlin, or a pharmaceutically acceptable salt, or a solvate, or an isomer, or a functional derivative thereof, is administered at from about 10-500 mg daily, optionally for 5-7 days in a 28- day cycle, or as needed based on symptoms resolution.

[0018]

[0019] 7. A method of treating a hemoglobinopathy in a subject in need thereof, comprising administering to the human Use of an effective amount of 4-((2R,3S,4R,5S)-3- (3-chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)-4-cyano-5-neopentylpyrrolidine-2- carboxamido)-3-methoxybenzoic acid (idasanutlin), or a pharmaceutically acceptable salt, or a solvate, or an isomer, or a functional derivative thereof, for treating a hemoglobinopathy in a human in need thereof, or in the manufacture of a medicament for treating a hemoglobinopathy in a human in need thereof, wherein the subject is a human

[0020] 8. The use of embodiment 7, further comprising the use of one or more of hydroxyurea, L-glutamine (Endari), crizanlizumab (Adakveo), or penicillin.

[0021] 9. The use of embodiment 7 or 8, wherein said hemoglobinopathy is sickle cell disease, thalassemia major, thalassemia intermedia, or acute and chronic hemolytic anemia.

[0022] 10. The use of embodiment 7, wherein said hemoglobinopathy is sickle cell disease.

[0023] 11 . The use of any one of embodiments 1- 5, wherein the subject is an adult or a child.

[0024] 12. The use of any one of embodiments 1 5, wherein the idasanutlin, or a pharmaceutically acceptable salt, or a solvate, or an isomer, or a functional derivative thereof, is for administration at from about 50-500 mg daily, optionally for 5-7 days in a 28-day cycle,

[0025] 13. A method of inducing HbF expression in a human cell, comprising said cell with of 4-((2R,3S,4R,5S)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)-4-cyano- 5-neopentylpyrrolidine-2-carboxamido)-3-methoxybenzoic acid (idasanutlin), or a pharmaceutically acceptable salt, or a solvate, or an isomer, or a functional derivative thereof.

[0026] 14. A pharmaceutical composition comprising idasanutlin and a pharmaceutically acceptable carrier for inducing fetal hemoglobin (HbF) during erythropoiesis in a subject in need thereof.

[0027] 15. The composition of embodiment 14, wherein the composition is formulated for oral, parenteral, administration.

[0028] 16. The composition of embodiment 14 or 15, wherein the composition is a modified-release, extended-release, or controlled-release formulation providing sustained plasma exposure of idasanutlin.

[0029] 17. A method of treating sickle cell disease in a subject, comprising administering to the subject a therapeutically effective amount of idasanutlin, thereby increasing HbF and reducing one or more clinical endpoints selected from frequency of vaso-occlusive crises, transfusion burden, hemolysis markers, or hospitalization rate.

[0030] 18. The method of embodiment 17, wherein idasanutlin exhibits reduced toxicity relative to hydroxyurea as measured by one or more parameters selected from neutropenia incidence, mucositis, gastrointestinal adverse events, or treatment discontinuation.

[0031] 19. .A method of preventing or reducing severity of sickle cell crisis in a subject with sickle cell disease, comprising administering idasanutlin prophylactically according to a dosing regimen sufficient to maintain elevated HbF.

[0032] 20. .A method of treating p-thalassemia, including transfusion-dependent |3- thalassemia, comprising administering idasanutlin to increase HbF and reduce transfusion frequency or volume.

[0033] 21. .A method of treating leukocytosis in a subject, comprising administering idasanutlin in an amount effective to reduce circulating leukocyte counts toward a target range while maintaining therapeutic HbF induction.

[0034] 22. A method of treating polycythemia in a subject, comprising administering idasanutlin in an amount effective to reduce hematocrit toward a target therapeutic range while maintaining therapeutic HbF induction.

[0035] 23. A method of treating a myeloproliferative disorder in a subject, comprising administering idasanutlin to modulate erythroid and / or myeloid proliferation and reduce one or more disease parameters selected from leukocytosis, thrombocytosis, splenomegaly, pruritus, constitutional symptoms, or phlebotomy frequency.

[0036] 24. The method of any one of embodiments 17-23, wherein the subject is pediatric, adolescent, adult, or pregnant, and the dosing is adjusted by body mass, renal / hepatic function, or pharmacogenomic profile.

[0037] 25 The method of any one of embodiments 17-23, wherein idasanutlin is administered in combination with one or more agents selected from L-glutamine,voxelotor, crizanlizumab, phlebotomy protocols, iron chelators, JAK inhibitors, or erythropoietin analogs, concurrently or sequentially.

[0038] 26. The composition of any one of embodiments 14-16, wherein the subject is a human.

[0039] 27. The method of any one of embodiments 17 to 25, wherein the subject is a human.

[0040] 28. Use of a pharmaceutical composition comprising idasanutlin and a pharmaceutically acceptable carrier for inducing fetal hemoglobin (HbF) during erythropoiesis in a subject in need thereof, or in the manufacture of a medicament for inducing fetal hemoglobin (HbF) during erythropoiesis in a subject in need thereof.

[0041] 29. The use of embodiment 28, wherein the composition is formulated for oral, parenteral, administration.

[0042] 30. The use of embodiment 28 or 29, wherein the composition is a modified- release, extended-release, or controlled-release formulation providing sustained plasma exposure of idasanutlin.

[0043] 31 . The use of a therapeutically effective amount of idasanutlin, for treating sickle cell disease in a subject thereby increasing HbF and reducing one or more clinical endpoints selected from frequency of vaso-occlusive crises, transfusion burden, hemolysis markers, or hospitalization rate, or in the manufacture of a medicament for treating sickle cell disease in a subject thereby increasing HbF and reducing one or more clinical endpoints selected from frequency of vaso-occlusive crises, transfusion burden, hemolysis markers, or hospitalization rate.

[0044] 32. The use of embodiment 31 , wherein idasanutlin exhibits reduced toxicity relative to hydroxyurea as measured by one or more parameters selected from neutropenia incidence, mucositis, gastrointestinal adverse events, or treatment discontinuation.

[0045] 33. .Use of idasanutlin prophylactically according to a dosing regimen sufficient to maintain elevated HbF for preventing or reducing severity of sickle cell crisis in a subject with sickle cell disease, or in the manufacture of a medicament for preventing or reducing severity of sickle cell crisis in a subject with sickle cell disease.

[0046] 34. Use of idasanutlin to increase HbF and reduce transfusion frequency or volume for treating p-thalassemia, including transfusion-dependent p-thalassemia, or in the manufacture of a medicament for treating p-thalassemia, including transfusiondependent p-thalassemia.

[0047] 35. .Use of idasanutlin in an amount effective to reduce circulating leukocyte counts toward a target range while maintaining therapeutic HbF induction for treating leukocytosis in a subject, or in the manufacture of a medicament treating leukocytosis in a subject.

[0048] 36. Use of idasanutlin in an amount effective to reduce hematocrit toward a target therapeutic range while maintaining therapeutic HbF induction for treating polycythemia in a subject, or in the manufacture of a medicament effective to reduce hematocrit toward a target therapeutic range while maintaining therapeutic HbF induction for treating polycythemia in a subject .

[0049] 37. Use of idasanutlin to modulate erythroid and / or myeloid proliferation and reduce one or more disease parameters selected from leukocytosis, thrombocytosis, splenomegaly, pruritus, constitutional symptoms, or phlebotomy frequency for treating a myeloproliferative disorder in a subject, or in the manufacture of a medicament for treating a myeloproliferative disorder in a subject.

[0050] 38. The use of any one of embodiments 31-37, wherein the subject is pediatric, adolescent, adult, or pregnant, and the dosing is adjusted by body mass, renal / hepatic function, or pharmacogenomic profile.

[0051] 39 The use of any one of embodiments 31-37, wherein idasanutlin is for use administration in combination with one or more agents selected from L-glutamine, voxelotor, crizanlizumab, phlebotomy protocols, iron chelators, JAK inhibitors, or erythropoietin analogs, concurrently or sequentially.

[0052] 40. The use of any one of embodiments 28-39, wherein the subject is a human.BRIEF DESCRIPTION OF THE FIGURES

[0053] Embodiments of the present disclosure will now be described, by way of example only, with reference to the attached Figures.

[0054] Figure 1 depicts affect of various HbF-inducing drugs in hematopoietic stem cells.

[0055] Figure 2 depicts the increase in HbF level in hematopoietic cells from idasanutlin (Figure 2A) compared with increase from hydroxyurea (Figure 2B).

[0056] Figure 3 depicts the increase in HbF level in erythroleukemia cells from idasanutlin is shown compared with increase from hydroxyurea.

[0057] Figure 4A and Figure 4B depict the increase in HbF level in hematopoietic cells from idasanutlin.

[0058] Figure 5A and Figure 5B depicts that that idasanutlin has cytostatic effect in erythroid cells at low concentrations.

[0059] Figure 6A and Figure 6B depicts idasanutlin reduces hypoxia-induced sickling in red blood cells.DETAILED DESCRIPTION

[0060] It is known that during embryonic development, fetal hemoglobin (HbF) is the major form and contains two a-globin and two y-globin subunits (a2y2), which is ultimately replaced with HbA during infancy as the expression of y-globin gradually decreases and P-globin increases. Hence, it stands to reason that pharmacological interventions to increase expression of y-globin instead of [3-globin may alleviate symptoms of sickle cell disease.

[0061] The treatment for children with sickle cell disease may include hydroxyurea (HU)- mediated induction of HbF. Mechanistically, HU reversibly inhibits ribonucleotide reductase, an enzyme for DNA synthesis, which ultimately impedes cell cycle progression through S-phase. Because temporarily arresting hematopoiesis alters erythroid kinetics upon recovery, stress-induced recruitment of early erythroid progenitors that maintain HbF-producing capacity is thought to increase HbF level. Although it is generally well tolerated, HU exposure may cause serious side effects such as myelosuppression and the long-term safety of treating young children with a DNA repair-modulating agent remains uncertain. While HU demonstrably reduces the occurrence of hospitalizations due to sickle cell crisis, it is not effective and does not prevent other complications of sickle cell disease. Other treatments may include procedures such as hematopoietic stem cell transplant. While hematopoietic stem cell transplant may offer a cure for sickle cell disease, its application is limited by the cost, risks, and lack of suitable donors.

[0062] Based on preliminary findings, a method to screen and validate HbF-inducing agents is provided. Specifically, it has been shown that idasanutlin increases HbF levels in multipotent erythroleukemia, hematopoietic stem cells, and sickle cell disease cells to provide another therapy for treatment of sickle cell disease.

[0063] Compounds which act as inhibitors of specific proteins are found to be involved in controlling cell proliferation. Accordingly, these compounds may be used for the treatment of sickle cell disease by controlling proliferation of hematopoietic stem cells and therebyaltering hematopoiesis to increase the number of fetal hemoglobin-expressing erythroid progenitors. In particular, the present disclosure relates to a compound of formula: 4- ((2R,3S,4R,5S)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)-4-cyano-5- neopentylpyrrolidine-2-carboxamido)-3-methoxybenzoic acid, referred to as idasanutlin (RG7388). The present disclosure provides a compound of formula (idasanutlin), or a pharmaceutically acceptable salt, or a solvate, or an isomer, or a functional derivative thereof. The present disclosure also encompasses idasanutlin in a physiologically or pharmacologically acceptable buffer, carrier, and / or excipient and in combination with hydroxyurea or other cytostatic drugs as approved therapies for the treatment of sickle cell disease.

[0064] In some example, the method further comprising the administration of one or more of hydroxyurea, L-glutamine (Endari), crizanlizumab (Adakveo), or penicillin in combination to optimize safety, tolerability and effectiveness for the desired outcome to reduce sickling crisis.

[0065] The term “subject”, as used herein, refers to an animal, and can include, for example, domesticated animals, such as cats, dogs, etc., livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.), mammals, non-human mammals, primates, non-human primates, rodents, birds, reptiles, amphibians, fish, and any other animal. In a specific example, the subject is a human. In another specific example, the human is a pediatric human or an adult human. In a specific example, the subject is a pediatric human (also referred to as a child).

[0066] The term “treatment”, “treat”, or “treating” as used herein, refers to obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission (whether partial or total), whether detectable or undetectable. "Treating" and "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0067] The term "amelioration" or "ameliorates" as used herein refers to a decrease, reduction or elimination of a condition, disease, disorder, or phenotype, including an abnormality or symptom.

[0068] The term "symptom" of a disease or disorder is any morbid phenomenon or departure from the normal in structure, function, or sensation, experienced by a subject and indicative of disease.

[0069] A "treatment regimen" as used herein refers to a combination of dosage, frequency of administration, or duration of treatment, with or without addition of a second medication, including chemotherapy or another immunotherapy such as immune checkpoint inhibitors.

[0070] The term “pharmaceutically effective amount” as used herein refers to the amount of a drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by a researcher or clinician. This amount can be a therapeutically effective amount.

[0071] Thus, as used herein, the term “therapeutically effective amount” refers to an amount that is effective for preventing, ameliorating, or treating a disease or disorder.

[0072] The term “pharmaceutically acceptable” as used herein refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0073] The term “pharmaceutically acceptable carrier” as used herein refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agents from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, for example the carrier does not decrease the impact of the agent on the treatment. In other words, a carrier is pharmaceutically inert. The terms “physiologically tolerable carriers” and “biocompatible delivery vehicles” are used interchangeably. Thus, the term “carrier” or “excipient” may refer to a non-toxic solid, semi-solid or liquid filler, diluent. The term includes solvents, dispersion, media, coatings, isotonic agents, and adsorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art.

[0074] As used herein, the term “pharmaceutically-acceptable salts” refers to the conventional nontoxic salts or quaternary ammonium salt. These salts can be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by separately reacting a compound in its free base or acid form with a suitable organic or inorganic acid or base, and isolating the salt thus formed during subsequent purification.Conventional nontoxic salts include those derived from inorganic acids such as sulfuric, sulfamic, phosphoric, nitric, and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic, sulfanilic, 2- acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isothionic, and the like.

[0075] In some examples, therapeutic formulations comprising the compounds or compositions as described herein may be prepared for by mixing compounds or compositions having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers, in the form of aqueous solutions, lyophilized or other dried formulations. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, histidine and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG).

[0076] A “pharmaceutical composition” as used herein refers to a chemical or biological composition suitable for administration to a subject. Such compositions may be specifically formulated for administration via one or more of a number of routes, including but not limited to, oral, parenteral, intravenous, intra-arterial, subcutaneous, intra-nasal, sublingual, intra-spinal, intra-cerebroventricular, and the like.

[0077] The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing the active compound into association with a carrier, which may constitute one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active compound with liquid carriers or finely divided solid carriers or both, and then if necessary, shaping the product.

[0078] The compounds and compositions may be administered to a subject by any convenient route of administration, whether systemically / peripherally or at the site of desired action, including but not limited to, oral (e.g. by ingestion); topical (including e.g. transdermal, intranasal, ocular, buccal, and sublingual); pulmonary (e.g. by inhalation or insufflation therapy using, e.g. an aerosol, e.g. through mouth or nose); rectal; vaginal; parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intra-arterial, intra-cardiac, intrathecal, intra-spinal, intra-capsular, sub- capsular, intra-orbital, intraperitoneal, intra-tracheal, subcuticular, intraarticular, subarachnoid, and intra-sternal; by implant of a depot I for example, subcutaneously or intramuscularly.

[0079] In some examples, idasanutlin may be used as a prophylaxis at lower doses to prevent disease complications. It can also be used in combination with hydroxyurea where there is very low response to hydroxyurea and to minimize the toxic effects of high- dose hydroxy urea.

[0080] In some examples, there is provided pharmaceutical compositions and uses of Idasanutlin for induction of fetal hemoglobin and treatment of hemoglobinopathies, myeloproliferation, leukocytosis, and polycythemia.

[0081] This disclosure relates to hematology and immunohematology, specifically to small-molecule or biologic modulators that induce fetal hemoglobin (HbF) during erythropoiesis and provide therapy and prophylaxis for sickle cell disease, p-thalassemia, leukocytosis, polycythemia, and myeloproliferative states.

[0082] Induction of HbF is a validated therapeutic mechanism in sickle cell disease (SCD), mitigating hemoglobin S polymerization, vaso-occlusion, and hemolysis; hydroxyurea, a standard HbF inducer, presents toxicity and tolerability limitations that motivate safer alternative

[0083] Idasanutlin can be used for (i) treating, preventing, and reducing severity of sickle cell crises; (ii) prophylaxis in SCD and transfusion-dependent p-thalassemia; and (iii) treating polycythemia, leukocytosis, and myeloproliferation. It increases HbF during erythropoiesis and exhibits reduced toxicity relative to hydroxyurea.

[0084] • “HbF induction” means an increase in gamma-globin (HBG1 / HBG2) mRNA and / or HbF protein levels in erythroid cells, measurable by standard assays.

[0085] • “Sickle cell crisis” includes vaso-occlusive pain crises and acute chest syndrome episodes.

[0086] • “Myeloproliferation” includes myeloproliferative neoplasms or reactive states characterized by increased myeloid lineage cells.

[0087] METHODS

[0088] Study samples were obtained from healthy adult donors and pediatric patients in collaboration with hematologists. During routine clinic visits, blood specimens were collected from pediatric patients with sickle cell disease following Research Ethics Board approval and informed consent. Blood samples were also collected from adult volunteers following informed consent.

[0089] Peripheral blood mononuclear cells (PBMCs) or white blood cells (WBCs) were isolated from blood samples, by density gradient centrifugation in Ficoll-Paque (Cytiva). CD34-positive hematopoietic stem cells were isolated from PBMCs or WBCs using CD34 microbeads and the MACS magnetic cell separation method (Miltenyi Biotec). Primary cells were used immediately or cryopreserved by initially freezing at -80°C in FBS containing 10% DMSO. These cells were used in assays described below.

[0090] Cells from KU812 and K562 erythroid leukemia cell lines were cultured in complete RPMI-1640 media containing 10% FBS and maintained at 37°C in a 5% CO2humidified incubator. Primary CD34-positive cells were cultured in STEMSPAN Serum- Free Expansion Medium (SFEM) with 1X STEMSPAN CD34-positive Expansion Supplement (STEMCELL Technologies), containing recombinant human fms-like tyrosine kinase 3 ligand (Flt3L), stem cell factor (SCF), interleukin 3 (IL-3), interleukin 6 (IL-6), and thrombopoietin (TPO). Cell viability was assessed by Alamar Blue assay or trypan blue staining and hematocytometer cell counting. To induce erythroid maturation and synthesis of hemoglobin, erythroblasts were supplemented with 10 ng / ml recombinant erythropoietin (EPO). For hypoxia-induced sickling, cells were treated with 1% sodium metabisulfite.

[0091] HbF mRNA and protein levels were determined by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) or immunoblot, respectively. For high-throughput screening (HTS), cells were seeded into 96-well plates and treated with individual molecules from a panel of FDA-approved drugs at 1 pM, followed by red blood cell lysis and quantification of HbF by ELISA. To validate top hits identified by HTS, total mRNA and protein were isolated from cells treated with increasing concentrations of candidate drugs, compared to HU as the standard of care treatment. RT-qPCR was performed with primers specific to HBG2, HBB, and HBA2 to determine the relative levels of fetal, adult, and total hemoglobin, respectively, normalized to GAPDH reference. Immunoblotting was performed with antibodies against y-globin, [3-globin , and a-globin to detect fetal, adult, and totalhemoglobin, respectively, compared to p-actin loading control along with appropriate negative and positive controls.

[0092] Data is presented as mean ± standard error of the mean (SEM) from three independent experiments. The one-way or two-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was performed at a significance of p<0.05. Analysis was conducted using GraphPad Prism 9.1 (GraphPad Software).

[0093] To gain a better understanding of the invention described herein, the following examples are set forth. It should be understood that these examples are for illustrative purposes only. Therefore, they should not limit the scope of this invention in anyway.

[0094] EXAMPLES

[0095] RESULTS

[0096] Referring to figure 1 , various HbF-inducing drugs in hematopoietic stem cells is shown. CD34-positive hematopoietic stem cells isolated from healthy donors were cultured under conditions to induce erythroid maturation and individually treated with a library containing over 200 FDA-approved drugs at 1 pM. In the present examples, the HbF level was assessed by ELISA. Candidates were filtered by drugs that increase HbF protein level greater than 2-fold (red), 1.5-fold (green), and 1.2-fold (blue), compared to HU and DMSO control.

[0097] Referring to figure 2, the increase in HbF level in hematopoietic cells from idasanutlin is shown compared with increase from hydroxyurea. CD34-positive hematopoietic stem cells isolated from healthy donors were cultured under conditions to induce erythroid maturation and treated with DMSO vehicle control, 125-500 nM idasanutlin (left panel), or 0.1-10 pM hydroxyurea (right panel) for 4 days and total mRNA was isolated for RT-qPCR analysis of HBA2, HBB, and HBG2, normalized to GAPDH levels.

[0098] Referring to figure 3, the increase in HbF level in erythroleukemia cells from idasanutlin is shown compared with increase from hydroxyurea. KU812 cell lysates treated with DMSO vehicle control, 125-500 nM idasanutlin, or 0.1-10 pM hydroxyurea (HU) for 4 days were immunoblotted for y-globin and a-globin to detect fetal hemoglobin (HbF) and total hemoglobin (HbA), respectively along with appropriate positive and negative controls

[0099] Referring to figures 4A and 4B, the increase in HbF level in hematopoietic cells from idasanutlin is shown. CD34-positive hematopoietic stem cells isolated from healthy donors (figure 4A) and patients with sickle cell disease (figure 4B) cultured under conditions to induce erythroid maturation and treated with DMSO vehicle control or 125-500 nM idasanutlin for 4 days were immunoblotted for y-globin to detect HbF level. |3- actin was used as loading control.

[0100] Referring to figure 5, studies show that idasanutlin has cytostatic effect in erythroid cells at low concentrations. Relative cell number and viability were assessed by trypan blue exclusion assay in KU812 erythroleukemia cells and CD34-positive hematopoietic stem cells isolated from healthy donors (HD) and patients with sickle cell disease after treatment in vitro with 0-1000 nM idasanutlin for 4 days.

[0101] Referring to figure 6, results show that idasanutlin reduces hypoxia- induced sickling in red blood cells. In this example, CD34-positive cells isolated from patients with sickle cell disease cultured to induce erythroid maturation and treated with DMSO vehicle control or idasanutlin were subjected to normoxia or hypoxic conditions. Representative images are shown (right panels, 6A). The percentage of sickle cells was quantified by phase-contrast microscopy and statistical significance given (left panel, 6B).

[0102] CONCLUSION

[0103] Individuals with sickle cell disease spend approximately $1 .7 million on disease-related medical expenses over their lifetime. Accordingly, this highlights the enormous economic burden affecting individuals living with sickle cell disease and support and validate the utility of the HbF-inducing agent idasanutlin. This agent can decrease the health care utilization of resources due to sickle cell disease and has the potential to generate $3 billion in revenue in North America every year.

[0104] The results show that idasanutlin may be used effectively in the treatment of hemoglobinopathies in which the pathogenesis, symptoms, or short-term and / or longterm consequences are mediated by sickle hemoglobin. These conditions or diseases include, but not limited to, sickle cell disease (SS and S bO), thalassemia major, thalassemia intermedia (Hemoglobin E / b thalassemia), and acute and chronic hemolytic anemia. Idasanutlin may be used as a treatment for both children and adults after the appearance of symptoms or as prophylaxis to prevent symptoms. In addition, idasanutlin may be used prior to routine immunizations to prevent infections, during imaging studies for stroke assessments, and during blood transfusions to prevent severe anemia. It may also be used in addition to recommendations for lifestyle changes, including avoiding smoking, hypoxia, and when seeking medical care for any signs of infection or other health issues. For these indications, idasanutlin may be used as a single agent or in combination with supportive care regimens using currently approved or in-development agents. These include hydroxyurea, L-glutamine (Endari), crizanlizumab (Adakveo), and penicillin.

[0105] Idasanutlin has been studied at various doses for different conditions. The dosing regimens may change based on the specific condition being treated, age and BSI of the patient, and whether idasanutlin was used as monotherapy or in combination with other drugs. Dose escalation studies will provide the amount to be used for each specific indication. In some examples, the initial dose regimen is 50-500 mg daily for 5-7 days in a 28-day cycle, or as needed based on symptoms resolution. This dose may be modified if idasanutlin is given with other agents and if it is given for prophylaxis or as treatment.

[0106] EXAMPLE 2

[0107] In come embodiments there is provided:

[0108] 1. A pharmaceutical composition comprising idasanutlin and a pharmaceutically acceptable carrier for inducing fetal hemoglobin (HbF) during erythropoiesis in a subject in need thereof.

[0109] 2. The composition of embodiment 1 , wherein the composition is formulated for oral, parenteral, administration.

[0110] 3. The composition of embodiment 1 or 2, wherein the composition is a modified-release, extended-release, or controlled-release formulation providing sustained plasma exposure of idasanutlin

[0111] 4. A method of treating sickle cell disease in a subject, comprising administering to the subject a therapeutically effective amount of idasanutlin, thereby increasing HbF and reducing one or more clinical endpoints selected from frequency of vaso-occlusive crises, transfusion burden, hemolysis markers, or hospitalization rate.

[0112] 5. The method of embodiment 4, wherein idasanutlin exhibits reduced toxicity relative to hydroxyurea as measured by one or more parameters selected from neutropenia incidence, mucositis, gastrointestinal adverse events, or treatment discontinuation.

[0113] 6. A method of preventing or reducing severity of sickle cell crisis in a subject with sickle cell disease, comprising administering idasanutlin prophylactically according to a dosing regimen sufficient to maintain elevated HbF.

[0114] 7. A method of treating p-thalassemia, including transfusiondependent |3-thalassemia, comprising administering idasanutlin to increase HbF and reduce transfusion frequency or volume.

[0115] 8. A method of treating leukocytosis in a subject, comprising administering idasanutlin in an amount effective to reduce circulating leukocyte counts toward a target range while maintaining therapeutic HbF induction.

[0116] 9. A method of treating polycythemia in a subject, comprising administering idasanutlin in an amount effective to reduce cell proliferation and hematocrit toward a target therapeutic range while maintaining therapeutic HbF induction.

[0117] 10. A method of treating a myeloproliferative disorder in a subject, comprising administering idasanutlin to modulate erythroid and / or myeloid proliferation and reduce one or more disease parameters selected from leukocytosis, thrombocytosis, splenomegaly, pruritus, constitutional symptoms, or phlebotomy frequency.

[0118] 11. The method of any one of embodiments 4-10, wherein the subject is pediatric, adolescent, adult, or pregnant, and the dosing is adjusted by body mass, renal / hepatic function, or pharmacogenomic profile.

[0119] 12. The method of any one of embodiments 4-10, wherein idasanutlin is administered in combination with one or more agents selected from L-glutamine, voxelotor, crizanlizumab, phlebotomy protocols, iron chelators, JAK inhibitors, or erythropoietin analogs, concurrently or sequentially.

[0120] Mechanism and Rationale

[0121] Idasanutlin increases HbF during erythropoiesis, providing clinical benefit by reducing HbS polymerization and downstream vaso-occlusion in SCD, and by compensating for deficient [3-globin in p-thalassemia, consistent with established HbF biology. Reduced toxicity relative to hydroxyurea addresses adherence and long-term safety limitations noted in current practice.

[0122] Indications

[0123] • Sickle cell disease: treatment and prophylaxis to reduce crisis frequency and severity, hemolysis, and hospitalizations.

[0124] • p-thalassemia major / intermedia: reduce transfusion burden via HbF induction, potentially in combination with standard care.

[0125] • Polycythemia, leukocytosis, myeloproliferation: modulation of erythroid / myeloid proliferation with a safety profile favorable to hydroxyurea-sensitive populations.

[0126] Combinations

[0127] Combination regimens include idasanutlin with L-glutamine, voxelotor, anti-P-selectin agents, iron chelators, or JAK inhibitors, and supportive modalities (e.g., phlebotomy) tailored to pathophysiology and patient subtype.

[0128] Patient Populations

[0129] Pediatric through adult; hydroxyurea-intolerant; pregnant where risk-benefit supports HbF induction; genotype-stratified SCD and p-thalassemia cohorts.

[0130] The details of one or more variations of the subject matter described herein are set forth in the accompanying drawings and the description below. Other features and advantages of the subject matter described herein will be apparent from the description and drawings, and from the claims. The disclosed subject matter is not, however, limited to any particular example disclosed.

[0131] The embodiments described herein are intended to be examples only. Alterations, modifications and variations can be effected to the particular embodiments by those of skill in the art. The scope of the claims should not be limited by the particular embodiments set forth herein, but should be construed in a manner consistent with the specification as a whole.

[0132] All publications, patents and patent applications mentioned in this Specification are indicative of the level of skill those skilled in the art to which this invention pertains and are herein incorporated by reference to the same extent as if each individual publication patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0133] The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modification as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method of treating a hemoglobinopathy in a subject in need thereof, comprising administering to the human an effective amount of 4-((2R,3S,4R,5S)-3-(3-chloro-2- fluorophenyl)-4-(4-chloro-2-fluorophenyl)-4-cyano-5-neopentylpyrrolidine-2-carboxamido)-3- methoxybenzoic acid (idasanutlin), or a pharmaceutically acceptable salt, or a solvate, or an isomer, or a functional derivative thereof, wherein the subject is a human.

2. The method of claim 1 , further comprising the administration of one or more of hydroxyurea, L-glutamine (Endari), crizanlizumab (Adakveo), or penicillin in combination to optimize safety, tolerability and effectiveness for the desired outcome to reduce sickling crisis.

3. The method of claim 1 or 2, wherein said hemoglobinopathy is sickle cell disease, thalassemia major, thalassemia intermedia, sickle cell beta thalassemia (Hb S / p Th), polycythemia, leukocytosis, myeloproliferative conditions or acute and chronic hemolytic anemia.

4. The method of claim 1 , wherein said hemoglobinopathy is sickle cell disease, thalassemia major, thalassemia intermedia, thalassemia major, sickle cell beta thalassemia (Hb S / p Th), polycythemia, leukocytosis, myeloproliferative conditions or acute and chronic hemolytic anemia.

5. The method of any one of claims 1- 5, wherein the subject is an adult or a child.

6. The method of any one of claims 1 -5 , wherein the idasanutlin, or a pharmaceutically acceptable salt, or a solvate, or an isomer, or a functional derivative thereof, is administered at from about 10-500 mg daily, optionally for 5-7 days in a 28-day cycle, or as needed based on symptoms resolution.

7. A method of treating a hemoglobinopathy in a subject in need thereof, comprising administering to the human Use of an effective amount of 4-((2R,3S,4R,5S)-3-(3-chloro-2- fluorophenyl)-4-(4-chloro-2-fluorophenyl)-4-cyano-5-neopentylpyrrolidine-2-carboxamido)-3- methoxybenzoic acid (idasanutlin), or a pharmaceutically acceptable salt, or a solvate, or an isomer, or a functional derivative thereof, for treating a hemoglobinopathy in a human in need thereof, or in the manufacture of a medicament for treating a hemoglobinopathy in a human in need thereof, wherein the subject is a human8. The use of claim 7, further comprising the use of one or more of hydroxyurea, L- glutamine (Endari), crizanlizumab (Adakveo), or penicillin.

9. The use of claim 7 or 8, wherein said hemoglobinopathy is sickle cell disease, thalassemia major, thalassemia intermedia, or acute and chronic hemolytic anemia.

10. The use of claim 7, wherein said hemoglobinopathy is sickle cell disease.11 . The use of any one of claims 1- 5, wherein the subject is an adult or a child.

12. The use of any one of claims 1 5, wherein the idasanutlin, or a pharmaceutically acceptable salt, or a solvate, or an isomer, or a functional derivative thereof, is for administration at from about 50-500 mg daily, optionally for 5-7 days in a 28-day cycle,13. A method of inducing HbF expression in a human cell, comprising said cell with of 4- ((2R,3S,4R,5S)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)-4-cyano-5- neopentylpyrrolidine-2-carboxamido)-3-methoxybenzoic acid (idasanutlin), or a pharmaceutically acceptable salt, or a solvate, or an isomer, or a functional derivative thereof.

14. A pharmaceutical composition comprising idasanutlin and a pharmaceutically acceptable carrier for inducing fetal hemoglobin (HbF) during erythropoiesis in a subject in need thereof.

15. The composition of claim 14, wherein the composition is formulated for oral, parenteral, administration.

16. The composition of claim 14 or 15, wherein the composition is a modified-release, extended-release, or controlled-release formulation providing sustained plasma exposure of idasanutlin.

17. A method of treating sickle cell disease in a subject, comprising administering to the subject a therapeutically effective amount of idasanutlin, thereby increasing HbF and reducing one or more clinical endpoints selected from frequency of vaso-occlusive crises, transfusion burden, hemolysis markers, or hospitalization rate.

18. The method of claim 17, wherein idasanutlin exhibits reduced toxicity relative to hydroxyurea as measured by one or more parameters selected from neutropenia incidence, mucositis, gastrointestinal adverse events, or treatment discontinuation.

19. .A method of preventing or reducing severity of sickle cell crisis in a subject with sickle cell disease, comprising administering idasanutlin prophylactically according to a dosing regimen sufficient to maintain elevated HbF.

20. .A method of treating p-thalassemia, including transfusion-dependent p-thalassemia, comprising administering idasanutlin to increase HbF and reduce transfusion frequency or volume.

21. .A method of treating leukocytosis in a subject, comprising administering idasanutlin in an amount effective to reduce circulating leukocyte counts toward a target range while maintaining therapeutic HbF induction.

22. A method of treating polycythemia in a subject, comprising administering idasanutlin in an amount effective to reduce hematocrit toward a target therapeutic range while maintaining therapeutic HbF induction.

23. A method of treating a myeloproliferative disorder in a subject, comprising administering idasanutlin to modulate erythroid and / or myeloid proliferation and reduce one or moredisease parameters selected from leukocytosis, thrombocytosis, splenomegaly, pruritus, constitutional symptoms, or phlebotomy frequency.

24. The method of any one of claims 17-23, wherein the subject is pediatric, adolescent, adult, or pregnant, and the dosing is adjusted by body mass, renal / hepatic function, or pharmacogenomic profile.25 The method of any one of claims 17-23, wherein idasanutlin is administered in combination with one or more agents selected from L-glutamine, voxelotor, crizanlizumab, phlebotomy protocols, iron chelators, JAK inhibitors, or erythropoietin analogs, concurrently or sequentially.

26. The composition of any one of claims 14-16, wherein the subject is a human.

27. The method of any one of claims 17 to 25, wherein the subject is a human.

28. Use of a pharmaceutical composition comprising idasanutlin and a pharmaceutically acceptable carrier for inducing fetal hemoglobin (HbF) during erythropoiesis in a subject in need thereof, or in the manufacture of a medicament for inducing fetal hemoglobin (HbF) during erythropoiesis in a subject in need thereof.

29. The use of claim 28, wherein the composition is formulated for oral, parenteral, administration.

30. The use of claim 28 or 29, wherein the composition is a modified-release, extended- release, or controlled-release formulation providing sustained plasma exposure of idasanutlin.31 . The use of a therapeutically effective amount of idasanutlin, for treating sickle cell disease in a subject thereby increasing HbF and reducing one or more clinical endpoints selected from frequency of vaso-occlusive crises, transfusion burden, hemolysis markers, or hospitalization rate, or in the manufacture of a medicament for treating sickle cell disease in a subject thereby increasing HbF and reducing one or more clinical endpoints selected from frequency of vaso-occlusive crises, transfusion burden, hemolysis markers, or hospitalization rate.

32. The use of claim 31 , wherein idasanutlin exhibits reduced toxicity relative to hydroxyurea as measured by one or more parameters selected from neutropenia incidence, mucositis, gastrointestinal adverse events, or treatment discontinuation.

33. .Use of idasanutlin prophylactically according to a dosing regimen sufficient to maintain elevated HbF for preventing or reducing severity of sickle cell crisis in a subject with sickle cell disease, or in the manufacture of a medicament for preventing or reducing severity of sickle cell crisis in a subject with sickle cell disease.

34. Use of idasanutlin to increase HbF and reduce transfusion frequency or volume for treating p-thalassemia, including transfusion-dependent p-thalassemia, or in themanufacture of a medicament for treating p-thalassemia, including transfusion-dependent |3- thalassemia.

35. .Use of idasanutlin in an amount effective to reduce circulating leukocyte counts toward a target range while maintaining therapeutic HbF induction for treating leukocytosis in a subject, or in the manufacture of a medicament treating leukocytosis in a subject.

36. Use of idasanutlin in an amount effective to reduce hematocrit toward a target therapeutic range while maintaining therapeutic HbF induction for treating polycythemia in a subject, or in the manufacture of a medicament effective to reduce hematocrit toward a target therapeutic range while maintaining therapeutic HbF induction for treating polycythemia in a subject .

37. Use of idasanutlin to modulate erythroid and / or myeloid proliferation and reduce one or more disease parameters selected from leukocytosis, thrombocytosis, splenomegaly, pruritus, constitutional symptoms, or phlebotomy frequency for treating a myeloproliferative disorder in a subject, or in the manufacture of a medicament for treating a myeloproliferative disorder in a subject.

38. The use of any one of claims 31-37, wherein the subject is pediatric, adolescent, adult, or pregnant, and the dosing is adjusted by body mass, renal / hepatic function, or pharmacogenomic profile.39 The use of any one of claims 31-37, wherein idasanutlin is for use administration in combination with one or more agents selected from L-glutamine, voxelotor, crizanlizumab, phlebotomy protocols, iron chelators, JAK inhibitors, or erythropoietin analogs, concurrently or sequentially.

40. The use of any one of claims 28-39, wherein the subject is a human.

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