Methods of treating anemia using salmeterol or a pharmaceutically acceptable salt thereof

Abstract

The present disclosure provides methods of treating anemia in a patient in need thereof, comprising administering to the patient in need thereof an effective amount of salmeterol or a pharmaceutically acceptable salt thereof. Salmeterol or a pharmaceutically acceptable salt thereof may be administered conjointly with an erythropoiesis-stimulating agent, optionally wherein the anemia is refractory to the erythropoiesis-stimulating agent. Also provided are methods of promoting differentiation of an erythroid progenitor cell toward a mature red blood cell in a patient in need thereof, comprising administering an effective amount of salmeterol or a pharmaceutically acceptable salt thereof. The present disclosure further provides methods comprising administering salbutamol or a pharmaceutically acceptable salt thereof. Salmeterol, salbutamol, or a pharmaceutically acceptable salt thereof may be administered conjointly with other FDA-approved drugs such as luspatercept, lenalidomide, daprodustat, vadadustat, an erythropoiesis-stimulating agent (ESA), and / or a hypomethylating agent.

Description

METHODS OF TREATING ANEMIA USING SALMETEROL ORA PHARMACEUTICALLY ACCEPTABLE SALT THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 641,831, filed May 2, 2024, the disclosure of which is incorporated by reference herein in its entirety.BACKGROUND

[0002] Anemia affects roughly twenty three percent of the world population and is a predominant co-morbidity in a wide range of hematologic disorders, such as myelodysplastic syndromes (MDS), bone marrow failure (BMF), anemia of inflammatory diseases, such as chronic kidney diseases, ribosomopathies, and leukemias such as acute myeloid leukemia (AML). For example, anemia is a predominant feature in -80-90% of MDS patients and its treatment remains the primary goal of designing new interventions (Castelli et al. (2018) Med. Oncol. 35:76; Feld et al. (2020) Ex / ?. Rev. Anticancer Ther. 20:465-482; Steensma (2018) B / c Cancer J. 8:47). Conventional erythropoiesis-stimulating agents are only effective in 50-60% of low-risk MDS patients (Schiavon et al. (2018)Afe Oncol. 35:76; Park et al. (2019) Br. J. Haematol. 184: 134-160). As a result, a major fraction of MDS patients eventually becomes transfusion-dependent and non-responsive to a handful of existing FDA-approved drugs, such as hypomethylating agents (Cheng et al. (2021) Hematol. 26:261-270; Kordella et al. (2021) Front. Oncol. 11 :650473; Schiffer et al. (2021) Ex / ?. Rev. Anticancer Ther. 21 :989-1002), lenalidomide (Hecht et al. (2021) Ann. Hematol. 100: 1463- 1471; ), or luspatercept (Chan et al. (2021) Fut. Oncol. 17: 1473-1481; Cheng et al. (2021) Hematol. 26: 261-270; Hecht et al. (2021) Ann Hematol. 100: 1463-1471; Kordella et al. (2021) Front. Oncol. 11 :650473; Kubasch et al. (2021) Blood Adv. 5:1565-1575; List et a / . (2021) J. Clin. Oncol. 39: 1001-1009), or progress to AML (-25-30% patients), if they are ineligible for the only curative treatment of allogeneic bone marrow transplant. Thus, there is a critical need to identify additional therapies to promote erythroid differentiation for alleviating anemia in these disorders.SUMMARY

[0003] The present disclosure is based, in part, on the discovery of a therapeutic approach of targeting anemia by administering salmeterol or a pharmaceutically acceptable salt thereof (e.g., salmeterol xinafoate). Salmeterol is a long-acting beta-2 adrenergic receptor agonist drug that is currently FDA-approved for the treatment of asthma and chronic obstructive pulmonary disease (COPD). The present disclosure encompasses a new and unexpected finding that salmeterol can increase red blood cell parameters and stimulate erythroid (red blood cell) differentiation.

[0004] The present disclosure also describes a therapeutic approach of targeting anemia by administering salbutamol (also referred to as albuterol) or a pharmaceutically acceptable salt thereof (e.g., salbutamol sulfate). Salbutamol and salbutamol sulfate are shortacting beta-2 adrenergic receptor agonist drugs that are currently FDA-approved for the treatment of asthma and chronic obstructive pulmonary disease (COPD).

[0005] Repurposing salmeterol and / or salbutamol has significant therapeutic benefits in the treatment of various hematologic disorders associated with anemia (e.g., such as anemia in hematologic malignancies, bone marrow failure disorders, inflammatory disorders, anemia in cancer patients receiving chemotherapy, and other disorders disclosed herein).

[0006] In some embodiments, provided herein are methods of treating anemia in a patient in need thereof, the method comprising administering to the patient in need thereof an effective amount of salmeterol or a pharmaceutically acceptable salt thereof (e.g., salmeterol xinafoate).

[0007] In some embodiments, provided herein are methods of treating anemia in a patient in need thereof, the method comprising administering to the patient in need thereof an effective amount of salbutamol or a pharmaceutically acceptable salt thereof (e.g., salbutamol sulfate).

[0008] In some embodiments, the anemia is selected from the group consisting of macrocytic anemia, hemolytic anemia, anemia caused by a ribosomopathy, anemia caused by insufficiency of serine / threonine-protein kinase RI0K2, anemia associated with chronic kidney disease (CKD), anemia caused by one or more mutations and / or deletions in human chromosome 5 or in an ortholog thereof, anemia caused by chromosomal translocations in the NUP98 gene or in an ortholog thereof, such as anemia caused by fusions of NUP98 with Abd-B group HOX genes (e.g., H0XD13), stress-induced anemia, anemia secondary to an intestinal cancer, Diamond Blackfan anemia, aplastic anemia, Schwachman-Diamondsyndrome, an anemia associated with an inflammatory disease, such as rheumatoid arthritis or multiple sclerosis, anemia secondary to chemotherapy in cancer patients, and anemia associated with a bone marrow failure syndrome. In some embodiments, the anemia is associated with a cancer. In some embodiments, the anemia is associated with a hematologic malignancy, such as a myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) or multiple myeloma (MM). In some embodiments, the anemia is associated with MDS. In some embodiments, the anemia is associated with an intestinal cancer, such as a colorectal cancer.

[0009] In some embodiments, provided herein are methods of promoting differentiation of an erythroid progenitor cell toward a mature red blood cell in a patient by administering an effective amount of salmeterol or a pharmaceutically acceptable salt thereof (e.g., salmeterol xinafoate). In some embodiments, provided herein are methods of promoting differentiation of a hematopoietic stem and progenitor cell (HSPC) or a multipotent progenitor (MPP) cell toward an erythroid progenitor cell in a patient by administering an effective amount of salmeterol or a pharmaceutically acceptable salt thereof (e.g., salmeterol xinafoate).

[0010] In some embodiments, provided herein are methods of promoting differentiation of an erythroid progenitor cell toward a mature red blood cell in a patient by administering an effective amount of salbutamol or a pharmaceutically acceptable salt thereof (e.g., salbutamol sulfate). In some embodiments, provided herein are methods of promoting differentiation of a hematopoietic stem and progenitor cell (HSPC) or a multipotent progenitor (MPP) cell toward an erythroid progenitor cell in a patient by administering an effective amount of salbutamol or a pharmaceutically acceptable salt thereof (e.g., salbutamol sulfate).

[0011] In some embodiments, salmeterol or a pharmaceutically acceptable salt thereof (e.g., salmeterol xinafoate) may be orally administered to the patient. In some embodiments, the patient is a human suffering from anemia. In some embodiments, a patient is a human that is at least 4 years of age (z.e., 4 years of age or older, > 4 years of age). In some embodiments, a patient is a human 4 to 11 years of age (i.e., > 4 years old and < 11 years old). In some embodiments, a patient is at least 4 years of age and less than 12 years of age (i.e., > 4 years of age and < 12 years of age). In some embodiments, a patient is a human that is at least 12 years of age (i.e., 12 years of age or older, > 12 years of age).

[0012] In some embodiments, salbutamol or a pharmaceutically acceptable salt thereof (e.g., salbutamol sulfate) may be orally administered to the patient. In some embodiments, the patient is a human suffering from anemia. In some embodiments, a patient is a human that is at least 2 years of age. In some embodiments, a patient is a human 2 to 6 years of age. In some embodiments, a patient is a human that is at least 4 years of age. In some embodiments, a patient is a human 4 to 11 years of age. In some embodiments, a patient is a human 6 to 12 years of age. In some embodiments, a patient is a human that is at least 12 years of age.

[0013] In some embodiments, the methods may further comprise administering (e.g., administering conjointly) to the patient in need thereof an effective amount of an erythropoiesis-stimulating agent (ESA) such as epoetin alfa or darbepoetin alfa, or other FDA-approved drug such as luspatercept, lenalidomide, daprodustat, vadadustat, and / or a hypomethylating agent, including but not limited to azacitidine or decitabine. In some embodiments, the erythropoiesis-stimulating agent comprises erythropoietin, epoetin alfa, epoetin beta, epoetin omega, epoetin zeta, or darbepoetin alfa.

[0014] In some embodiments, a patient who may be treated with the provided methods may be receiving or has received an effective amount of an erythropoiesisstimulating agent (ESA) such as epoetin alfa or darbepoetin alfa, or other FDA-approved drug such as luspatercept, lenalidomide, daprodustat, vadadustat, and / or a hypomethylating agent, including but not limited to azacitidine or decitabine and combinations thereof. In some embodiments, the erythropoiesis-stimulating agent comprises erythropoietin, epoetin alfa, epoetin beta, epoetin omega, epoetin zeta, or darbepoetin alfa and combinations thereof.

[0015] In some embodiments, provided herein are methods of treating anemia in a patient in need thereof, comprising administering to the patient in need thereof an effective amount of salmeterol or a pharmaceutically acceptable salt thereof (e.g., salmeterol xinafoate) conjointly with an erythropoiesis-stimulating agent, wherein the anemia is refractory to the erythropoiesis-stimulating agent. In some embodiments, the erythropoiesis-stimulating agent comprises erythropoietin, epoetin alfa, epoetin beta, epoetin omega, epoetin zeta, or darbepoetin alfa. In some embodiments, salmeterol or a pharmaceutically acceptable salt thereof (e.g., salmeterol xinafoate) may be administered conjointly with other FDA-approved drugs such as luspatercept, lenalidomide, daprodustat, vadadustat, and / or a hypomethylating agent, such as azacitidine or decitabine. The anemia may be selected from the group consisting of macrocytic anemia, hemolytic anemia, anemia caused by a ribosomopathy,anemia caused by insufficiency of serine / threonine-protein kinase RI0K2, anemia associated with chronic kidney disease (CKD), anemia caused by one or more mutations and / or deletions in human chromosome 5 or in an ortholog thereof, anemia caused by chromosomal translocations in the NUP98 gene or in an ortholog thereof, such as anemia caused by fusions of NUP98 with Abd-B group HOX genes (e.g., H0XD13), stress-induced anemia, anemia secondary to an intestinal cancer, Diamond Blackfan anemia, aplastic anemia, Schwachman- Diamond syndrome, an anemia associated with an inflammatory disease, such as rheumatoid arthritis or multiple sclerosis, anemia secondary to chemotherapy in cancer patients, and anemia associated with a bone marrow failure syndrome.

[0016] In some embodiments, provided herein are methods of treating anemia in a patient in need thereof, comprising administering to the patient in need thereof an effective amount of salmeterol or a pharmaceutically acceptable salt thereof e.g., salmeterol xinafoate), wherein the patient is receiving, has received or will receive treatment with an erythropoiesis-stimulating agent.

[0017] In some embodiments, the anemia is associated with a cancer. In some embodiments, the cancer is a hematologic malignancy, such as a myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) or multiple myeloma (MM). In some embodiments, the anemia is associated with an intestinal cancer, such as colorectal cancer. In some embodiments, the anemia is associated with MDS.

[0018] Throughout the description, where methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are methods according to the present disclosure that consist essentially of, or consist of, the recited steps.

[0019] It should be understood that the order of steps or order for performing certain action is immaterial so long as the invention remains operable. Moreover, two or more steps or actions may be conducted simultaneously.

[0020] Any of the embodiments described herein can be used in conjunction with one another, unless otherwise indicated or apparent from the context. Other embodiments will become apparent to those skilled in the art from a review of the ensuing description.BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The Drawings included herein, which is composed of the following Figures, are for illustration purposes only and not for limitation.

[0022] FIG. 1 shows that salmeterol (Sei) at a dose of 0.3 mg / kg does not increase hemoglobin (Hb) in phenylhydrazine (PHZ)-treated anemic mice. Salmeterol treatment at 0.3 mg / kg daily (i.p.) does not elevate hemoglobin (Hb) in wild-type mice treated with sublethal dose of phenylhydrazine (60 mg / kg) that induces hemolytic anemia. n=5 mice per group. Comparisons are performed with respect to vehicle-treated mice as a control. 2-way ANOVA. ns: non-significant.

[0023] FIG. 2 shows that salmeterol (Sei) at a dose of 0.6 mg / kg elevates hemoglobin (Hb) in phenylhydrazine (PHZ)-treated anemic mice. Salmeterol treatment at 0.6 mg / kg daily (i.p.) elevates hemoglobin in wild-type mice treated with sublethal dose of phenylhydrazine (60 mg / kg) that induces hemolytic anemia. n=5 mice per group. Comparisons are performed with respect to vehicle-treated mice as a control. **** p < 0.0001, 2-way ANOVA.

[0024] FIG. 3A - FIG. 3D show that salmeterol (Sei) at a dose of 0.6 mg / kg elevates red blood cell (RBC) parameters, but not white blood cell (WBC) parameters in phenylhydrazine (PHZ)-treated anemic mice. Salmeterol treatment at 0.6 mg / kg daily (i.p.) increases hemoglobin (Hb) (FIG. 3 A) and hematocrit% (HCT%)(FIG. 3B), but not platelets (FIG. 3C) or WBCs (FIG. 3D), in wild-type mice treated with a sublethal dose of phenylhydrazine (60 mg / kg) that induces hemolytic anemia. n=5 mice per group.Comparisons are performed with respect to vehicle-treated mice as a control. ** p < 0.01, student’s t-test. ns: non-significant.

[0025] FIG. 4A - FIG. 4D show that salmeterol (Sei) at a dose of 0.6 mg / kg elevates erythroid progenitors, but not granulocytic monocytic progenitors (GMP) or megakaryocytic progenitors (MkP) in phenylhydrazine (PHZ)-treated anemic mice. Salmeterol treatment at 0.6 mg / kg daily (i.p.) elevates total erythroid progenitors (FIG. 4A), and terminal erythroid progenitors (FIG. 4B), but not GMP (FIG. 4C) or MkP (FIG. 4D) in the bone marrow (BM) of wild-type mice treated with a sublethal dose of phenylhydrazine (60 mg / kg) to induce hemolytic anemia. n=5 mice per group. Comparisons are performed with respect to vehicle- treated mice as a control. * p < 0.05, ** p < 0.01, student’s t-test. ns: non-significant.

[0026] FIG. 5 shows an exemplary experimental plan to analyze the effect of salmeterol (Sei) treatment (Sei: 0.6 mg / kg ip 5x weekly) in MDS model mice (NUP98 / HOXD 13 transgenic mice) over 4 weeks of treatment.

[0027] FIG. 6 shows that salmeterol treatment mildly reverses anemia in MDS mouse model. MDS model mice (NUP98 / HOXD13 transgenic mice) and littermate controls were treated in accordance with FIG. 5 (Sei: 0.6 mg / kg ip 5x weekly for 4 weeks). FIG. 6 depicts agraph of change in hematocrit over time. MDS model mice show a mild increase in hematocrit (HCT%) in peripheral blood relative to vehicle-treated (DMSO) MDS mice. * p < 0.05, *** p < 0.001, ANOVA. n=5 mice per group, ns: non-significant.

[0028] FIG. 7 shows an exemplary experimental plan to analyze the effect of once- daily salbutamol treatment in phenylhydrazine (PHZ)-treated anemic mice. Wild-type mice were treated with a sublethal dose of PHZ (60 mg / kg) that induces hemolytic anemia, followed by treatment with salbutamol at once daily dosing of 0.3 mg / kg ip.

[0029] FIG. 8 shows that salbutamol (Sal) once-daily treatment does not increase hemoglobin (Hb) in phenylhydrazine (PHZ)-treated anemic mice. Salbutamol treatment at 0.3 mg / kg once daily (i.p.) does not elevate hemoglobin (Hb) in wild-type mice treated with a sublethal dose of PHZ (60 mg / kg) that induces hemolytic anemia. n=5 mice per group. Comparisons are performed with respect to vehicle-treated mice as a control.

[0030] FIG. 9 shows that salbutamol (Sal) once-daily treatment does not increase erythroid progenitors in the bone marrow of phenylhydrazine (PHZ)-treated anemic mice. Sal at 0.3 mg / kg once daily (i.p.) does not elevate terminal erythroid progenitors in wild-type mice in response to PHZ-mediated hemolytic stress. Comparisons are performed with respect to vehicle-treated mice as a control. n=5 mice per group. Student’s t-test. ns: non-significant.

[0031] FIG. 10 shows an exemplary experimental plan to analyze the effect of twice- daily SABA (Salbutamol (Sal) / Salbutamol sulfate (SS)) treatment in phenylhydrazine (PHZ)-treated anemic mice. Wild-type mice are treated with a sublethal dose of PHZ (60 mg / kg) that induces hemolytic anemia, followed by twice-daily SABA (Salbutamol (Sal) / Salbutamol sulfate (SS)) treatment of 0.3 mg / kg ip.

[0032] FIG. HA and FIG. HB show that Salbutamol (Sal) and Salbutamol sulfate (SS) treatments increase red blood cell (RBC) parameters in mice with phenylhydrazine (PHZ)-driven hemolytic anemia. Sal / SS twice-daily treatments of 0.3 mg / kg / dose (i.p.) elevates hemoglobin (Hb) (FIG. 11 A) and hematocrit% (HCT%) (FIG. 1 IB) in wild-type mice in response to PHZ-mediated hemolytic stress. Comparisons are performed with respect to vehicle-treated mice as a control. n=5 mice per group. ****p < 0.0001, one-way ANOVA.

[0033] FIG. 12A and FIG. 12B show that Salbutamol (Sal) and Salbutamol sulfate (SS) treatments increase red blood cell (RBC) parameters in mice with phenylhydrazine (PHZ)-driven hemolytic anemia. Sal / SS twice-daily treatments of 0.3 mg / kg / dose (i.p.) elevates absolute values of hemoglobin (Hb) (FIG. 12A) and hematocrit% (HCT%) (FIG. 12B) in wild-type mice in response to PHZ-mediated hemolytic stress after 13 days oftreatment. Comparisons are performed with respect to vehicle (DMSO) treated mice as a control. n=5 mice per group. ****p < 0.0001, one-way ANOVA.

[0034] FIG. 13A and FIG. 13B show that Salbutamol (Sal) and Salbutamol sulfate (SS) treatments do not affect white blood cell (WBC) parameters in mice with phenylhydrazine (PHZ)-driven hemolytic anemia. Sal / SS twice-daily treatments of 0.3 mg / kg / dose (i.p.) do not influence platelets (FIG. 13A) and WBCs (FIG. 13B) in wild-type mice in response to PHZ-mediated hemolytic stress after 13 days of treatment. Comparisons are performed with respect to vehicle (DMSO) treated mice as a control. n=5 mice per group, ns: non-significant, one-way ANOVA.

[0035] FIG. 14A and FIG. 14B show that Salbutamol (Sal) and Salbutamol sulfate (SS) treatments, respectively enhance erythroid progenitors in the bone marrow of mice with phenylhydrazine (PHZ)-driven hemolytic anemia. Sal (FIG. 14 A) and SS (FIG. 14B) twice- daily treatments of 0.3 mg / kg / dose (i.p.) elevate terminal erythroid progenitors in the bone marrow (BM) of wild-type mice in response to PHZ-mediated hemolytic stress after 13 days of treatment. All comparisons are performed with respect to vehicle (DMSO) treated mice as a control. n=5 mice per group. **p < 0.01, student’s t-test.

[0036] FIG. 15A and FIG. 15B show that Salbutamol (Sal) and Salbutamol sulfate (SS) treatments do not affect granulocytic monocytic progenitors (GMP) or megakaryocytic progenitors (MkP), respectively, in the bone marrow of mice with phenylhydrazine (PHZ)- driven hemolytic anemia. Sal and SS twice-daily treatments of 0.3 mg / kg / dose (i.p.) do not affect GMP (FIG. 15 A) or MkP (FIG. 15B) in the bone marrow (BM) of wild-type mice in response to PHZ-mediated hemolytic stress after 13 days of treatment. All comparisons are performed with respect to vehicle (DMSO) treated mice as a control. n=5 mice per group. One-way ANOVA. ns: non-significant.DETAILED DESCRIPTIONDefinitions

[0037] In order for the present invention(s) to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification. The publications and other reference materials referenced herein to describe the background of the disclosure and to provide additional detail regarding its practice are hereby incorporated by reference.

[0038] In this application, unless otherwise clear from context, (i) the terms “a” and “an” are used herein to refer to one or to more than one (z.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0039] The terms “cancer” or “tumor” or “hyperproliferative” refer to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features.

[0040] Cancer cells are often in the form of a tumor, but such cells may exist alone within an animal, or may be a non-tumorigenic cancer cell, such as a leukemia cell. As used herein, the term “cancer” includes premalignant as well as malignant cancers. Cancers include, but are not limited to, B cell cancer, e.g., myelomas like multiple myeloma, Waldenstrom’s macroglobulinemia, the heavy chain diseases, such as, for example, alpha chain disease, gamma chain disease, and mu chain disease, benign monoclonal gammopathy, and immunocytic amyloidosis, melanomas, breast cancer, lung cancer, bronchus cancer, colorectal cancer, prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, cancer of hematologic tissues, and the like. Other nonlimiting examples of types of cancers applicable to the methods encompassed by the present disclosure include human sarcomas and carcinomas, e.g., fibrosarcoma, myosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing’s tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, liver cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms’ tumor, cervical cancer, bone cancer, brain tumor, testicular cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and acute myelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronic leukemia (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin’s disease and non-Hodgkin’s disease), myeloma, multiple myeloma, Waldenstrom’s macroglobulinemia, and heavy chain disease. In some embodiments, cancers are epithelial in nature and include but are not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecologic cancers, renal cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer. In other embodiments, the cancer is breast cancer, prostate cancer, lung cancer, or colon cancer. In still other embodiments, the epithelial cancer is non-small-cell lung cancer, nonpapillary renal cell carcinoma, cervical carcinoma, ovarian carcinoma (e.g., serous ovarian carcinoma), or breast carcinoma. The epithelial cancers may be characterized in various other ways including, but not limited to, serous, endometrioid, mucinous, clear cell, Brenner, or undifferentiated.

[0041] The term “erythroid progenitor cell” refers to the hematopoietic stem cell- derived progenitor cell that gives rise to erythrocytes (red blood cells) after terminal differentiation.

[0042] As used herein, the term “anemia” includes macrocytic anemia, hemolytic anemia, anemia associated with inflammation such as chronic kidney disease (CKD) and other inflammatory diseases such as autoimmune disorders (e.g., rheumatoid arthritis and multiple sclerosis), anemia caused by insufficiency of serine / threonine-protein kinase Right Open Reading Frame Kinase 2 (RIOK2), anemia caused by one or more mutations and / or deletions in human chromosome 5 or in an ortholog thereof, anemia caused by chromosomal translocations in the NUP98 gene or in an ortholog thereof, such as anemia caused by fusions of NUP98 with Abd-B group HOX genes (e.g., H0XD13 stress-induced anemia, aplastic anemia, anemia secondary to an intestinal cancer, Diamond Blackfan anemia, Schwachman- Diamond syndrome or anemia secondary to chemotherapy treatment in cancer patients. The anemia may be an anemia associated with a cancer, optionally wherein the cancer is a hematologic malignancy (e.g, myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) or multiple myeloma (MM)). The anemia may be an anemia associated with an intestinal cancer, such as a colorectal cancer. For example,the anemia may be the result of intestinal adenoma caused by familial adenomatous polyposis (FAP). Adenomatous polyposis coli (APC) is a tumor suppressor gene mutated in colorectal cancers. Alterations in the APC gene generate truncated gene products, leading to activation of the Wnt signaling pathway and deregulation of multiple other cellular processes contributing to tumorigenesis. For more details, please see Su et al. Multiple intestinal neoplasia caused by a mutation in the murine homolog of the APC gene. Science. 1992 May l;256(5057):668-70 and Moser et al. A dominant mutation that predisposes to multiple intestinal neoplasia in the mouse. Science. 1990 Jan 19;247(4940):322-4. The anemia may be an anemia associated with a bone marrow failure disorder. The anemia may be an anemia caused or associated with a ribosomopathy. As used herein “ribosomopathies” are diseases caused by defects in ribosomal constituents or in factors with a role in ribosome assembly that trigger defects in ribosome biogenesis. Congenital ribosomopathies display a paradoxical transition from early symptoms due to cellular hypo-proliferation to an elevated cancer risk later in life. More details re: ribosomopathies can be found in Kim R Kampen et al.(2020). Nucleic Acids Res. 48(3): 1013-1028. Mutations that disrupt ribosome biogenesis often affect tissues that rely on cell division for their function. Many ribosomopathies have an anemia component, as blood cell production from the bone marrow relies heavily on cell division. Examples of ribosomapthaies include, but are not limited to, Diamond-Blackfan anemia (DBA), 5q-syndrome, Schwachman-Diamond syndrome (SDS), X-linked dyskeratosis congenita (DC), Cartilage-hair hypoplasia (CHH), Treacher-Collins syndrome (TCS), Bowen-Conradi syndrome, and North American Indian childhood cirrhosis.

[0043] As used herein, myelodysplastic syndromes (MDS), include, but are not limited to, a heterogeneous group of myeloid neoplasms, which are characterized in common by manifestations of bone marrow failure with abnormal cell morphology and, in some cases, a propensity to acute myeloid leukemia (AML). In some instances, MDS is caused by mutations or deletions on human chromosome 5 or chromosomal translocations in the NUP98 gene (e.g., translocations leading to fusions of NUP98 with Abd-B group HOX genes (e.g., H0XD13)). For more details regarding chromosomal translocations in the NUP98 gene, please see Lin et al. NUP98-HOXD13 transgenic mice develop a highly penetrant, severe myelodysplastic syndrome that progresses to acute leukemia. Blood. 2005 Jul l;106(l):287- 95.

[0044] The term “patient”, as used herein, refers to a human suffering from anemia.

[0045] As used herein, the phrase “conjoint administration” refers to any form of administration of two or more different therapeutic agents such that the second agent is administered while the previously administered therapeutic agent is still effective in the body (e.g., the two agents are simultaneously effective in the subject, which may include synergistic effects of the two agents). For example, the different therapeutic agents can be administered either in the same formulation or in separate formulations, either concomitantly or sequentially. In certain embodiments, the different therapeutic agents can be administered within about one hour, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, or about a week of one another. Thus, a subject who receives such treatment can benefit from a combined effect of different therapeutic agents. As used herein, any two agents and / or additional agents may be conjointly administered according to the methods provided herein.

[0046] The term “therapeutic effect” refers to a local or systemic effect in humans, caused by a pharmacologically active substance. The term thus means any substance intended for use in the diagnosis, cure, mitigation, or treatment of disease or in the enhancement of desirable physical or mental development and conditions in a patient (e.g., a human suffering from anemia).

[0047] The terms “therapeutically-effective amount” and “effective amount” refers to an amount necessary (for example, at dosages and for periods of time and for the means of enteral or oral administration) to achieve the desired therapeutic result of treating anemia. An effective amount of the agonist of p2-adrenoreceptor may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the monoclonal antibody to elicit a desired response in the individual. An effective amount is also one in which medical provider, e.g, the attending physician, determines that any toxic or detrimental effects of the agonist are outweighed by the therapeutically beneficial effects.Salmeterol

[0048] Salmeterol is a long-acting beta-2 adrenergic receptor agonist drug that is currently prescribed for the treatment of asthma and chronic obstructive pulmonary disease (COPD). The present disclosure provides a new use for salmeterol in the treatment of anemia. The present disclosure encompasses a new and unexpected finding that salmeterol can increase red blood cell parameters and stimulate erythroid (red blood cell) differentiation.

[0049] In some embodiments, provided herein are methods of treating anemia in a patient in need thereof, the method comprising administering to the patient in need thereof an effective amount of salmeterol or a pharmaceutically acceptable salt thereof (e.g., salmeterol xinafoate). In some embodiments, provided herein are methods of promoting differentiation of an erythroid progenitor cell toward a mature red blood cell in a patient in need thereof, comprising administering an effective amount of salmeterol or a pharmaceutically acceptable salt thereof (e.g., salmeterol xinafoate). In some embodiments, provided herein are methods of increasing hemoglobulin and / or hematocrit in a patient in need thereof, the method comprising administering to the patient in need thereof an effective amount of salmeterol or a pharmaceutically acceptable salt thereof (e.g., salmeterol xinafoate). In some embodiments, the methods may further comprise administering to the patient in need thereof an effective amount of an erythropoiesis-stimulating agent. In some embodiments, provided herein are methods of treating anemia in a patient in need thereof, comprising administering to the patient in need thereof an effective amount of salmeterol or a pharmaceutically acceptable salt thereof (e.g., salmeterol xinafoate) conjointly with an erythropoiesis-stimulating agent, wherein the anemia is refractory to the erythropoiesis-stimulating agent (such as erythropoietin, epoetin alfa, epoetin beta, epoetin omega, epoetin zeta, or darbepoetin alfa). In some embodiments, salmeterol or a pharmaceutically acceptable salt thereof (e.g., salmeterol xinafoate) may be administered conjointly with other FDA-approved drugs such as luspatercept, lenalidomide, daprodustat, vadadustat, and / or a hypomethylating agent, such as azacitidine or decitabine.

[0050] In some embodiments, salmeterol or a pharmaceutically acceptable salt thereof (e.g., salmeterol xinafoate) is provided herein for use in treating anemia in a patient. In some embodiments, provided herein are uses of salmeterol or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of anemia in a patient. In some embodiments, provided herein are pharmaceutical compositions comprising salmeterol or a pharmaceutically acceptable salt thereof (e.g., salmeterol xinafoate) for use in the treatment of anemia in a patient.

[0051] In some embodiments, the anemia is selected from the group consisting of macrocytic anemia, hemolytic anemia, anemia caused by a ribosomopathy, anemia caused by insufficiency of serine / threonine-protein kinase RI0K2, anemia associated with chronic kidney disease (CKD), anemia caused by one or more mutations and / or deletions in human chromosome 5 or in an ortholog thereof, anemia caused by chromosomal translocations in theNUP98 gene or in an ortholog thereof, such as anemia caused by fusions of NUP98 with Abd-B group HOX genes (e.g., H0XD13 stress-induced anemia, anemia secondary to an intestinal cancer, Diamond Blackfan anemia, aplastic anemia, Schwachman-Diamond syndrome, an anemia associated with an inflammatory disease, such as rheumatoid arthritis or multiple sclerosis, anemia associated with intestinal adenoma, anemia associated with a bone marrow failure syndrome and anemia secondary to chemotherapy in cancer patients. In some embodiments, the anemia is associated with a cancer, optionally wherein the cancer is a hematologic malignancy, such as a myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), or any hematologic malignancy disclosed herein.

[0052] In some embodiments, methods and uses disclosed herein enhance erythropoiesis in bone marrow cells and therefore may improve erythroid differentiation defects, for example, in hematological malignancies such as acute myeloid leukemia and other diseases disclosed herein, such as bone marrow failure disorders, including but not limited to Diamond-Blackfan anemia and aplastic anemia. In some embodiments, provided methods and uses disclosed herein have the benefit of once daily dosing of salmeterol or a pharmaceutically acceptable salt thereof (e.g., salmeterol xinafoate).

[0053] In some embodiments, methods and uses disclosed herein do not affect (increase or decrease) white blood cell count and / or platelets. In some embodiments, methods and uses disclosed herein do not affect (increase or decrease) granulocytic monocytic progenitors (GMP) or megakaryocytic progenitors (MkP).

[0054] In some embodiments, the disclosure provides herein new uses of salmeterol or a pharmaceutically acceptable salt thereof (e.g., salmeterol xinafoate) for oral administration for treatment of anemia as described further herein.Salbutamol

[0055] Salbutamol is a short-acting beta-2 adrenergic receptor agonist drug that is currently prescribed for the treatment of asthma and chronic obstructive pulmonary disease (COPD). The present disclosure provides a new use for salbutamol in the treatment of anemia. The present disclosure encompasses a new and unexpected finding that salbutamol can increase red blood cell parameters and stimulate erythroid (red blood cell) differentiation.

[0056] In some embodiments, provided herein are methods of treating anemia in a patient in need thereof, the method comprising administering to the patient in need thereof an effective amount of salbutamol or a pharmaceutically acceptable salt thereof (e.g., salbutamolsulfate). In some embodiments, provided herein are methods of promoting differentiation of an erythroid progenitor cell toward a mature red blood cell in a patient in need thereof, comprising administering an effective amount of salbutamol or a pharmaceutically acceptable salt thereof (e.g., salbutamol sulfate). In some embodiments, provided herein are methods of increasing hemoglobin and / or hematocrit in a patient in need thereof, the method comprising administering to the patient in need thereof an effective amount of salbutamol or a pharmaceutically acceptable salt thereof (e.g., salbutamol sulfate). In some embodiments, the methods may further comprise administering to the patient in need thereof an effective amount of an erythropoiesis-stimulating agent. In some embodiments, provided herein are methods of treating anemia in a patient in need thereof, comprising administering to the patient in need thereof an effective amount of salbutamol or a pharmaceutically acceptable salt thereof (e.g., salbutamol sulfate) conjointly with an erythropoiesis-stimulating agent, wherein the anemia is refractory to the erythropoiesis-stimulating agent (such as erythropoietin, epoetin alfa, epoetin beta, epoetin omega, epoetin zeta, or darbepoetin alfa). In some embodiments, salbutamol or a pharmaceutically acceptable salt thereof (e.g., salbutamol sulfate) may be administered conjointly with other FDA-approved drugs such as luspatercept, lenalidomide, daprodustat, vadadustat, and / or a hypomethylating agent, such as azacitidine or decitabine.

[0057] In some embodiments, salbutamol or a pharmaceutically acceptable salt thereof (e.g., salbutamol sulfate) is provided herein for use in treating anemia in a patient. In some embodiments, provided herein are uses of salbutamol or a pharmaceutically acceptable salt thereof (e.g., salbutamol sulfate) in the manufacture of a medicament for the treatment of anemia in a patient. In some embodiments, provided herein are pharmaceutical compositions comprising salbutamol or a pharmaceutically acceptable salt thereof (e.g., salbutamol sulfate) for use in the treatment of anemia in a patient.

[0058] In some embodiments, the anemia is selected from the group consisting of macrocytic anemia, hemolytic anemia, anemia caused by a ribosomopathy, anemia caused by insufficiency of serine / threonine-protein kinase RI0K2, anemia associated with chronic kidney disease (CKD), anemia caused by one or more mutations and / or deletions in human chromosome 5 or in an ortholog thereof, anemia caused by chromosomal translocations in the NUP98 gene or in an ortholog thereof, such as anemia caused by fusions of NUP98 with Abd-B group HOX genes (e.g., H0XD13 stress-induced anemia, anemia secondary to an intestinal cancer, Diamond Blackfan anemia, aplastic anemia, Schwachman-Diamondsyndrome, an anemia associated with an inflammatory disease, such as rheumatoid arthritis or multiple sclerosis, anemia associated with a bone marrow failure syndrome and anemia secondary to chemotherapy in cancer patients. In some embodiments, the anemia is associated with a cancer, optionally wherein the cancer is a hematologic malignancy, such as a myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), or any hematologic malignancy disclosed herein.

[0059] In some embodiments, methods and uses disclosed herein enhance erythropoiesis in bone marrow cells and therefore may improve erythroid differentiation defects, for example, in hematological malignancies such as acute myeloid leukemia and other diseases disclosed herein, such as bone marrow failure disorders, including but not limited to Diamond-Blackfan anemia and aplastic anemia.

[0060] In some embodiments, methods and uses disclosed herein do not affect (increase or decrease) white blood cell count and / or platelets. In some embodiments, methods and uses disclosed herein do not affect (increase or decrease) granulocytic monocytic progenitors (GMP) or megakaryocytic progenitors (MkP).

[0061] In some embodiments, the disclosure provides herein new uses of salbutamol or a pharmaceutically acceptable salt thereof (e.g., salbutamol sulfate) for oral administration for treatment of anemia as described further herein.Patients

[0062] A patient may be any human afflicted by anemia. In some embodiments, terms “individual” or “patient” are used and are intended to be interchangeable with “subject.” A patient may be a human subject of any age. In some embodiments, a patient is an adult. In some embodiments, a patient is an adolescent. In some embodiments, a patient is a child. In some embodiments, a patient is an infant.

[0063] In some embodiments, a patient to be treated by methods, compositions, and uses described herein may be a human that is at least 4 years of age (z.e., 4 years of age or older). In some embodiments, a patient is 4 to 11 years of age (i.e., > 4 years old and < 11 years old). In some embodiments, a patient is at least 4 years of age and less than 12 years of age (i.e., > 4 years of age and < 12 years of age). In some embodiments, a patient is a human that is at least 12 years of age i.e., 12 years of age or older, > 12 years of age).

[0064] In some embodiments, a patient to be treated by methods, compositions, and uses described herein may be a human that is at least 2 years of age (i.e., 2 years of age orolder, > 2 years of age). In some embodiments, a patient is 2 to 6 years of age. In some embodiments, a patient is a human that is at least 4 years of age (z.e., 4 years of age or older, > 4 years of age). In some embodiments, a patient is a human 4 to 11 years of age (i.e., > 4 years old and < 11 years old). In some embodiments, a patient is at least 4 years of age and less than 12 years of age (z.e., > 4 years of age and < 12 years of age). In some embodiments, a patient is between 6 and 12 years of age. In some embodiments, a patient is > 6 years old and < 12 years old. In some embodiments, a patient is 12 years of age or older (z.e., 12 years of age or older, > 12 years of age).

[0065] In some embodiments, a patient may be an aging patient or any patient afflicted by a disorder associated with aging that display anemia.

[0066] Methods and compositions encompassed by the present disclosure can be used in myelodysplastic syndromes (MDS) and anemias, such as, anemia caused by insufficiency of serine / threonine-protein kinase RI0K2, anemia caused by mutations or deletions on human chromosome 5, anemia caused by chromosomal translocations in the NUP98 gene or in an ortholog thereof, such as anemia caused by fusions of NUP98 with Abd-B group HOX genes (e.g., H0XD13), macrocytic anemia, anemia associated with inflammatory disorders, such as rheumatoid arthritis or multiple sclerosis, anemia associated with chronic kidney disease (CKD), stress-induced anemia, chemotherapy-induced anemia in cancer patients, aplastic anemia, anemia secondary to an intestinal cancer, Diamond Blackfan anemia, and Shwachman-Diamond syndrome. Similarly, the methods and compositions encompassed by the present disclosure can be used across bone marrow failure syndromes since it has been determined that Right Open reading frame Kinase 2 (RIOK2) regulates blood cell development and agonists of RIOK2 activity reverse anemia associated with bone marrow failure syndromes, such as aplastic anemia, Diamond Blackfan anemia, dyskeratosis congenita (DC), fanconi anemia (FA), Pearson syndrome, severe congenital neutropenia (SCN), Shwachman-Diamond syndrome (SDS), and others.

[0067] In some embodiments, a patient has anemia associated with a cancer (e.g., anemia associated with a cancer disclosed herein). In some embodiments, a patient has myelodysplastic syndromes (MDS).

[0068] The present disclosure encompasses a recognition that mitochondrial defects may contribute to the pathogenesis of hematological disorders. Mitochondrial dysfunction is often associated with the pathogenesis of hematological disorders, such as MDS and AML (Fontenay et al. (2006) Oncogene 25:4757-4767). For example, altered mitochondrialtranscription (Schildgen et al. (2011) Exp. HematoL 39:666-675), deregulated HIFla expression (Liu et al. (2019) Oncol. Lett. 17:5395-5402; Stergiouc / al. (2021) Int. J. Mol. Set. 22), presence of isocitrate dehydrogenase (IDH) mutations expressing oncogenic metabolite 2-hydroxy-glutarate (2-HG) (Gonzalez-Menendez et al. (2021) Cell Rep. 34: 108723;Intlekofer et al. (2018) Nature 559: 125-129; Testa et al. (2020) Cancers 12:2427), elevation of mitochondrial oxidative stress markers (Saigo et al. (2011) J. Int. Med. Res. 39: 1941- 1945), and presence of aberrant oxidation and mutations in mitochondrial DNA (mtDNA) (Coelho-Silva et al. (2021) Set. Rep. 11 :1675; Schildgen et al. (2011) Exp. HematoL 39:666- 675; Ward et al. (2021) Blood Adv. 5:2216-2228; Wulfert et al. (2008) Exp. HematoL 36:577-586) strongly implicate defective mitochondrial functions as a critical player in MDS pathogenesis. In some embodiments, a patient for treatment by methods disclosed herein may have a hematological disorder associated with a mitochondrial defect or dysfunction.Administration of Agents

[0069] In some embodiments, salmeterol or a pharmaceutically acceptable salt thereof (e.g., salmeterol xinafoate) can be administered to patients disclosed herein according to methods encompassed by the present disclosure in a biologically compatible form suitable for pharmaceutical administration in vivo. By “biologically compatible form suitable for administration in vivo ' is meant a form to be administered in which any toxic effects are outweighed by the therapeutic effects. Administration of salmeterol or a pharmaceutically acceptable salt thereof (e.g., salmeterol xinafoate) can be in any pharmacological form including a therapeutically active amount of an agent alone or in combination with a pharmaceutically acceptable carrier.

[0070] Salmeterol is a long-acting beta-2 adrenergic receptor agonist drug. B2-AR are cell-membrane spanning receptors for adrenaline (epinephrine) that mediate smooth muscle relaxation and bronchodilation via adenylate cyclase stimulation (Abosamak and Shahin (2021). In StatPearls (Treasure Island (FL)); Johnson (2006) J Allergy Clin Immunol 117, 18- 24; quiz 25; Yang et al. (2021) Life Sci 265: 118864). B2-AR is encoded by the ADRB2 gene.

[0071] Salmeterol has a molecular formula C25H37NO4. Exemplary molecular forms of salmeterol include, but are not limited to CAS reference 89365-50-4 and IUPAC name 2- (hydroxymethyl)-4-[l-hydroxy-2-[6-(4-phenylbutoxy)hexylamino]ethyl]phenol. In some embodiments, salmeterol is or comprises a molecule corresponding to Formula I below.

[0072] Formula I

[0073] Commercially available forms of salmeterol include, without limitation, PI- 26968 from Pi Chemicals, AR003BM6 from Aaron Chemicals LLC, and sc-224277 from Santa Cruz Biotechnology, Inc.

[0074] Exemplary salt forms of salmeterol include, but are not limited to, salmeterol xinafoate (molecular formula C36H45NO7), CAS reference 94749-08-3, and TUPAC name 2- (hydroxymethyl)-4-[l -hydroxy -2-[6-(4-phenylbutoxy)hexylamino]ethyl]phenol;l- hydroxynaphthalene-2-carboxylic acid. Commercially available forms of salmeterol xinafoate include, without limitation, R483281 from RR Scientific, 1609603 USP from Sigma- Aldrich, and AR00IJ75 from Aaron Chemicals LLC.

[0075] Salmeterol is well known in the art, see e.g., US 4,992,474 and US 5,126,375, which are each incorporated by reference herein in their entirety. Other methods of formulations, described in Remington’s Pharmaceutical Sciences, 21stEdition, University of the Sciences in Philadelphia, Philadelphia, Pa., USA (2006) can be employed in implementing embodiments of the present disclosure. As used herein, salmeterol includes all stereoisomers (e.g., (R)- and (S)-isomers), including all enantiomers (R;R and S;S) and all diastereomers (R;S and S;R).

[0076] In some embodiments, salbutamol or a pharmaceutically acceptable salt thereof (e.g., salbutamol sulfate) can be administered to patients disclosed herein according to methods encompassed by the present disclosure in a biologically compatible form suitable for pharmaceutical administration in vivo. By “biologically compatible form suitable for administration in vivo'' is meant a form to be administered in which any toxic effects are outweighed by the therapeutic effects. Administration of salbutamol or a pharmaceutically acceptable salt thereof (e.g., salbutamol sulfate) can be in any pharmacological form including a therapeutically active amount of an agent alone or in combination with a pharmaceutically acceptable carrier.

[0077] Salbutamol is a short-acting beta-2 adrenergic receptor agonist drug. P2-AR are cell-membrane spanning receptors for adrenaline (epinephrine) that mediate smoothmuscle relaxation and bronchodilation via adenylate cyclase stimulation (Abosamak and Shahin (2021). In StatPearls (Treasure Island (FL)); Johnson (2006) J Allergy Clin Immunol 117, 18-24; quiz 25; Yang et al. (2021) Life Sci 265'. 118864). P2- AR is encoded by the ADRB2 gene.

[0078] Salbutamol, also referred to as albuterol, has a molecular formula C13H21NO3. Exemplary molecular forms of salbutamol includes, but are not limited to CAS reference 18559-94-9 and IUPAC name 4-[2-(tert-butylamino)-l-hydroxyethyl]-2- (hydroxymethyl)phenol. In some embodiments, salbutamol is or comprises a molecule corresponding to Formula II below.

[0079] Formula II

[0080] Commercially available forms of salbutamol include, without limitation, S5494 from Selleckchem, 1012600 from Sigma Aldrich, and sc-253527 from Santa Cruz Biotechnology, Inc.

[0081] Exemplary salt forms of salbutamol include, but is not limited to, salbutamol sulfate (molecular formula C26H44N2O10S), albuterol sulfate, CAS reference 51022-70-9, salbutamol hemisulfate, and venetlin, and IUPAC name 4-[2-( / c / 7-butylamino)- l - hydroxyethyl]-2-(hydroxymethyl)phenol;sulfuric acid. Commercially available forms of salbutamol sulfate include, without limitation, 1012633 from Sigma Aldrich, sc-203373 from Santa Cruz Biotechnology, Inc., and R199247 from RR Scientific.

[0082] Salbutamol is well known in the art, being first described in Svenska lakartidningen (1962), 59, 3384-5. Salbutamol compositions and formulations are also well described, such as in US 3,644,353 A, US 4,594,359 A and US 4,499,108 A, which are each incorporated by reference herein in their entirety. Other methods of formulations, described in Remington's Pharmaceutical Sciences, 21st Edition, University of the Sciences in Philadelphia, Philadelphia, Pa., USA (2006) can be employed in implementing embodiments of the present disclosure. As used herein, salbutamol includes all stereoisomers (e.g., (R)- and (S)-isomers), including all enantiomers (R;R and S;S) and all diastereomers (R;S and S;R).

[0083] In some embodiments, salbutamol may be formulated as a racemic mixture of the R- and S-isomers. The R-isomer has 150 times greater affinity for the beta2-receptor thanthe S-isomer and the S-isomer has been associated with toxicity. In some embodiments, salbutamol is enriched for the R-isomer. In some certain embodiments, salbutamol is levalbuterol, comprising the single R-isomer of salbutamol.

[0084] Agents encompassed by the present disclosure can be administered either alone or conjointly with an additional therapy. In the conjoint therapy, an agent encompassed by the present disclosure and another agent, such as an erythropoiesis-stimulating agent (e.g., erythropoietin, epoetin alfa, epoetin beta, epoetin omega, epoetin zeta, darbepoetin alfa), or other FDA-approved drugs such as luspatercept, lenalidomide, daprodustat, vadadustat, and / or a hypomethylating agent, such as azacitidine or decitabine, can be delivered to the same or different cells and can be delivered at the same or different times. In some embodiments, an erythropoiesis-stimulating agent is a hypoxia-inducible factor-prolyl hydroxylase (HIF-PH) inhibitor. HIF-PH inhibitors are a class of oral drugs designed to boost production of red blood cells by mimicking the body’s response at high altitudes. In some embodiments, a HIF-PH is daprodustat or vadadustat. The agents encompassed by the present disclosure can be incorporated into pharmaceutical compositions suitable for administration. Such compositions can comprise one or more agents or one or more molecules that result in the production of such one or more agents and a pharmaceutically acceptable carrier.

[0085] The therapeutic agent described herein (e.g., salmeterol or a pharmaceutically acceptable salt thereof, such as salmeterol xinafoate) can be administered in a convenient manner such as by oral administration, injection (subcutaneous, intravenous, intraperitoneal (i.p.) etc.), inhalation, transdermal application, or rectal administration. Depending on the route of administration, the active compound can be coated in a material to protect the compound from the action of enzymes, acids and other natural conditions which can inactivate the compound. For example, for administration of agents, by other than parenteral administration, it can be desirable to coat the agent with, or co-administer the agent with, a material to prevent its inactivation.

[0086] As described in detail below, the pharmaceutical compositions encompassed by the present disclosure (e.g., a composition comprising salmeterol or a pharmaceutically acceptable salt thereof, such as salmeterol xinafoate) can be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes; or (2) parenteral administration, for example, bysubcutaneous, intramuscular, intraperitoneal (i.p.) or intravenous injection as, for example, a sterile solution or suspension.

[0087] The phrase “pharmaceutically acceptable” is employed herein to refer to those agents, 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 without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0088] The phrase “pharmaceutically-acceptable carrier” as used herein means 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 chemical 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 and not injurious to the patient. Those skilled in the art will recognize, or be able to employ suitable pharmaceutically-acceptable carriers for the therapeutic agent disclosed herein (e.g., salmeterol or a pharmaceutically acceptable salt thereof, such as salmeterol xinafoate) from the pharmaceutically-acceptable carriers known in pharmaceutical science (See, for example, Adejare, Adeboye, ed. Remington: the science and practice of pharmacy. Academic Press, 2020).

[0089] The term “pharmaceutically-acceptable salts” refers to the relatively nontoxic, inorganic and organic acid addition salts of the therapeutic agent disclosed herein (z.e., salmeterol, the pharmaceutically-acceptable salts of which include, for example, salmeterol xinafoate). These salts can be prepared in situ during the final isolation and purification of the therapeutic agents, or by separately reacting a purified therapeutic agent in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed. Those skilled in the art will recognize, or be able to employ suitable pharmaceutically-acceptable salts for the therapeutic agents disclosed herein (e.g., salmeterol, the pharmaceutically-acceptable salts of which include, for example, salmeterol xinafoate) from the pharmaceutically-acceptable salts known in pharmaceutical science (See, for example, Berge et al. (1977) J. Pharm. Sci. 66: 1- 19).

[0090] Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0091] Examples of pharmaceutically-acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0092] Formulations useful in the methods encompassed by the present disclosure include those suitable for oral administration, intravenous administration and / or administration by injection (e.g., intraperitoneal (i.p.) injection). The formulations can conveniently be presented in unit dosage form and can be prepared by any methods well- known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. In some embodiments, salmeterol or a pharmaceutically acceptable salt thereof (e.g., salmeterol xinafoate) is in a form formulated for oral administration. Exemplary oral forms of salmeterol can be found in the art. In some embodiments, the agent provided herein is an oral formulation of salmeterol or a pharmaceutically acceptable salt thereof (e.g., salmeterol xinafoate).

[0093] In general, the formulations are prepared by uniformly and intimately bringing into association a therapeutic agent with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0094] Formulations suitable for oral administration can be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and / or as mouth washes and the like, each containing a predetermined amount of a therapeutic agent as an active ingredient. A compound can also be administered as a bolus, electuary or paste.

[0095] In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically-acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose and / or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, acetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions can also comprise buffering agents. Solid compositions of a similar type can also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

[0096] A tablet can be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surfaceactive or dispersing agent. Molded tablets can be made by molding in a suitable machine a mixture of the powdered peptide or peptidomimetic moistened with an inert liquid diluent.

[0097] Tablets, and other solid dosage forms, such as dragees, capsules, pills and granules, can optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well-known in the pharmaceutical-formulating art. They can also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and / or microspheres. They can be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions, which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions can also optionally contain opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions, which can be used include polymeric substances and waxes. The active ingredient can also be in micro- encapsulated form, if appropriate, with one or more of the above-described excipients.

[0098] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In additionto the active ingredient, the liquid dosage forms can contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, com, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

[0099] Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0100] Suspensions, in addition to the active agent can contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

[0101] Pharmaceutical compositions encompassed by the present disclosure suitable for parenteral administration comprise one or more therapeutic agents in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which can be reconstituted into sterile injectable solutions or dispersions just prior to use, which can contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

[0102] These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It can also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.

[0103] In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This can be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, can depend upon crystal size and crystalline form. Alternatively,delayed absorption of a parenterally-administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

[0104] Injectable depot forms are made by forming microencapsule matrices of salmeterol or a pharmaceutically acceptable salt thereof (e.g., salmeterol xinafoate), in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug 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 formulations are also prepared by entrapping the drug in liposomes or microemulsions, which are compatible with body tissue.

[0105] When the therapeutic agents encompassed by the present disclosure are administered as pharmaceuticals, to humans, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.

[0106] It will be understood that the total daily dosage of the compounds of the present disclosure will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the anemia being treated and the severity of the anemia; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and plasma half-life of the specific compound employed; the duration of the treatment; drugs used conjointly or coincidental with the specific agonist employed; and like factors well known in the medical arts. The daily dosage of the active ingredient may include, but is not limited to, about 0.1 to 500 pg per adult per day. Typically, the pharmaceutical compositions contain 0.1, 1.0, 5.0, 10.0, 12.0, 15.0, 20.0, 50, 75, 100, 150, 200, 250, 300, 400, 500 pg of salmeterol or a pharmaceutically acceptable salt thereof (e.g., salmeterol xinafoate), preferably 1 to 100 pg. An effective amount of the drug is ordinarily supplied in a dose and route of administration to achieve an effective amount of salmeterol or a pharmaceutically acceptable salt thereof (e.g., salmeterol xinafoate) to treat anemia and plasma levels of 0.03-150 pg / mL, preferably 0.3-30 pg / mL.

[0107] Actual dosage levels of the active ingredients in pharmaceutical compositions encompassed by the present disclosure can be determined by the methods known in the art to obtain an amount of the active ingredient that is effective to achieve the desired therapeuticresponse for a particular patient, composition, and mode of administration, without being toxic to the patient.Numbered Illustrative Embodiments

[0108] Embodiment 1. A method of treating anemia in a patient in need thereof, the method comprising administering to the patient an effective amount of salmeterol or a pharmaceutically acceptable salt thereof.

[0109] Embodiment 2. The method of embodiment 1, wherein the anemia is selected from the group consisting of macrocytic anemia, hemolytic anemia, anemia caused by a ribosomopathy, anemia caused by insufficiency of serine / threonine-protein kinase RIOK2, anemia associated with chronic kidney disease (CKD), anemia caused by one or more mutations and / or deletions in human chromosome 5 or in an ortholog thereof, stress-induced anemia, Diamond Blackfan anemia, aplastic anemia, Schwachman-Diamond syndrome, an anemia associated with an inflammatory disease, such as rheumatoid arthritis or multiple sclerosis, anemia secondary to intestinal cancer or to chemotherapy in cancer patients, and anemia associated with a bone marrow failure syndrome.

[0110] Embodiment 3. The method of embodiment 1 or embodiment 2, wherein the anemia is associated with a cancer, optionally wherein the cancer is a hematologic malignancy, such as a myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) or multiple myeloma (MM).[OHl] Embodiment 4. The method of embodiment 1 or embodiment 2, wherein the anemia is associated with a cancer, optionally wherein the cancer is an intestinal cancer, such as colorectal cancer.

[0112] Embodiment 5. A method of promoting differentiation of a hematopoietic stem and progenitor cell (HSPC) or a multipotent progenitor (MPP) cell toward an erythroid progenitor cell or an erythroid progenitor cell toward a mature red blood cell in a patient in need thereof, comprising administering to the patient in need thereof an effective amount of salmeterol or a pharmaceutically acceptable salt thereof.

[0113] Embodiment 6. The method of embodiment 5, wherein the patient is a human suffering from anemia.

[0114] Embodiment 7. The method of embodiment 6, wherein the anemia is selected from the group consisting of macrocytic anemia, hemolytic anemia, anemia caused by a ribosomopathy, anemia caused by insufficiency of serine / threonine-protein kinase RIOK2,anemia associated with chronic kidney disease (CKD), anemia caused by one or more mutations and / or deletions in human chromosome 5 or in an ortholog thereof, stress-induced anemia, Diamond Blackfan anemia, aplastic anemia, Schwachman-Diamond syndrome, an anemia associated with an inflammatory disease, such as rheumatoid arthritis or multiple sclerosis, anemia secondary to chemotherapy in cancer patients, and anemia associated with a bone marrow failure syndrome.

[0115] Embodiment 8. The method of embodiment 6 or embodiment 7, wherein the anemia is associated with a cancer, optionally wherein the cancer is a hematologic malignancy, such as a myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) or multiple myeloma (MM).

[0116] Embodiment 9. The method of embodiment 6 or embodiment 7, wherein the anemia is associated with a cancer, optionally wherein the cancer is an intestinal cancer, such as colorectal cancer.

[0117] Embodiment 10. The method of any one of embodiments 1 to 9, further comprising administering to the patient in need thereof an effective amount of an erythropoiesis-stimulating agent.

[0118] Embodiment 11. The method of embodiment 10, wherein the erythropoiesisstimulating agent comprises erythropoietin, epoetin alfa, epoetin beta, epoetin omega, epoetin zeta, or darbepoetin alfa.

[0119] Embodiment 12. A method of treating anemia in a patient in need thereof, the method comprising administering to the patient an effective amount of salmeterol or a pharmaceutically acceptable salt thereof conjointly with an erythropoiesis-stimulating agent, wherein the anemia is refractory to the erythropoiesis-stimulating agent.

[0120] Embodiment 13. The method of embodiment 12, wherein the anemia is selected from the group consisting of macrocytic anemia, hemolytic anemia, anemia caused by a ribosomopathy, anemia caused by insufficiency of serine / threonine-protein kinase RIOK2, anemia associated with chronic kidney disease (CKD), anemia caused by one or more mutations and / or deletions in human chromosome 5 or in an ortholog thereof, stress- induced anemia, Diamond Blackfan anemia, aplastic anemia, Schwachman-Diamond syndrome, an anemia associated with an inflammatory disease, such as rheumatoid arthritis or multiple sclerosis, anemia secondary to chemotherapy in cancer patients, and anemia associated with a bone marrow failure syndrome.

[0121] Embodiment 14. The method of embodiment 12 or embodiment 13, wherein the anemia is associated with a cancer, optionally wherein the cancer is a hematologic malignancy, such as a myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) or multiple myeloma (MM).

[0122] Embodiment 15. The method of embodiment 12 or embodiment 13, wherein the anemia is associated with a cancer, optionally wherein the cancer is an intestinal cancer, such as colorectal cancer.

[0123] Embodiment 16. The method of any one of embodiments 12 to 15, wherein the erythropoiesis-stimulating agent comprises erythropoietin, epoetin alfa, epoetin beta, epoetin omega, epoetin zeta, or darbepoetin alfa.

[0124] Embodiment 17. The method of any one of embodiments 1 to 16, wherein salmeterol or the pharmaceutically acceptable salt thereof is orally administered to the patient.

[0125] Embodiment 18. The method of any one of embodiments 1 to 17, wherein salmeterol or the pharmaceutically acceptable salt thereof is salmeterol xinafoate.

[0126] Embodiment 19. The method of any one of embodiments 1 to 18, wherein salmeterol or the pharmaceutically acceptable salt thereof is administered conjointly with luspatercept, lenalidomide, daprodustat, vadadustat, erythropoiesis-stimulating agents (ESAs) such as epoetin alfa or darbepoetin alfa, and / or a hypomethylating agent, wherein the hypomethylating agent is optionally azacitidine and / or decitabine.

[0127] Embodiment 20. The method of any one of embodiments 1 to 19, wherein the patient is a human that is 4 to 11 years of age.

[0128] Embodiment 21. The method of any one of embodiments 1 to 19, wherein the patient is a human that is at least 12 years of age.

[0129] Embodiment 22. A method of treating anemia in a patient in need thereof, the method comprising administering to the patient an effective amount of salbutamol or a pharmaceutically acceptable salt thereof.

[0130] Embodiment 23. The method of embodiment 22, wherein the anemia is selected from the group consisting of macrocytic anemia, hemolytic anemia, anemia caused by a ribosomopathy, anemia caused by insufficiency of serine / threonine-protein kinase RIOK2, anemia associated with chronic kidney disease (CKD), anemia caused by one or more mutations and / or deletions in human chromosome 5 or in an ortholog thereof, stress- induced anemia, Diamond Blackfan anemia, aplastic anemia, Schwachman-Diamondsyndrome, an anemia associated with an inflammatory disease, such as rheumatoid arthritis or multiple sclerosis, anemia secondary to chemotherapy in cancer patients, and anemia associated with a bone marrow failure syndrome.

[0131] Embodiment 24. The method of embodiment 22 or embodiment 23, wherein the anemia is associated with a cancer, optionally wherein the cancer is a hematologic malignancy, such as a myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) or multiple myeloma (MM).

[0132] Embodiment 25. The method of embodiment 22 or embodiment 23, wherein the anemia is associated with a cancer, optionally wherein the cancer is an intestinal cancer, such as colorectal cancer.

[0133] Embodiment 26. A method of promoting differentiation of a hematopoietic stem and progenitor cell (HSPC) or a multipotent progenitor (MPP) cell toward an erythroid progenitor cell or an erythroid progenitor cell toward a mature red blood cell in a patient in need thereof, comprising administering to the patient in need thereof an effective amount of salbutamol or a pharmaceutically acceptable salt thereof.

[0134] Embodiment 27. The method of embodiment 26, wherein the patient is a human suffering from anemia.

[0135] Embodiment 28. The method of embodiment 27, wherein the anemia is selected from the group consisting of macrocytic anemia, hemolytic anemia, anemia caused by a ribosomopathy, anemia caused by insufficiency of serine / threonine-protein kinase RIOK2, anemia associated with chronic kidney disease (CKD), anemia caused by one or more mutations and / or deletions in human chromosome 5 or in an ortholog thereof, stress- induced anemia, Diamond Blackfan anemia, aplastic anemia, Schwachman-Diamond syndrome, an anemia associated with an inflammatory disease, such as rheumatoid arthritis or multiple sclerosis, anemia secondary to chemotherapy in cancer patients, and anemia associated with a bone marrow failure syndrome.

[0136] Embodiment 29. The method of embodiment 27 or embodiment 28, wherein the anemia is associated with a cancer, optionally wherein the cancer is a hematologic malignancy, such as a myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) or multiple myeloma (MM).

[0137] Embodiment 30. The method of embodiment 27 or embodiment 28, wherein the anemia is associated with a cancer, optionally wherein the cancer is an intestinal cancer, such as colorectal cancer.

[0138] Embodiment 31. The method of any one of embodiments 22 to 30, further comprising administering to the patient in need thereof an effective amount of an erythropoiesis-stimulating agent.

[0139] Embodiment 32. The method of embodiment 31, wherein the erythropoiesisstimulating agent comprises erythropoietin, epoetin alfa, epoetin beta, epoetin omega, epoetin zeta, or darbepoetin alfa.

[0140] Embodiment 33. A method of treating anemia in a patient in need thereof, the method comprising administering to the patient an effective amount of salbutamol or a pharmaceutically acceptable salt thereof conjointly with an erythropoiesis-stimulating agent, wherein the anemia is refractory to the erythropoiesis-stimulating agent.

[0141] Embodiment 34. The method of embodiment 33, wherein the anemia is selected from the group consisting of macrocytic anemia, hemolytic anemia, anemia caused by a ribosomopathy, anemia caused by insufficiency of serine / threonine-protein kinase RI0K2, anemia associated with chronic kidney disease (CKD), anemia caused by one or more mutations and / or deletions in human chromosome 5 or in an ortholog thereof, stress- induced anemia, Diamond Blackfan anemia, aplastic anemia, Schwachman-Diamond syndrome, an anemia associated with an inflammatory disease, such as rheumatoid arthritis or multiple sclerosis, anemia secondary to chemotherapy in cancer patients, and anemia associated with a bone marrow failure syndrome.

[0142] Embodiment 35. The method of embodiment 33 or embodiment 34, wherein the anemia is associated with a cancer, optionally wherein the cancer is a hematologic malignancy, such as a myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) or multiple myeloma (MM).

[0143] Embodiment 36. The method of embodiment 33 or embodiment 34, wherein the anemia is associated with a cancer, optionally wherein the cancer is an intestinal cancer, such as colorectal cancer.

[0144] Embodiment 37. The method of any one of embodiments 33 to 36, wherein the erythropoiesis-stimulating agent comprises erythropoietin, epoetin alfa, epoetin beta, epoetin omega, epoetin zeta, or darbepoetin alfa.

[0145] Embodiment 39. The method of any one of embodiments 22 to 37, wherein salbutamol or the pharmaceutically acceptable salt thereof is orally administered to the patient.

[0146] Embodiment 39. The method of any one of embodiments 22 to 38, wherein salbutamol or the pharmaceutically acceptable salt thereof is salbutamol sulfate.

[0147] Embodiment 40. The method of any one of embodiments 22 to 39, wherein salbutamol or the pharmaceutically acceptable salt thereof is administered conjointly with luspatercept, lenalidomide, erythropoiesis-stimulating agents (ESAs) such as epoetin alfa or darbepoetin alfa, vadadustat, and / or a hypomethylating agent, wherein the hypomethylating agent is optionally azacitidine and / or decitabine.

[0148] Embodiment 41. The method of any one of embodiments 22 to 40, wherein the patient is a human 2 to 6 years of age.

[0149] Embodiment 42. The method of any one of embodiments 22 to 40, wherein the patient is a human 6 to 12 years of age.

[0150] Embodiment 43. The method of any one of embodiments 22 to 40, wherein the patient is a human that is at least 12 years of age.EXAMPLES

[0151] Described herein is a therapeutic approach of targeting anemia using salmeterol, an FDA-approved p2-adrenoreceptor agonist for COPD and asthma treatment. Administration of salmeterol significantly enhanced erythroid differentiation and RBC parameters in wild-type mice under both steady state and stress-induced hemolytic anemia settings. Thus, use of an existing FDA-approved drug is uncovered to reverse anemia and offer therapeutic benefits in a spectrum of human diseases, including but not limited to, hematologic malignancies, aplastic anemia, anemia of chronic kidney diseases, ribosomopathies, anemia secondary to chemotherapeutic agents in cancer patients, and bone marrow failure (BMF) disorders.Example 1: Salmeterol alleviates anemia in vivo

[0152] It was hypothesized that administration of salmeterol, a long-acting selective P2-AR agonist, might promote erythroid differentiation. The present example analyzes whether salmeterol can alleviate anemia in an in vivo mouse model. The murine P2- adrenoreceptor has 87.08% similarity to the protein composition of the human analog. The ability of salmeterol to treat anemia was investigated in a phenylhydrazine-mediated mouse hemolytic stress model of anemia.Mouse experiments

[0153] C57BL / 6J mice were obtained from Jackson laboratories and were housed for at least 2 weeks at the Dana-Farber Cancer Institute (DFCI) animal resources facility before beginning experiments.Statistical tests

[0154] Data are presented as mean ± SEM. Unpaired two-tailed / -test was used for comparing two groups. Analysis of variance (ANOVA) with Tukey’s correction or Kruskal- Wallis test with Dunn’s correction was used for comparisons amongst multiple groups, wherever applicable as per requirements of data and quantification. GraphPad Prism v8.0 / 9.0 (GraphPad Software Inc., San Diego, CA) was used to perform statistical analyses. Sample size was not predetermined.Results

[0155] The possible effects of salmeterol in mice undergoing sublethal hemolytic anemia were determined. To assess this, sublethal dose of phenylhydrazine (PHZ: 60 mg / kg) was first administered in 10-12 week old mice followed by daily i.p. injections of 0.3 mg / kg salmeterol or vehicle (0.3%DMSO in saline). PHZ is a strong oxidant that readily oxidizes hemoglobin in RBCs triggering their immediate lysis, thus causing hemolytic anemia.

[0156] Salmeterol treatment at a dose 0.3 mg / kg once daily did not significantly enhance hemoglobulin (Hb) production as compared to the vehicle-treated group (FIG. 1). These mice were sacrificed and their bone marrow (BM) progenitors were analyzed.

[0157] The effect of salmeterol treatment at a higher dose was then assessed. As shown in FIG. 2, at a dose of 0.6 mg / kg once daily salmeterol significantly increased Hb production relative to vehicle-treated anemic mice.

[0158] Salmeterol treatment consistently improved RBC parameters, such as Hb (FIG. 3A) and HCT% (FIG. 3B) in the anemic mice as compared to vehicle-treated controls. No significant impact was observed in platelets (FIG. 3C) or WBCs (FIG. 3D) in the salmeterol-treated groups, even at a dose of 0.6 mg / kg once daily. This dose of salmeterol increased erythroid progenitors in the BM of mice (FIG. 4A), as well as terminal erythroid progenitors (FIG. 4B), but did not significantly increase granulocyte monocyte progenitors (GMP) (FIG. 4C) or megakaryocytic progenitors (MkP) (FIG. 4D).

[0159] Taken together, these data support that salmeterol can have a dose-dependent role in erythroid differentiation and can alleviate anemia at moderate doses, but not low doses.

[0160] Example 2: Salmeterol alleviates anemia in MDS mouse modelThe present example analyzes the effects of salmeterol in an in vivo mouse model of myelodysplastic syndrome (MDS). MDS refers to a group of myeloid neoplasms that are characterized by ineffective hematopoiesis and abnormal cell morphology; anemia is a co-morbidity of MDS. MDS typically has one of three outcomes: patients can survive for an extended period of time with the disease, patients can die as a result of complications of severe cytopenia, or the disease can transform to an acute leukemia. Lin et al. Blood. 2005 Jul l;106(l):287-95, which is incorporated herein by reference in its entirety.

[0161] The effect of salmeterol was assessed in a mouse model of MDS having a NUP98-HOXD 13 fusion gene as described in Lin et al. (2005) supra. This mouse has been reported to faithfully recapitulate all of the key features of MDS, including peripheral blood cytopenias, bone marrow dysplasia, and apoptosis, and transformation to acute leukemia. Id. To assess the effects of salmeterol, an experimental plan as depicted in FIG. 5 was conducted. Specifically, ~ 8 month old anemic MDS model mice were administered 5 times per week i.p. injections of 0.6 mg / kg salmeterol or vehicle (0.3%DMSO in saline).Salmeterol treatment mildly, but significantly increased HCT% (FIG. 6) relative to vehicle- treated MDS model mice. Thus, salmeterol treatment can alleviate anemia in an in vivo MDS model.

[0162] This dose of salmeterol also showed a slight, but not statistically significant increase in erythroid progenitors in the BM of MDS mice (data not shown). A variable effect of salmeterol was observed for granulocyte monocyte progenitors (GMP) and megakaryocytic progenitors (MkP) at this dose (data not shown). Without wishing to be bound by theory, it is believed that higher doses of salmeterol may be needed to see a more pronounced effect on blood cell parameters in MDS model mice.

[0163] The effect of higher doses of salmeterol are to be assessed in the MDS mouse model. Stepwise doses of salmeterol increasing in 0.3 mg / kg intervals (ie., doses of 0.9 mg / kg salmeterol, 1.2 mg / kg salmeterol, or 1.5 mg / kg salmeterol) are to be administered to ~ 8 month old anemic MDS mice 5 times per week by i.p. injections, and results compared to vehicle treated MDS mice and littermate controls. Based on the results above, it is expected that salmeterol will significantly improve red blood cell parameters, including increase erythroid progenitors, at higher doses.Example 3: Salbutamol alleviates anemia in vivo

[0164] It was hypothesized that administration of salbutamol, a short-acting selective P2-AR agonist, might promote erythroid differentiation. The present example analyzes whether salbutamol alleviates anemia in an in vivo mouse model. The murine P2- adrenoreceptor has 87.08% similarity to the protein composition of the human analog. The ability of salbutamol to treat anemia was investigated in a phenylhydrazine-mediated hemolytic stress mouse model of anemia.Mouse experiments

[0165] C57BL / 6J mice were obtained from Jackson laboratories and were housed for at least 2 weeks at the Dana-Farber Cancer Institute (DFCI) animal resources facility before beginning experiments.Statistical tests

[0166] Data are presented as mean ± SEM. Unpaired two-tailed / -test was used for comparing two groups. Analysis of variance (ANOVA) with Tukey’s correction or Kruskal- Wallis test with Dunn’s correction was used for comparisons amongst multiple groups, wherever applicable as per requirements of data and quantification. GraphPad Prism v8.0 / 9.0 (GraphPad Software Inc., San Diego, CA) was used to perform statistical analyses. Sample size was not predetermined.Results

[0167] The possible effects of salbutamol in mice undergoing sublethal hemolytic anemia were determined. To assess this, an experimental plan as depicted in FIG. 7 was conducted. Specifically, a sublethal dose of phenylhydrazine (PHZ: 60 mg / kg) was first administered in 10-12 week old mice followed by daily i.p. injections of 0.3 mg / kg salbutamol or vehicle (0.3%DMSO in saline). PHZ is a strong oxidant that readily oxidizes hemoglobin in RBCs triggering their immediate lysis, thus causing hemolytic anemia.

[0168] Salbutamol treatment at a dose 0.3 mg / kg once daily did not significantly enhance hemoglobulin (Hb) production as compared to the vehicle-treated group (FIG. 8) and also did not significantly increase terminal erythroid progenitors in the BM of these mice (FIG. 9). Thus, salbutamol treatment at a low, once daily dose of 0.3 mg / kg was insufficient to alleviate anemia in this mouse model.

[0169] Given that salbutamol is a short-acting P2-AR agonist, the effect of salbutamol and salbutamol sulfate with increased frequency of dosing was then assessed, as outlined in FIG. 10. Specifically, 0.3 mg / kg salbutamol and salbutamol sulfate were each administeredi.p. twice daily. Twice daily salbutamol and salbutamol sulfate treatment both significantly increased Hb production (FIG. 11 A) and HCT% (FIG. 11B) relative to vehicle-treated anemic mice.

[0170] Salbutamol treatment and salbutamol sulfate treatment each consistently improved RBC parameters, such as Hb (FIG. 12A) and HCT% (FIG. 12B) in the anemic mice as compared to vehicle-treated controls. No significant impact was observed in platelets (FIG. 13A) or WBCs (FIG. 13B) in either of the salbutamol-treated groups, even at twice daily dosing. Twice daily dosing of salbutamol and salbutamol sulfate each increased erythroid progenitors in the BM of mice (FIG. 14A), as well as terminal erythroid progenitors (FIG. 14B), but did not significantly increase granulocyte monocyte progenitors (GMP) (FIG. 15A) or megakaryocytic progenitors (MkP) (FIG. 15B).

[0171] Taken together, these data support that salbutamol and salbutamol sulfate dosing of an appropriate frequency can promote erythroid differentiation and alleviate anemia.INCORPORATION BY REFERENCE

[0172] All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.EQUIVALENTS

[0173] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

CLAIMSWhat is claimed is:

1. A method of treating anemia in a patient in need thereof, the method comprising administering to the patient an effective amount of salmeterol or a pharmaceutically acceptable salt thereof.

2. The method of claim 1, wherein the anemia is selected from the group consisting of macrocytic anemia, hemolytic anemia, anemia caused by a ribosomopathy, anemia caused by insufficiency of serine / threonine-protein kinase RI0K2, anemia associated with chronic kidney disease (CKD), anemia caused by one or more mutations and / or deletions in human chromosome 5 or in an ortholog thereof, stress-induced anemia, Diamond Blackfan anemia, aplastic anemia, Schwachman-Diamond syndrome, an anemia associated with an inflammatory disease, such as rheumatoid arthritis or multiple sclerosis, anemia secondary to chemotherapy in cancer patients, and anemia associated with a bone marrow failure syndrome.

3. The method of claim 1 or claim 2, wherein the anemia is associated with a cancer, wherein the cancer is a hematologic malignancy selected from a myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) or multiple myeloma (MM).

4. The method of claim 1 or claim 2, wherein the anemia is associated with a cancer, wherein the cancer is an intestinal cancer.

5. A method of promoting differentiation of a hematopoietic stem and progenitor cell (HSPC) or a multipotent progenitor (MPP) cell toward an erythroid progenitor cell or an erythroid progenitor cell toward a mature red blood cell in a patient in need thereof, comprising administering to the patient in need thereof an effective amount of salmeterol or a pharmaceutically acceptable salt thereof.

6. The method of claim 5, wherein the patient is a human suffering from anemia.

7. The method of claim 6, wherein the anemia is selected from the group consisting of macrocytic anemia, hemolytic anemia, anemia caused by a ribosomopathy, anemia caused by insufficiency of serine / threonine-protein kinase RI0K2, anemia associated with chronic kidney disease (CKD), anemia caused by one or more mutations and / or deletions in human chromosome 5 or in an ortholog thereof, stress-induced anemia, Diamond Blackfan anemia, aplastic anemia, Schwachman-Diamond syndrome, an anemia associated with an inflammatory disease, such as rheumatoid arthritis or multiple sclerosis, anemia secondary to chemotherapy in cancer patients, and anemia associated with a bone marrow failure syndrome.

8. The method of claim 6 or claim 7, wherein the anemia is associated with a cancer, wherein the cancer is a hematologic malignancy, such as a myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) or multiple myeloma (MM).

9. The method of claim 6 or claim 7, wherein the anemia is associated with a cancer, wherein the cancer is an intestinal cancer, such as colorectal cancer.

10. The method of any one of claims 1 to 9, further comprising administering to the patient in need thereof an effective amount of an erythropoiesis-stimulating agent.

11. The method of claim 10, wherein the erythropoiesis-stimulating agent comprises erythropoietin, epoetin alfa, epoetin beta, epoetin omega, epoetin zeta, or darbepoetin alfa.

12. A method of treating anemia in a patient in need thereof, the method comprising administering to the patient an effective amount of salmeterol or a pharmaceutically acceptable salt thereof conjointly with an erythropoiesis-stimulating agent, wherein the anemia is refractory to the erythropoiesis-stimulating agent.

13. The method of claim 12, wherein the anemia is associated with a cancer, wherein the cancer is a hematologic malignancy, such as a myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) or multiple myeloma (MM).

14. The method of claim 12, wherein the anemia is associated with a cancer, wherein the cancer is an intestinal cancer, such as colorectal cancer.

15. The method of any one of claims 12 to 14, wherein the erythropoiesis-stimulating agent comprises erythropoietin, epoetin alfa, epoetin beta, epoetin omega, epoetin zeta, or darbepoetin alfa.

16. The method of any one of claims 1 to 15, wherein salmeterol or the pharmaceutically acceptable salt thereof is orally administered to the patient.

17. The method of any one of claims 1 to 16, wherein salmeterol or the pharmaceutically acceptable salt thereof is salmeterol xinafoate.

18. The method of any one of claims 1 to 17, wherein salmeterol or the pharmaceutically acceptable salt thereof is administered conjointly with luspatercept, lenalidomide, daprodustat, vadadustat, erythropoiesis-stimulating agents (ESAs) such as epoetin alfa or darbepoetin alfa, and / or a hypomethylating agent, wherein the hypomethylating agent is optionally azacitidine and / or decitabine.

19. The method of any one of claims 1 to 18, wherein the patient is a human that is 4 to 11 years of age.

20. The method of any one of claims 1 to 18, wherein the patient is a human that is at least 12 years of age.

Images