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

Abstract

The present invention provides a method of treating anemia in a patient in need thereof, comprising administering to the patient an effective amount of formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate). Formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) can be administered in combination with an erythropoiesis-stimulating agent, optionally wherein the anemia is refractory to the erythropoiesis-stimulating agent. The present invention further provides a method of promoting differentiation of erythroid progenitor cells into mature red blood cells in a patient in need thereof, comprising administering an effective amount of formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 422,210, filed November 3, 2022, U.S. Provisional Application No. 63 / 455,540, filed March 29, 2023, and U.S. Provisional Application No. 63 / 537,307, filed September 8, 2023, the disclosures of each of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Anemia affects approximately one-third of the world's population (Chaparro and Suchdev (2019). Ann. N.Y.Acad. Sci. 1450:15-31) and is a major comorbidity in a wide range of hematological disorders, including myelodysplastic syndromes (MDS), bone marrow failure (BMF), anemia in inflammatory diseases such as chronic kidney disease, ribosomal diseases, and leukemias such as acute myeloid leukemia (AML). For example, anemia is a major feature in approximately 80-90% of MDS patients, and its treatment remains a primary goal for designing novel interventions (Castelli et al. (2018) Med. Oncol. 35:76; Feld et al. (2020) Exp. Rev. Anticancer Ther. 20:465-482; Steensma (2018) Blood Cancer J. 8:47). Conventional erythropoiesis-stimulating agents are effective in only 50–60% of patients with low-risk MDS (Schiavon et al. (2018) Med. Oncol. 35:76; Park et al. (2019) Br. J. Haematol. 184:134–160).As a result, the majority of MDS patients eventually become transfusion-dependent and require treatment with hypomethylating agents (Cheng et al. (2021) Hematol. 26:261-270; Kordella et al. (2021) Front. Oncol. 11:650-473; Schiffer et al. (2021) Exp. 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 Approximately 25-30% of patients either become unresponsive to the few currently available FDA-approved drugs (e.g., Kubasch et al. (2021) Front. Oncol. 11:650-473; Kubasch et al. (2021) Blood Adv. 5:1565-1575; List et al. (2021) J. Clin. Oncol. 39:1001-1009) or progress to AML if they do not receive the only curative treatment, allogeneic bone marrow transplantation. Therefore, there is a critical need to identify additional novel therapies that promote erythroid differentiation to alleviate anemia in these diseases. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Chaparro and Suchdev(2019).Ann.NYAcad.Sci.1450:15-31 [Non-patent document 2] Castelli et al. (2018)Med.Oncol.35:76 [Non-patent document 3] Feld et al.(2020)Exp.Rev.Anticancer Ther.20:465-482 [Non-patent document 4] Steensma(2018)Blood Cancer J.8:47

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Summary of the Invention

Means for Solving the Problems

[0004] The inventions described herein are based in part on the discovery of a novel therapeutic approach that targets anemia (e.g., anemia in hematological malignancies such as MDS, cancer patients undergoing chemotherapy, and other diseases disclosed herein) by administering formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate (FF) or arformoterol tartrate (Arf)). It is shown herein that FF / Arf simultaneously enhances mitochondrial biogenesis and erythroid differentiation of primary human hematopoietic stem and progenitor cells (HSPCs). FF / Arf treatment significantly enhances erythropoiesis in bone marrow cells from MDS patients, thereby providing a potential therapeutic strategy for ameliorating erythroid differentiation defects in hematological malignancies such as acute myeloid leukemia and other diseases disclosed herein, such as bone marrow failure diseases, including, but not limited to, Diamond-Blackfan anemia and aplastic anemia. Reclaiming formoterol or its pharmaceutically acceptable salts (e.g., formoterol fumarate or arformoterol tartrate) to stimulate erythroid differentiation has significant therapeutic effects in the treatment of various anemia-related blood disorders, such as aplastic anemia, Diamond-Blackfan anemia, Shwachman-Diamond syndrome, MDS, anemia of inflammatory diseases such as chronic kidney disease, ribosomal diseases, hematological malignancies including, but not limited to, acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and multiple myeloma (MM), anemia secondary to chemotherapy in cancer patients, anemia secondary to intestinal cancer, as well as general anemia and age-related anemia. In some aspects, provided herein are methods of treating anemia in a patient in need thereof, the methods comprising administering to a patient in need thereof an effective amount of formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate).

[0005] In some embodiments, the effective amount of formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) is a dose of 100 μg or less. In some embodiments, the dose of formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) is administered daily (e.g., once daily). In some embodiments, the effective amount of formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) is a dose of 0.1 μg / day to 100 μg / day. In some embodiments, the effective amount of formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) is a dose of 1 μg / day to 60 μg / day.

[0006] In some embodiments, the effective amount of formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) is for a total daily dose of 100 μg or less. In some embodiments, the effective amount of formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) is for a total daily dose of 0.1 μg to 100 μg. In some preferred embodiments, the effective amount of formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) is for a total daily dose of 1 μg to 60 μg.

[0007] In some embodiments, the anemia is selected from the group consisting of macrocytic anemia, hemolytic anemia, anemia caused by ribosomal diseases, anemia caused by dysfunction of the 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 its homologous genes, anemia caused by chromosomal translocations in the NUP98 gene or its homologous genes (such as anemia caused by a fusion of NUP98 with an Abd-B group HOX gene (e.g., HOXD13)), stress-induced anemia, anemia secondary to intestinal cancer, anemia associated with inflammatory diseases such as Diamond-Blackfan anemia, aplastic anemia, Shwachman-Diamond syndrome, rheumatoid arthritis, or multiple sclerosis, anemia secondary to chemotherapy in cancer patients, and anemia associated with bone marrow failure syndromes. In some embodiments, the anemia is associated with cancer, optionally wherein the cancer is a hematological malignancy such as myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), or multiple myeloma (MM). In some embodiments, the anemia is associated with intestinal cancer, such as colon cancer.

[0008] In some aspects, provided herein are methods for promoting differentiation of erythroid progenitor cells into mature erythrocytes in a patient by administering an effective amount of formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate). In some embodiments, the effective amount of formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) is administered at a dose of 100 μg or less. In some embodiments, the dose of formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) is administered daily (e.g., once daily). In some embodiments, the effective amount of formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) is administered at a dose of 0.1 μg / day to 100 μg / day. In some embodiments, the effective amount of formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) is in a dosage of 1 μg / day to 60 μg / day.

[0009] In some embodiments, the effective amount of formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) is for a total daily dose of 100 μg or less. In some embodiments, the effective amount of formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) is for a total daily dose of 0.1 μg to 100 μg. In some preferred embodiments, the effective amount of formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) is for a total daily dose of 1 μg to 60 μg.

[0010] In some embodiments, formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) may be orally administered to the patient. In some embodiments, the patient is a human suffering from anemia. In some embodiments, the method may further comprise administering (e.g., co-administering) to the patient in need thereof an effective amount of an erythropoiesis-stimulating agent (ESA) or other FDA-approved agent, such as luspatercept, lenalidomide, and / or a hypomethylating agent, including, but not limited to, epoetin alfa or darbepoetin alfa, azacitidine, or decitabine. In some embodiments, the erythropoiesis-stimulating agent comprises erythropoietin, epoetin alfa, epoetin beta, epoetin omega, epoetin zeta, or darbepoetin alfa.

[0011] In some aspects, 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 formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) in combination 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, formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) can be administered in combination with other FDA-approved agents, such as luspatercept, lenalidomide, and / or hypomethylating agents, such as azacitidine or decitabine. The anemia is selected from the group consisting of macrocytic anemia, hemolytic anemia, anemia caused by ribosomal diseases, anemia caused by dysfunction of the 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 its homologous genes, anemia caused by chromosomal translocations in the NUP98 gene or its homologous genes (such as anemia caused by a fusion of NUP98 with an Abd-B group HOX gene (e.g., HOXD13)), stress-induced anemia, anemia secondary to intestinal cancer, anemia associated with inflammatory diseases such as Diamond-Blackfan anemia, aplastic anemia, Shwachman-Diamond syndrome, rheumatoid arthritis, or multiple sclerosis, anemia secondary to chemotherapy in cancer patients, and anemia associated with bone marrow failure syndromes.

[0012] In some embodiments, the patient is a patient who would benefit from weight gain. In some embodiments, the patient is a patient who would benefit from increased bone density. In some embodiments, the patient is a patient who would benefit from increased muscle mass. In some embodiments, the patient is suffering from anemia associated with weight loss, decreased bone density, and / or muscle wasting. In some embodiments, the anemia associated with weight loss, decreased bone density, and / or muscle wasting is a cancer-associated anemia disclosed herein.

[0013] In some embodiments, methods are provided for treating anemia and increasing the weight of a subject in need thereof. In some embodiments, the patient is suffering from anemia associated with weight loss (e.g., cancer-associated anemia as disclosed herein).

[0014] In some embodiments, methods are provided for treating anemia and increasing bone density in a subject in need thereof. In some embodiments, the patient is suffering from anemia associated with decreased bone density (e.g., cancer-associated anemia as disclosed herein).

[0015] In some embodiments, methods are provided for treating anemia and increasing muscle mass in a subject in need thereof. In some embodiments, the patient is suffering from anemia associated with loss of muscle mass (e.g., cancer-associated anemia as disclosed herein).

[0016] In some embodiments, the anemia is associated with cancer. In some embodiments, the cancer is a hematological malignancy such as myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), or multiple myeloma (MM). In some embodiments, the anemia is associated with intestinal cancer, such as colon cancer.

[0017] The drawings contained herein, which comprise the following figures, are for illustration purposes only and not for limitation: [Brief explanation of the drawings]

[0018] [Figure 1A] Formoterol fumarate induces erythropoiesis. Administration of formoterol fumarate (FF) to primary human HSPCs dose-dependently enhances erythroid differentiation (CD235a). n=3 HSPCs from healthy donors. **p<0.01, ***p<0.001, one-way analysis of variance (ANOVA). When statistical significance is not indicated, "Ns" (not significant) is applied. Data are expressed as mean ± SEM. All comparisons were made relative to the control (0 μM: vehicle (DMSO) treatment). [Figure 1B] Formoterol fumarate induces erythropoiesis. Administration of formoterol fumarate (FF) to primary human HSPCs does not affect viability. n=3 healthy donor-derived HSPCs. **p<0.01, ***p<0.001, one-way analysis of variance (ANOVA). When statistical significance is not indicated, "Ns" (not significant) is applied. Data are expressed as mean ± SEM. All comparisons were made relative to the control (0 μM: vehicle (DMSO) treatment). [Figure 1C] Formoterol fumarate induces erythropoiesis. Administration of formoterol fumarate (FF) to primary human HSPCs does not affect megakaryocytes (CD41-61). n=3 HSPCs from healthy donors. **p<0.01, ***p<0.001, one-way analysis of variance (ANOVA). When statistical significance is not indicated, "Ns" (not significant) is applied. Data are expressed as mean ± SEM. All comparisons were made relative to the control (0 μM: vehicle (DMSO) treatment). [Figure 1D] Formoterol fumarate induces erythropoiesis. Administration of formoterol fumarate (FF) to primary human HSPCs does not affect myeloid (CD11b) differentiation (Figure 1D). n=3 healthy donor-derived HSPCs. **p<0.01, ***p<0.001, one-way analysis of variance (ANOVA). When statistical significance is not indicated, "Ns" (not significant) is applied. Data are expressed as mean ± SEM. All comparisons were made relative to the control (0 μM: vehicle (DMSO) treatment). [Figure 2] Formoterol fumarate (FF) and arformoterol tartrate (Arf) induce erythropoiesis. Administration of 1 / 5 μM formoterol fumarate (FF) or arformoterol tartrate (Arf) to primary human HSPCs dose-dependently induces erythroid differentiation (CD235a). Representative of HSPCs from n=5 healthy donors, n=4 within each donor with four technical replicates. **p<0.01, ***p<0.001, ****p<0.0001, one-way analysis of variance (ANOVA). Data are presented as mean ± SEM. All comparisons were made relative to control (DMSO treatment). ns=not significant. [Figure 3A] Figure 1 shows that formoterol fumarate increases erythropoiesis. Administration of formoterol fumarate (FF) from Selleckchem enhances erythroid differentiation in primary human HSPCs isolated from n=4 healthy donors. **p<0.01, ***p<0.001, one-way analysis of variance (ANOVA). When statistical significance is not indicated, "Ns" (not significant) is applied. Data are expressed as mean ± SEM. All comparisons were made relative to the control (0 μM: vehicle (DMSO) treatment). [Figure 3B] Figure 1 shows that formoterol fumarate increases erythropoiesis. Administration of formoterol fumarate (FF) from Sigma enhances erythroid differentiation in primary human HSPCs isolated from n=4 healthy donors. **p<0.01, ***p<0.001, one-way analysis of variance (ANOVA). When statistical significance is not indicated, "Ns" (not significant) is applied. Data are expressed as mean ± SEM. All comparisons were made relative to the control (0 μM: vehicle (DMSO) treatment). [Figure 4]Treatment of primary human HSPCs with formoterol fumarate (FF) enhances the formation of red blood cell (RBC) precursors, burst-forming units-erythroid (BFU-E) and colony-forming units-erythroid (CFU-E). n=4 HSPCs from healthy donors. *p<0.05, Student's t-test. Data are presented as mean ± SEM. All comparisons were made against the control, i.e., vehicle (DMSO treatment). [Figure 5A] Formoterol fumarate (FF) enhances mitochondrial function. Treatment of primary human HSPCs with formoterol fumarate (FF) enhances the expression of mitochondrial DNA-encoded genes MT-ND1, MT-CytB, MT-CO1, and MT-ATP6. *p<0.05, **p<0.01, one-way analysis of variance (ANOVA) / Student's t-test. When statistical significance is not indicated, "Ns" (not significant) is applied. Data are expressed as mean ± SEM. All comparisons were made relative to the control (0 μM / vehicle (DMSO) treatment). [Figure 5B] Formoterol fumarate (FF) enhances mitochondrial function. Treatment of primary human HSPCs with formoterol fumarate (FF) enhances mitochondrial membrane potential (TMRE staining). *p<0.05, **p<0.01, one-way analysis of variance (ANOVA) / Student's t-test. When statistical significance is not indicated, "Ns" (not significant) is applied. Data are expressed as mean ± SEM. All comparisons were made relative to control (0 μM / vehicle (DMSO) treatment). [Figure 5C]Figure 1 shows that formoterol fumarate (FF) enhances mitochondrial function. FF treatment increases mitochondrial mass in HSPCs (MitoTracker® staining). *p<0.05, **p<0.01, one-way analysis of variance (ANOVA) / Student's t-test. When statistical significance is not indicated, "Ns" (not significant) is applied. Data are expressed as mean ± SEM. All comparisons were made relative to the control (0 μM / vehicle (DMSO) treatment). [Figure 6A] Figure 1 shows that formoterol fumarate (FF) stimulates erythroid differentiation of RIOK2 knockdown (KD) and RPS14 KD HSPCs, but not ADRB2-deficient HSPCs. Figure 2 shows that intraperitoneal (ip) injection of formoterol fumarate (FF) increases erythroid differentiation (CD235a) of RIOK2 KD HSPCs compared to vehicle (DMSO) treatment. *p<0.05, **p<0.01, ***p<0.001, one-way analysis of variance (ANOVA). Ns: not significant. Data are presented as mean ± SEM. All comparisons were made relative to vehicle (DMSO) treatment. [Figure 6B] Figure 1 shows that formoterol fumarate (FF) stimulates erythroid differentiation of RIOK2 knockdown (KD) and RPS14 KD HSPCs, but not ADRB2-deficient HSPCs. Figure 2 shows that intraperitoneal (ip) injection of formoterol fumarate (FF) increases erythroid differentiation (CD235a) of RIOK2 KD HSPCs compared to vehicle (DMSO) treatment. *p<0.05, **p<0.01, ***p<0.001, one-way analysis of variance (ANOVA). Ns: not significant. Data are presented as mean ± SEM. All comparisons were made relative to vehicle (DMSO) treatment. [Figure 6C]Figure 1 shows that formoterol fumarate (FF) stimulates erythroid differentiation of RIOK2 knockdown (KD) and RPS14 KD HSPCs, but not ADRB2-deficient HSPCs. Intraperitoneal injection of FF increases erythroid differentiation (CD235a) in RPS14 KD HSPCs compared to vehicle (DMSO) treatment. *p<0.05, **p<0.01, ***p<0.001, one-way analysis of variance (ANOVA). Ns: not significant. Data are presented as mean ± SEM. All comparisons were made relative to vehicle (DMSO) treatment. [Figure 6D] Figure 1 shows that formoterol fumarate (FF) stimulates erythroid differentiation of RIOK2 knockdown (KD) and RPS14 KD HSPCs, but not ADRB2-deficient HSPCs. Intraperitoneal injection of FF increases erythroid differentiation (CD235a) in RPS14 KD HSPCs compared to vehicle (DMSO) treatment. *p<0.05, **p<0.01, ***p<0.001, one-way analysis of variance (ANOVA). Ns: not significant. Data are presented as mean ± SEM. All comparisons were made relative to vehicle (DMSO) treatment. [Figure 6E] Figure 1 shows that formoterol fumarate (FF) stimulates erythroid differentiation of RIOK2 knockdown (KD) and RPS14 KD HSPCs, but not ADRB2-deficient HSPCs. The absence of the ADRB2 gene, which encodes the β2-AR, indicates that FF is unable to induce erythroid differentiation. *p<0.05, **p<0.01, ***p<0.001, one-way analysis of variance (ANOVA). Ns: not significant. Data are presented as mean ± SEM. All comparisons were made against vehicle (DMSO) treatment. [Figure 6F]Figure 1 shows that formoterol fumarate (FF) stimulates erythroid differentiation of RIOK2 knockdown (KD) and RPS14 KD HSPCs, but not ADRB2-deficient HSPCs. The absence of the ADRB2 gene, which encodes the β2-AR, indicates that FF is unable to induce erythroid differentiation. *p<0.05, **p<0.01, ***p<0.001, one-way analysis of variance (ANOVA). Ns: not significant. Data are presented as mean ± SEM. All comparisons were made against vehicle (DMSO) treatment. [Figure 7] An exemplary workflow for studying the effects of formoterol fumarate (FF) on MDS patient-derived cells using liquid culture is shown. [Figure 8A] These results demonstrate that formoterol fumarate (FF) enhances erythroid differentiation in bone marrow cells from MDS patients. Erythroid differentiation (CD235a) in bone marrow cells from MDS patients treated with vehicle (DMSO) and FF (Selleck). n=40 de-identified MDS patients. Vehicle: DMSO, FF: formoterol fumarate. ****p<0.0001, non-parametric Wilcoxon paired signed-rank test. All comparisons were made against vehicle (DMSO)-treated controls. [Figure 8B] These results show that formoterol fumarate (FF) enhances erythroid differentiation in bone marrow cells from MDS patients. Erythroid differentiation (CD235a) in bone marrow cells from MDS patients treated with vehicle (DMSO) and FF (Sigma). n=40 de-identified MDS patients. Vehicle: DMSO, FF: formoterol fumarate. ****p<0.0001, non-parametric Wilcoxon paired signed-rank test. All comparisons were made against vehicle (DMSO)-treated controls. [Figure 9] 1 shows the results of a liquid culture (ex vivo) experiment demonstrating that formoterol fumarate (FF) does not affect myelopoiesis / megakaryopoiesis in 40 MDS patient-derived cells. [Figure 10] An exemplary workflow for studying the effects of formoterol fumarate (FF) on hematopoietic progenitor cells isolated from MDS patients is shown. [Figure 11A] Figure 1 shows that formoterol fumarate (FF) enhances the differentiation of erythroid progenitors in bone marrow cells from MDS patients. Photographs show an increase in burst-forming units-erythroid (BFU-E) in MDS bone marrow (BM) cells treated with Selleckchem FF and Sigma FF compared to vehicle-treated BM cells. **p<0.01, ****p<0.0001, nonparametric Wilcoxon paired signed-rank test. Ns: not significant. All comparisons were made against vehicle (DMSO)-treated controls. [Figure 11B] This shows that formoterol fumarate (FF) enhances the differentiation of erythroid progenitor cells in bone marrow cells from MDS patients. The numbers of erythroid progenitor cells BFU-E and CFU-E (colony-forming units erythroid) in MDS bone marrow cells treated with vehicle (DMSO) and FF are shown, n=23 MDS patients. Vehicle: DMSO, FF: formoterol fumarate. **p<0.01, ****p<0.0001, nonparametric Wilcoxon paired signed-rank test. Ns: not significant. All comparisons were made against vehicle (DMSO)-treated controls. [Figure 11C] This shows that formoterol fumarate (FF) enhances the differentiation of erythroid progenitor cells in bone marrow cells from MDS patients. The numbers of erythroid progenitor cells BFU-E and CFU-E (colony-forming units erythroid) in MDS bone marrow cells treated with vehicle (DMSO) and FF are shown, n=23 MDS patients. Vehicle: DMSO, FF: formoterol fumarate. **p<0.01, ****p<0.0001, nonparametric Wilcoxon paired signed-rank test. Ns: not significant. All comparisons were made against vehicle (DMSO)-treated controls. [Figure 11D]These results show that formoterol fumarate (FF) enhances the differentiation of erythroid progenitor cells in bone marrow cells from MDS patients. FF treatment does not affect the viability, myelopoiesis (CFU-GM), or megakaryopoiesis (CFU-Mk) of bone marrow cells from MDS patients. **p<0.01, ****p<0.0001, nonparametric Wilcoxon paired signed-rank test. Ns: not significant. All comparisons were made against vehicle (DMSO)-treated controls. [Figure 12] This shows that β2-adrenergic receptor agonists and antagonists do not affect megakaryocytopoiesis. Treatment of primary human HSPCs with 1 / 5 μM formoterol fumarate (FF) or arformoterol tartrate (Arf) does not affect megakaryocytic differentiation (CD41 / 61). Representative of HSPCs from n=5 healthy donors, n=4 from four technical replicates within each donor. One-way analysis of variance (ANOVA). Data are presented as mean ± SEM. All comparisons were made against control (DMSO) treatment. ns=not significant. [Figure 13] 1 shows an exemplary workflow for a pilot study on formoterol fumarate (FF) treatment in vivo. [Figure 14A] Figure 1 shows that formoterol fumarate (FF) treatment mildly increases RBC parameters in wild-type mice at steady state. Graphs showing the % change in mouse body weight with vehicle or FF treatment at doses of 0.1 / 0.3 / 0.5 / 1.0 mg / kg are shown. *p<0.05, **p<0.01, ANOVA. When statistical significance is not indicated, "Ns" (not significant) is applied. n=3 mice per group. All comparisons were made against vehicle-treated (DMSO) controls. [Figure 14B]Formoterol fumarate (FF) treatment mildly increases RBC parameters in wild-type mice at steady state. After 14 days of daily intraperitoneal (ip) injection, FF administration enhances mitochondrial biogenesis, as observed by MitoTracker® staining of peripheral blood mononuclear cells (PBMCs). *p<0.05, **p<0.01, ANOVA. When statistical significance is not indicated, "Ns" (not significant) is applied. n=3 mice per group. All comparisons were made against vehicle-treated (DMSO) controls. [Figure 14C] Formoterol fumarate (FF) treatment mildly increases RBC parameters in wild-type mice at steady state. After 14 days of daily intraperitoneal injection, FF treatment mildly increases RBC parameters such as RBC count, hematocrit (HCT) % and hemoglobin (Hb) in peripheral blood. *p<0.05, **p<0.01, ANOVA. When statistical significance is not indicated, "Ns" (not significant) is applied. n=3 mice per group. All comparisons were made against vehicle-treated (DMSO) controls. [Figure 14D] Formoterol fumarate (FF) treatment mildly increases RBC parameters in wild-type mice at steady state. After 14 days of daily intraperitoneal injection, FF treatment mildly increases RBC parameters such as RBC count, hematocrit (HCT) % and hemoglobin (Hb) in peripheral blood. *p<0.05, **p<0.01, ANOVA. When statistical significance is not indicated, "Ns" (not significant) is applied. n=3 mice per group. All comparisons were made against vehicle-treated (DMSO) controls. [Figure 14E]Formoterol fumarate (FF) treatment mildly increases RBC parameters in wild-type mice at steady state. After 14 days of daily intraperitoneal injection, FF treatment mildly increases RBC parameters such as RBC count, hematocrit (HCT) % and hemoglobin (Hb) in peripheral blood. *p<0.05, **p<0.01, ANOVA. When statistical significance is not indicated, "Ns" (not significant) is applied. n=3 mice per group. All comparisons were made against vehicle-treated (DMSO) controls. [Figure 14F] Figure 1 shows that formoterol fumarate (FF) treatment mildly increases RBC parameters in wild-type mice at steady state. FF treatment does not affect white blood cell (WBC) or monocyte counts in peripheral blood 14 days after daily intraperitoneal injection. *p<0.05, **p<0.01, ANOVA. When statistical significance is not indicated, "Ns" (not significant) is applied. n=3 mice per group. All comparisons were made against vehicle-treated (DMSO) controls. [Figure 14G] Figure 1 shows that formoterol fumarate (FF) treatment mildly increases RBC parameters in wild-type mice at steady state. FF treatment does not affect white blood cell (WBC) or monocyte counts in peripheral blood 14 days after daily intraperitoneal injection. *p<0.05, **p<0.01, ANOVA. When statistical significance is not indicated, "Ns" (not significant) is applied. n=3 mice per group. All comparisons were made against vehicle-treated (DMSO) controls. [Figure 15] Figure 1 shows the results of complete blood count (CBC) on day 8 of formoterol fumarate (FF) treatment by intraperitoneal injection in wild-type C57BL / 6J mice. If statistical significance is not indicated, "Ns" (not significant) is applied. [Figure 16] Figure 1 shows reticulocyte counts on day 8 after daily intraperitoneal injection of formoterol fumarate (FF) treatment in wild-type C57BL / 6J mice. *p<0.05, ANOVA. If statistical significance is not indicated, "Ns" (not significant) is applied. [Figure 17] 1 shows an exemplary workflow for phenylhydrazine (PHZ) studies and in vivo formoterol fumarate (FF) treatment with sublethal doses (50 mg / kg). [Figure 18A] Formoterol fumarate (FF) treatment significantly increased RBC parameters in mice (10-12 weeks old) with phenylhydrazine (PHZ)-mediated stress-induced anemia. FF treatment at doses of 0.1 / 0.3 mg / kg increased absolute body weight and % change in body weight in mice after sublethal PHZ-induced hemolytic anemia. PHZ dose: 50 mg / kg. *p<0.05, **p<0.01, ****p<0.0001, one-way ANOVA and two-way ANOVA. When statistical significance was not indicated, "Ns" (not significant) was applied. n=4 female mice per group. All comparisons were made relative to vehicle (DMSO)-treated controls. [Figure 18B] Formoterol fumarate (FF) treatment significantly increased RBC parameters in mice (10-12 weeks old) with phenylhydrazine (PHZ)-mediated stress-induced anemia. FF administration enhanced and maintained peripheral blood RBC parameters, including hemoglobin (Hb), hematocrit (HCT) % and RBC count, after PHZ-induced hemolytic anemia. *p<0.05, **p<0.01, ****p<0.0001, one-way ANOVA and two-way ANOVA. When statistical significance was not indicated, "Ns" (not significant) was applied. n=4 female mice per group. All comparisons were made relative to vehicle (DMSO)-treated controls. [Figure 18C]Formoterol fumarate (FF) treatment significantly increased RBC parameters in mice (10-12 weeks old) with phenylhydrazine (PHZ)-mediated stress-induced anemia. FF treatment increased WBC and monocyte counts in peripheral blood after 4 days of daily intraperitoneal injection, but the effect did not persist over 14 days. Platelet counts were not statistically significant. *p<0.05, **p<0.01, ****p<0.0001, one-way ANOVA and two-way ANOVA. When statistical significance was not indicated, "Ns" (not significant) was applied. n=4 female mice per group. All comparisons were made relative to vehicle (DMSO)-treated controls. [Figure 19A] Formoterol fumarate (FF) treatment significantly enhanced erythroid differentiation in the bone marrow (BM) of mice (10-12 weeks old) with phenylhydrazine (PHZ)-mediated stress-induced anemia. Flow plots show that FF treatment at doses of 0.1 / 0.3 mg / kg significantly increased RI, RII, RIII, and RIV erythroid progenitors in the BM of mice after sublethal PHZ-induced hemolytic anemia. PHZ dose: 50 mg / kg. *p<0.05, **p<0.01, ***p<0.001, ANOVA. n=4 female mice per group. All comparisons were made relative to the vehicle (DMSO) control. [Figure 19B] Figure 19 shows that formoterol fumarate (FF) treatment significantly enhances erythroid differentiation in the bone marrow (BM) of mice (10-12 weeks old) with phenylhydrazine (PHZ)-mediated stress-induced anemia. Quantification of data shown in Figure 19A shows the absolute numbers of RI, RII, RIII, and RIV erythroid progenitors per million BM cells in mice after sublethal PHZ-induced hemolytic anemia. *p<0.05, **p<0.01, ***p<0.001, ANOVA. n=4 female mice per group. All comparisons were made against vehicle (DMSO) control. [Figure 20] 1 shows an exemplary workflow for phenylhydrazine (PHZ) studies and in vivo formoterol fumarate (FF) treatment with sublethal doses (60 mg / kg). [Figure 21] Figure 1 shows that formoterol fumarate (FF) treatment increases MitoTracker® staining, indicative of mitochondrial mass, in peripheral blood mononuclear cells (PBMCs) 4 days after PHZ treatment. [Figure 22A] Formoterol fumarate (FF) treatment significantly increased RBC parameters in mice (10-12 weeks old) with phenylhydrazine (PHZ)-mediated stress-induced anemia. FF treatment at doses of 0.1 / 0.3 mg / kg increased absolute body weight and % change in body weight in male mice after sublethal PHZ-induced hemolytic anemia. PHZ dose: 60 mg / kg. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, two-way ANOVA. When statistical significance is not indicated, "Ns" (not significant) is applied. n=5 male mice per group. All comparisons were made relative to vehicle (DMSO)-treated controls. [Figure 22B] Formoterol fumarate (FF) treatment significantly increased RBC parameters in mice (10-12 weeks old) with phenylhydrazine (PHZ)-mediated stress-induced anemia. FF administration significantly enhanced and maintained peripheral blood RBC parameters, including hemoglobin (Hb), hematocrit (HCT) % and RBC count, after sublethal PHZ-induced hemolytic anemia. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, two-way ANOVA. When statistical significance was not indicated, "Ns" (not significant) was applied. n=5 male mice per group. All comparisons were made relative to vehicle (DMSO)-treated controls. [Figure 22C]Formoterol fumarate (FF) treatment significantly increased RBC parameters in mice (10-12 weeks old) with phenylhydrazine (PHZ)-mediated stress-induced anemia. FF treatment increased WBC and monocyte counts in peripheral blood after 4 days of daily intraperitoneal injection, but the effect did not persist over 14 days. Platelet counts were not statistically significant. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, two-way ANOVA. When statistical significance is not indicated, "Ns" (not significant) is applied. n=5 male mice per group. All comparisons were made relative to vehicle (DMSO)-treated controls. [Figure 23A] Formoterol fumarate (FF) treatment significantly enhanced BM progenitor cell viability and mitochondrial biogenesis in mice (10-12 weeks old) with phenylhydrazine (PHZ)-mediated stress-induced anemia. FF treatment at doses of 0.1 / 0.3 mg / kg significantly increased BM cell viability in mice after sublethal PHZ-induced hemolytic anemia (PHZ dose: 60 mg / kg). *p<0.05, ****p<0.0001, ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO)-treated controls. [Figure 23B] Formoterol fumarate (FF) treatment significantly enhanced BM progenitor cell viability and mitochondrial biogenesis in mice (10-12 week old) with phenylhydrazine (PHZ)-mediated stress-induced anemia. FF treatment enhanced mitochondrial biogenesis as observed by MitoTracker® staining in BM cells after PHZ-induced hemolytic anemia. *p<0.05, ****p<0.0001, ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO)-treated controls. [Figure 23C]Formoterol fumarate (FF) treatment significantly enhanced BM progenitor cell viability and mitochondrial biogenesis in mice (10-12 week old) with phenylhydrazine (PHZ)-mediated stress-induced anemia. FF administration significantly reduced mitochondrial superoxide production, demonstrating mitochondrial fitness as observed by MitoSox® staining in BM cells after PHZ-induced hemolytic anemia. *p<0.05, ****p<0.0001, ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO)-treated controls. [Figure 24] Flow cytometry plots for studying myeloid progenitor cells (BMPs) are shown. [Figure 25A] Figure 1 shows that formoterol fumarate (FF) enhances LS-K cells in bone marrow (BM). *p<0.05, **p<0.01, ANOVA. Ns: not significant. [Figure 25B] Figure 1 shows that formoterol fumarate (FF) enhances LS-K cells in bone marrow (BM). *p<0.05, **p<0.01, ANOVA. Ns: not significant. [Figure 26A] Formoterol fumarate (FF) treatment significantly increased megakaryocytic erythroid progenitors (MEPs), but not common myeloid progenitors (CMPs) or granulocytic monocytic progenitors (GMPs), in the BM of mice (10-12 weeks old) with phenylhydrazine (PHZ)-mediated stress-induced anemia. Flow plots show that FF treatment at doses of 0.1 / 0.3 mg / kg significantly increased MEPs in the BM of mice after PHZ-induced sublethal hemolytic anemia. CMPs and GMPs were unaffected. LS-K: lineage-Sca1-cKit+ BM cells. PHZ dose: 60 mg / kg. **p<0.01, ***p<0.001, ANOVA. Ns: not significant. n=5 male mice per group. All comparisons were made relative to vehicle (DMSO-treated) controls. [Figure 26B]Figure 26 shows that formoterol fumarate (FF) treatment significantly increased megakaryocytic erythroid progenitors (MEPs), but not common myeloid progenitors (CMPs) or granulocytic monocytic progenitors (GMPs), in the BM of mice (10-12 week-old mice) with phenylhydrazine (PHZ)-mediated stress-induced anemia. Quantification of the data shown in Figure 26A, which shows the absolute numbers of CMP, GMP, and MEP progenitors per million BM cells in mice after PHZ-induced hemolytic anemia, is shown. **p<0.01, ***p<0.001, ANOVA. Ns: not significant. n=5 male mice per group. All comparisons were made against vehicle (DMSO-treated) controls. [Figure 26C] Figure 26 shows that formoterol fumarate (FF) treatment significantly increased megakaryocytic erythroid progenitors (MEPs), but not common myeloid progenitors (CMPs) or granulocytic monocytic progenitors (GMPs), in the BM of mice (10-12 week-old mice) with phenylhydrazine (PHZ)-mediated stress-induced anemia. Quantification of the data shown in Figure 26A, which shows the absolute numbers of CMP, GMP, and MEP progenitors per million BM cells in mice after PHZ-induced hemolytic anemia, is shown. **p<0.01, ***p<0.001, ANOVA. Ns: not significant. n=5 male mice per group. All comparisons were made against vehicle (DMSO-treated) controls. [Figure 26D] Figure 26 shows that formoterol fumarate (FF) treatment significantly increased megakaryocytic erythroid progenitors (MEPs), but not common myeloid progenitors (CMPs) or granulocytic monocytic progenitors (GMPs), in the BM of mice (10-12 week-old mice) with phenylhydrazine (PHZ)-mediated stress-induced anemia. Quantification of the data shown in Figure 26A, which shows the absolute numbers of CMP, GMP, and MEP progenitors per million BM cells in mice after PHZ-induced hemolytic anemia, is shown. **p<0.01, ***p<0.001, ANOVA. Ns: not significant. n=5 male mice per group. All comparisons were made against vehicle (DMSO-treated) controls. [Figure 27]Formoterol fumarate (FF) does not affect megakaryocytic progenitor cells in the BM of mice with phenylhydrazine (PHZ)-mediated stress-induced anemia (10-12 week old mice). Flow plots show that FF treatment at doses of 0.1 / 0.3 mg / kg does not affect megakaryocytic progenitor cells (MKPs) in the BM of mice after PHZ-induced sublethal hemolytic anemia. LS-K: Lineage-Sca1-cKit+ BM cells. PHZ dose: 60 mg / kg. n = 5 male mice per group. [Figure 28A] Formoterol fumarate (FF) selectively enriches pre-CFU-E cells in the BM. *p<0.05, ***p<0.001, ANOVA. [Figure 28B] Formoterol fumarate (FF) selectively enriches pre-CFU-E cells in the BM. *p<0.05, ***p<0.001, ANOVA. [Figure 29A] We show that formoterol fumarate (FF) selectively enriches proerythroblasts in the BM. [Figure 29B] We show that formoterol fumarate (FF) selectively enriches proerythroblasts in the BM. [Figure 30A] Formoterol fumarate (FF) treatment significantly enhanced erythroid differentiation in the BM of mice (10-12 weeks old) with phenylhydrazine (PHZ)-mediated stress-induced anemia. Flow plots show that FF treatment at doses of 0.1 / 0.3 mg / kg significantly increased RI, RII, RIII, and RIV erythroid progenitors in the BM of mice after PHZ-induced hemolytic anemia. PHZ dose: 60 mg / kg. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ANOVA. n=5 male mice per group. All comparisons were made relative to vehicle (DMSO)-treated controls. [Figure 30B]Figure 3 shows that formoterol fumarate (FF) treatment significantly enhances erythroid differentiation in the BM of mice (10-12 weeks old) with phenylhydrazine (PHZ)-mediated stress-induced anemia. Quantification of data shown in Figure 30A shows the absolute numbers of RI, RII, RIII, and RIV erythroid progenitors per million BM cells in mice after PHZ-induced hemolytic anemia. *p<0.05, **p<0.01, ***P<0.001, ****P<0.0001, ANOVA. n=5 male mice per group. All comparisons were made to vehicle (DMSO)-treated controls. [Figure 31] Schematic diagram of intraperitoneal FF / Arf treatment in phenylhydrazine (PHZ)-treated mice at a sublethal dose of 60 mg / kg. [Figure 32] Intraperitoneal FF / Arf treatment increases body weight in mice treated with a sublethal dose (60 mg / kg) of phenylhydrazine (PHZ). ns = not significant. [Figure 33A] Figure 1 shows that intraperitoneal FF / Arf treatment does not affect WBC parameters in mice treated with a sublethal dose (60 mg / kg) of phenylhydrazine (PHZ). FF / Arf treatment does not affect platelets in peripheral blood after intraperitoneal administration. ***p<0.001, ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO)-treated controls. Ns=not significant. [Figure 33B] Figure 1 shows that intraperitoneal FF / Arf treatment does not affect WBC parameters in mice treated with a sublethal dose (60 mg / kg) of phenylhydrazine (PHZ). FF / Arf treatment does not affect WBC in peripheral blood after intraperitoneal administration. ***p<0.001, ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO)-treated controls. Ns=not significant. [Figure 34A]Intraperitoneal FF / Arf treatment mildly increases RBC parameters in mice treated with a sublethal dose (60 mg / kg) of phenylhydrazine (PHZ). *p<0.05, ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO)-treated controls. Ns=not significant. [Figure 34B] Figure 1 shows that intraperitoneal FF / Arf treatment mildly increases RBC parameters in mice treated with a sublethal dose (60 mg / kg) of phenylhydrazine (PHZ). FF / Arf treatment increases RBC parameters such as hematocrit (HCT) % and RBC in peripheral blood 7 days after intraperitoneal administration. *p<0.05, ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO)-treated controls. NS=not significant. [Figure 35A] Figure 1 shows that intraperitoneal FF / Arf treatment increases RBC parameters in mice treated with a sublethal dose (60 mg / kg) of phenylhydrazine (PHZ). FF and Arf treatment increases hemoglobin (Hb) in peripheral blood 7 days after intraperitoneal administration. *p<0.05, ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO)-treated controls. Ns=not significant. [Figure 35B] Figure 1 shows that intraperitoneal FF / Arf treatment increases RBC parameters in mice treated with a sublethal dose (60 mg / kg) of phenylhydrazine (PHZ). FF and Arf treatment increases hemoglobin (Hb) in peripheral blood 14 days after intraperitoneal administration. *p<0.05, ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO)-treated controls. Ns=not significant. [Figure 36A]Figure 1 shows that intraperitoneal FF / Arf treatment increases LSK, LS-K, and mitochondrial activity in the BM of mice treated with a sublethal dose (60 mg / kg) of phenylhydrazine (PHZ). FF and Arf treatment increases LS-K in bone marrow progenitor cells (BMP) 14 days after intraperitoneal administration. *p<0.05, **p<0.01, ***p<0.001, ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO)-treated controls. NS=not significant. [Figure 36B] Figure 1 shows that intraperitoneal FF / Arf treatment increases LSK, LS-K, and mitochondrial activity in the BM of mice treated with a sublethal dose (60 mg / kg) of phenylhydrazine (PHZ). FF and Arf treatment increases LSK in bone marrow progenitor cells (BMP) 14 days after intraperitoneal administration. *p<0.05, **p<0.01, ***p<0.001, ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO)-treated controls. NS=not significant. [Figure 36C] Figure 1 shows that intraperitoneal FF / Arf treatment increases LSK, LS-K, and mitochondrial activity in the BM of mice treated with a sublethal dose (60 mg / kg) of phenylhydrazine (PHZ). FF and Arf treatment increases mitochondrial membrane potential (TMRE staining) in bone marrow progenitor cells (BMP) 14 days after intraperitoneal administration. *p<0.05, **p<0.01, ***p<0.001, ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO)-treated controls. NS=not significant. [Figure 37A] Figure 1 shows that intraperitoneal treatment with FF / Arf increases megakaryocytic progenitor cells (MEPs) but not granulocyte-monocyte progenitor cells (GMPs) in the BM of mice treated with a sublethal dose (60 mg / kg) of phenylhydrazine (PHZ). FF and Arf treatment increases MEPs in myeloid progenitor cells (BMPs) 14 days after intraperitoneal administration. *p<0.05, ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO)-treated controls. NS=not significant. [Figure 37B] Figure 1 shows that intraperitoneal treatment with FF / Arf increases megakaryocytic progenitors (MEPs) but not granulocyte-monocyte progenitors (GMPs) in the BM of mice treated with a sublethal dose (60 mg / kg) of phenylhydrazine (PHZ). FF and Arf treatment does not increase GMPs in myeloid progenitors (BMPs) 14 days after intraperitoneal administration. *p<0.05, ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO)-treated controls. NS=not significant. [Figure 38A] Figure 1 shows that intraperitoneal FF / Arf treatment consistently enriches erythroid progenitors in the bone marrow (BM) of mice treated with sublethal doses (60 mg / kg) of phenylhydrazine (PHZ). Flow plots show that FF / Arf treatment at a dose of 0.3 mg / kg affects RI, RII, RIII, and RIV erythroid progenitors in the BM of mice. *p<0.05, **p<0.01, ***P<0.001, ****P<0.0001, ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO)-treated controls. NS=not significant. [Figure 38B] Figure 38 shows that intraperitoneal FF / Arf treatment consistently enriches erythroid progenitors in the bone marrow (BM) of mice treated with a sublethal dose (60 mg / kg) of phenylhydrazine (PHZ). Quantification of data shown in Figure 38A shows the absolute numbers of RI, RII, RIII, and RIV erythroid progenitors per million BM cells in mice. *p<0.05, **p<0.01, ***P<0.001, ****P<0.0001, ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO)-treated controls. NS=not significant. [Figure 39A]Figure 1 shows that intraperitoneal FF treatment enhances colony-forming units-erythroid (CFU-e) in the bone marrow (BM) of mice treated with a sublethal dose (60 mg / kg) of phenylhydrazine (PHZ). Graph showing the long-term effect of 0.3 mg / kg FF treatment on mouse BM to generate CFU-e. *p<0.05, ***P<0.001, ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO)-treated controls. NS=not significant. [Figure 39B] Figure 1 shows that intraperitoneal FF treatment enhances colony-forming units-erythroid (CFU-e) in the bone marrow (BM) of mice treated with a sublethal dose (60 mg / kg) of phenylhydrazine (PHZ). Graphs show enhanced transferrin receptor expression in BMP-derived CFU-e from FF / Arf-treated mice. *p<0.05, ***P<0.001, ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO)-treated controls. NS=not significant. [Figure 40] Flow cytometry plots for studying erythroid progenitor cells (EryPs) in the BM of mice are shown. [Figure 41] We show that formoterol fumarate (FF) does not affect granulocytic progenitor cells (GMP) in the bone marrow (BM). [Figure 42A]This figure shows that formoterol fumarate (FF) treatment confers a significant survival benefit in phenylhydrazine (PHZ)-mediated lethal hemolytic anemia in mice (10-12 week old). Kaplan-Meier survival plots show the survival benefit of FF treatment at 0.1 / 0.3 mg / kg doses in male mice after PHZ-induced lethal hemolytic anemia (PHZ-150 mg / kg). n = 5 mice per group. In the vehicle-treated group (gray circle), one mouse died on day 1, and four mice died on day 2 after the lethal PHZ dose. In the FF 0.1 mg / kg group (orange circle), two mice died on day 2, and three mice still survived the lethal PHZ dose. In the FF 0.3 mg / kg group (maroon circle), one mouse died on day 2, and four mice still survived the lethal PHZ dose. *p<0.05, log-rank (Mantel-Cox) test. n=5 mice per group. All comparisons were made against vehicle (DMSO)-treated controls. [Figure 42B] This figure shows that formoterol fumarate (FF) treatment confers a significant survival benefit in phenylhydrazine (PHZ)-mediated lethal hemolytic anemia in mice (10-12 week old mice). FF treatment at 0.1 / 0.3 mg / kg doses steadily increases body weight in male mice after PHZ-induced lethal hemolytic anemia. *p<0.05, log-rank (Mantel-Cox) test. n=5 mice per group. All comparisons were made against vehicle (DMSO)-treated controls. [Figure 42C]This figure shows that formoterol fumarate (FF) treatment confers a significant survival benefit in phenylhydrazine (PHZ)-mediated lethal hemolytic anemia in mice (10-12 week old). Kaplan-Meier survival plots show the survival benefit of FF treatment at 0.1 / 0.3 mg / kg doses in female mice after PHZ-induced lethal hemolytic anemia (PHZ-120 mg / kg). n = 5 mice per group. In the vehicle-treated group (gray circles), three mice died on day 2 and two mice died on day 3 after the lethal PHZ dose. In the FF 0.1 mg / kg group (orange circles), one mouse died on day 2, and four mice still survived the lethal PHZ dose. In the FF 0.3 mg / kg group (maroon circles), two mice died on day 2 and three mice still survived the lethal PHZ dose. *p<0.05, log-rank (Mantel-Cox) test. n=5 mice per group. All comparisons were made against vehicle (DMSO)-treated controls. [Figure 42D] This figure shows that formoterol fumarate (FF) treatment confers a significant survival benefit in phenylhydrazine (PHZ)-mediated lethal hemolytic anemia in mice (10-12 week old mice). FF treatment at 0.1 / 0.3 mg / kg doses steadily increases body weight in female mice after PHZ-induced lethal hemolytic anemia. *p<0.05, log-rank (Mantel-Cox) test. n=5 mice per group. All comparisons were made against vehicle (DMSO)-treated controls. [Figure 43] 1 shows flow cytometry plots of formoterol fumarate (FF) on MDS patient-derived cells using semi-solid methylcellulose culture. [Figure 44] 1 shows flow cytometry plots of formoterol fumarate (FF) on MDS patient-derived cells using semi-solid methylcellulose culture. [Figure 45]Formoterol fumarate (FF) significantly restores erythropoiesis in human hematopoietic stem and progenitor cells with RIOK2 knockdown (RIOK2 KD HSPCs) in vitro. *p<0.05, **p<0.01, ANOVA. [Figure 46] Figure 1 shows that formoterol fumarate (FF) modestly restores erythropoiesis in human hematopoietic stem and progenitor cells with RPS14 and APC knockdown (RPS14 and APC KD HSPCs) in vitro. *p<0.05, **p<0.01, ANOVA. Ns: not significant. [Figure 47] FIG. 1 shows a schematic diagram of FF treatment by oral gavage in wild-type mice at steady state. [Figure 48A] Figure 1 shows that FF treatment by oral gavage increases RBC parameters in wild-type mice at steady state. FF treatment increases RBC parameters such as hematocrit (HCT) % in peripheral blood after 14 days of daily oral gavage (og). *p<0.05, ANOVA. N=5 mice per group. All comparisons were made against vehicle-treated (DMSO) controls. Ns=not significant. [Figure 48B] Figure 1 shows that FF treatment by oral gavage increases RBC parameters in wild-type mice at steady state. FF treatment increases RBC parameters such as RBC count in peripheral blood after 14 days of daily oral gavage (og). *p<0.05, ANOVA. N=5 mice per group. All comparisons were made against vehicle-treated (DMSO) controls. Ns=not significant. [Figure 48C] Figure 1 shows that FF treatment by oral gavage increases RBC parameters in wild-type mice at steady state. FF treatment increases RBC parameters such as hemoglobin (Hb) in peripheral blood after 14 days of daily oral gavage (og). *p<0.05, ANOVA. N=5 mice per group. All comparisons were made against vehicle-treated (DMSO) controls. Ns=not significant. [Figure 49A]Figure 1 shows that FF treatment by oral gavage does not alter WBC parameters or platelets in wild-type mice at steady state. FF treatment does not affect WBC parameters such as WBC in peripheral blood after 14 days of daily oral gavage (og). N=5 mice per group for ANOVA. All comparisons were made against vehicle-treated (DMSO) controls. ns=not significant. [Figure 49B] Figure 1 shows that FF treatment by oral gavage does not alter WBC parameters or platelets in wild-type mice at steady state. FF treatment does not affect WBC parameters such as monocytes in peripheral blood after 14 days of daily oral gavage (og). N=5 mice per group for ANOVA. All comparisons were made against vehicle-treated (DMSO) controls. ns=not significant. [Figure 49C] Figure 1 shows that FF treatment by oral gavage does not alter WBC parameters or platelets in wild-type mice at steady state. FF treatment does not affect WBC parameters such as platelets in peripheral blood after 14 days of daily oral gavage (og). N=5 mice per group for ANOVA. All comparisons were made against vehicle-treated (DMSO) controls. ns=not significant. [Figure 50] Panels A and B show that FF treatment by oral gavage enhances bone marrow progenitor (BMP) viability and mitochondrial activity in wild-type mice at steady state. FF treatment increases BMP viability and mitochondrial membrane potential (measured via TMRE staining) after 14 days of daily oral gavage (og). *p<0.05, **p<0.01, ****p<0.0001, ANOVA. n=5 mice per group. All comparisons were made against vehicle-treated (DMSO) controls. ns=not significant. [Figure 51]Panels A-C show that FF treatment by oral gavage enhances MEPs and MkPs, but not GMPs, in the bone marrow (BM) of wild-type mice at steady state. FF treatment does not affect granulocyte-monocyte progenitors (GMPs) but increases BMPs, megakaryocytic erythroid progenitors (MEPs) and megakaryocytic progenitors (MkPs), after 14 days of daily oral gavage (og). ***p<0.001, ****p<0.0001, ANOVA. n=5 mice per group. All comparisons were made against vehicle-treated (DMSO) controls. ns=not significant. [Figure 52A] Figure 1 shows that oral FF treatment by gavage enhances erythroid progenitor cells in the bone marrow (BM) of wild-type mice at steady state. Flow plots show that FF treatment at doses of 0.1 / 0.5 / 1.0 mg / kg significantly increased RI, RII, RIII, and RIV erythroid progenitor cells in the BM of mice. *p<0.05, **p<0.01, ***P<0.001, ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO)-treated controls. ns=not significant. [Figure 52B] Figure 52 shows that oral FF treatment enhances erythroid progenitor cells in the bone marrow (BM) of wild-type mice at steady state. Quantification of data shown in Figure 52A shows the absolute numbers of RI, RII, RIII, and RIV erythroid progenitors per million BM cells in mice. *p<0.05, **p<0.01, ***P<0.001, ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO)-treated controls. ns=not significant. [Figure 53] Figure 1 shows that FF treatment mildly increases mouse body weight at steady state after FF treatment by oral gavage (og). The graph shows that FF treatment by oral gavage mildly increases the % change in mouse body weight. *p<0.05, **p<0.01, two-way ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO) treated controls. ns=not significant. [Figure 54]This figure shows that oral FF treatment confers a significant survival benefit to mice (10-12 weeks old) given a lethal dose of 135 mg / kg phenylhydrazine (PHZ). Kaplan-Meier survival plots show the survival benefit of oral FF treatment at 0.1 / 0.5 mg / kg doses in mice after PHZ-induced lethal hemolytic anemia (PHZ-135 mg / kg). Log-rank (Mantel-Cox) test. n = 5 mice per group. All comparisons were made against vehicle (DMSO)-treated controls. [Figure 55] Schematic diagram of FF treatment by oral gavage in phenylhydrazine (PHZ)-treated mice at a sublethal dose of 60 mg / kg. [Figure 56] FF treatment by oral gavage (og) does not affect the body weight of mice treated with a sublethal dose (60 mg / kg) of phenylhydrazine (PHZ). ns = not significant. [Figure 57A] Figure 1 shows that FF treatment by oral gavage increases RBC parameters in mice treated with a sublethal dose (60 mg / kg) of phenylhydrazine (PHZ). FF treatment increases RBC parameters such as hematocrit (HCT) % in peripheral blood after oral gavage (og). *p<0.05, **p<0.01, ***P<0.001, ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO)-treated controls. ns=not significant. [Figure 57B] Figure 1 shows that FF treatment by oral gavage increases RBC parameters in mice treated with a sublethal dose (60 mg / kg) of phenylhydrazine (PHZ). FF treatment increases RBC parameters such as hemoglobin (Hb) in peripheral blood after oral gavage (og). *p<0.05, **p<0.01, ***P<0.001, ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO)-treated controls. ns=not significant. [Figure 58A]Figure 1 shows that FF treatment by oral gavage does not affect WBC parameters in mice treated with a sublethal dose (60 mg / kg) of phenylhydrazine (PHZ). FF treatment does not affect WBC in peripheral blood after oral gavage (og). ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO) treated controls. ns=not significant. [Figure 58B] Figure 1 shows that FF treatment by oral gavage does not affect WBC parameters in mice treated with a sublethal dose (60 mg / kg) of phenylhydrazine (PHZ). FF treatment does not affect platelets in peripheral blood after oral gavage (og). ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO) treated controls. ns=not significant. [Figure 59A] Figure 1 shows that FF treatment by oral gavage enhances bone marrow progenitor cell (BMP) viability and mitochondrial activity in mice treated with a sublethal dose (60 mg / kg) of phenylhydrazine (PHZ). FF treatment increases BMP viability 14 days after daily oral gavage (og). *p<0.05, **p<0.01, ***p<0.001, ANOVA. n=5 mice per group. All comparisons were made against vehicle-treated (DMSO) controls. ns=not significant. [Figure 59B] Figure 1 shows that FF treatment by oral gavage enhances bone marrow progenitor cell (BMP) viability and mitochondrial activity in mice treated with a sublethal dose (60 mg / kg) of phenylhydrazine (PHZ). FF treatment increases mitochondrial membrane potential (measured via TMRE staining) 14 days after daily oral gavage (og). *p<0.05, **p<0.01, ***p<0.001, ANOVA. n=5 mice per group. All comparisons were made against vehicle-treated (DMSO) controls. ns=not significant. [Figure 60A]Figure 1 shows that oral FF treatment enhances MkPs, but not GMPs, in the bone marrow (BM) of mice treated with a sublethal dose (60 mg / kg) of phenylhydrazine (PHZ). FF treatment does not affect granulocyte-monocyte progenitor cells (GMPs) after 14 days of daily oral gavage. ***p<0.001, ****p<0.0001, ANOVA. n=5 mice per group. All comparisons were made against vehicle-treated (DMSO) controls. ns=not significant. [Figure 60B] Figure 1 shows that oral FF treatment enhances MkPs, but not GMPs, in the bone marrow (BM) of mice treated with a sublethal dose (60 mg / kg) of phenylhydrazine (PHZ). FF treatment increases megakaryocytic progenitor cells (MkPs) in BMPs 14 days after daily oral gavage (og). ***p<0.001, ****p<0.0001, ANOVA. n=5 mice per group. All comparisons were made against vehicle-treated (DMSO) controls. ns=not significant. [Figure 61A] Figure 1 shows that oral FF treatment by gavage enhances erythroid progenitor cells in the bone marrow (BM) of mice treated with a sublethal dose (60 mg / kg) of phenylhydrazine (PHZ). Figure 2 shows flow plots demonstrating that FF treatment at doses of 0.1 / 0.3 / 0.5 / 1.0 mg / kg significantly increased RI, RII, RIII, and RIV erythroid progenitors in the BM of mice. *p<0.05, **p<0.01, ***P<0.001, ****P<0.0001, ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO)-treated controls. ns=not significant. [Figure 61B]Figure 61 shows that oral FF treatment enhances erythroid progenitor cells in the bone marrow (BM) of mice treated with a sublethal dose (60 mg / kg) of phenylhydrazine (PHZ). Quantification of the data shown in Figure 61A shows the absolute numbers of RI, RII, RIII, and RIV erythroid progenitors per million BM cells in mice. *p<0.05, **p<0.01, ***P<0.001, ****P<0.0001, ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO)-treated controls. ns=not significant. [Figure 62] A schematic diagram of the experimental setup for analyzing the effects of formoterol fumarate (FF) in naive mice without external stress due to phenylhydrazine (PHZ) is shown. [Figure 63] Figure 62 shows that FF treatment increases the body weight of wild-type mice at steady state. The graph shows the % change in body weight of mice treated with vehicle or FF at doses of 0.1 / 0.3 / 0.5 mg / kg. **p<0.01, "ns" = not significant. The 0.1 and 0.3 mg / kg groups showed a clear increase in body weight compared to the vehicle-treated group, although this did not show statistical significance in Graphpad PRISM. [Figure 64A] Figure 62 shows that FF treatment significantly increases red blood cell (RBC) parameters in wild-type mice (10-12 week old male mice). FF administration enhances and maintains RBC parameters, such as RBC count, in peripheral blood. The 0.1 and 0.5 mg / kg groups did not show statistical significance in HCT% according to Graphpad PRISM. *p<0.05, **p<0.01, ***p<0.001, ****P<0.0001, "ns"=not significant, one-way ANOVA and two-way ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO)-treated controls. [Figure 64B]Figure 62 shows that FF treatment significantly increases red blood cell (RBC) parameters in wild-type mice (10-12 week old male mice). FF administration enhances and maintains RBC parameters such as hemoglobin (Hb) in peripheral blood. The 0.1 and 0.5 mg / kg groups did not show statistical significance in HCT% according to Graphpad PRISM. *p<0.05, **p<0.01, ***p<0.001, ****P<0.0001, "ns" = not significant, one-way ANOVA and two-way ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO)-treated controls. [Figure 64C] Figure 62 shows that FF treatment significantly increases red blood cell (RBC) parameters in wild-type mice (10-12 week old male mice). FF administration enhances and maintains RBC parameters such as hematocrit (HCT)% in peripheral blood. The 0.1 and 0.5 mg / kg groups did not show statistical significance in HCT% according to Graphpad PRISM. *p<0.05, **p<0.01, ***p<0.001, ****P<0.0001, "ns"=not significant, one-way ANOVA and two-way ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO)-treated controls. [Figure 65A] Figure 62 shows that FF treatment does not affect total white blood cell (non-RBC) parameters in wild-type mice (10-12 week old male mice). FF administration does not affect white blood cell (WBC) counts. *p<0.05, **p<0.01, ***p<0.001, ****P<0.0001, "ns"=not significant, one-way ANOVA and two-way ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO) treated controls. [Figure 65B]Figure 62 shows that FF treatment does not affect total white blood cell (non-RBC) parameters in wild-type mice (10-12 week old male mice). FF administration modestly affects monocyte counts in the peripheral blood of naive mice. *p<0.05, **p<0.01, ***p<0.001, ****P<0.0001, "ns" = not significant, one-way ANOVA and two-way ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO)-treated controls. [Figure 65C] Figure 62 shows that FF treatment does not affect total white blood cell (non-RBC) parameters in wild-type mice (10-12 week old male mice). FF administration does not affect platelet counts. *p<0.05, **p<0.01, ***p<0.001, ****P<0.0001, "ns" = not significant, one-way ANOVA and two-way ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO) treated controls. [Figure 66A] Figure 62 shows that FF treatment significantly enhances mitochondrial biogenesis in bone marrow progenitor cells (BMPs) of wild-type mice (10-12 week old mice). FF administration enhances mitochondrial biogenesis as observed by MitoTracker staining in BMPs of naive mice. *p<0.05, **p<0.01, ***p<0.001, ****P<0.0001, "ns" = not significant. n=5 male mice per group. All comparisons were made against vehicle (DMSO)-treated controls. [Figure 66B] Figure 62 shows that FF treatment significantly enhances mitochondrial biogenesis in bone marrow progenitor cells (BMP) from wild-type mice (10-12 week old mice). FF administration enhances mitochondrial membrane potential as observed by TMRE staining in BMP from naive mice. *p<0.05, **p<0.01, ***p<0.001, ****P<0.0001, "ns" = not significant. n=5 male mice per group. All comparisons were made against vehicle (DMSO)-treated controls. [Figure 66C]Figure 62 shows that FF treatment significantly enhanced mitochondrial biogenesis in bone marrow progenitor cells (BMPs) from wild-type mice (10-12 week old mice). FF administration significantly reduced mitochondrial superoxide production, indicating mitochondrial fitness as observed by MitoSox staining in BMPs from naive mice. *p<0.05, **p<0.01, ***p<0.001, ****P<0.0001, "ns" = not significant. n=5 male mice per group. All comparisons were made against vehicle (DMSO)-treated controls. [Figure 67A] Figure 62 shows that FF treatment significantly enhances bone marrow (BM) progenitor cell viability and LS-K cells (lineage-Sca1-cKit+) viability in wild-type mice (10-12 week old mice). FF treatment at doses of 0.1 / 0.3 / 0.5 mg / kg significantly increases BM viability in naive mice. *p<0.05, ****P<0.0001, ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO)-treated controls. [Figure 67B] Figure 62 shows that FF treatment significantly enhances bone marrow (BM) progenitor cell viability and LS-K cells (lineage-Sca1-cKit+) in wild-type mice (10-12 week old mice). FF treatment at doses of 0.1 / 0.3 / 0.5 mg / kg significantly increases LS-K cells. *p<0.05, ****P<0.0001, ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO) treated controls. [Figure 68A]Figure 62 shows that FF treatment significantly increased megakaryocytic erythroid progenitors (MEPs) and megakaryocytic progenitors (MKPs), but not granulocyte-monocyte progenitors (GMPs), in the bone marrow (BM) of naive mice (10-12 week-old mice). Flow plots show that FF treatment at doses of 0.1 / 0.3 / 0.5 mg / kg significantly increased megakaryocytic erythroid progenitors (MEPs) in the BM of wild-type mice. LS-K: lineage-Sca1-cKit+ BM cells. **p<0.01, ***p<0.001, "ns" = not significant, ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO-treated) controls. [Figure 68B] Figure 62 shows that FF treatment significantly increased megakaryocytic erythroid progenitors (MEPs) and megakaryocytic progenitors (MKPs), but not granulocyte-monocyte progenitors (GMPs), in the bone marrow (BM) of naive mice (10-12 week-old mice). Flow plots show that FF treatment at doses of 0.1 / 0.3 / 0.5 mg / kg significantly increased megakaryocytic progenitors (MKPs). LS-K: Lineage-Sca1-cKit+ BM cells. **p<0.01, ***p<0.001, "ns" = not significant, ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO-treated) controls. [Figure 68C] Figure 62 shows that FF treatment significantly increased megakaryocyte erythroid progenitors (MEPs) and megakaryocyte progenitors (MKPs) in the bone marrow (BM) of naive mice (10-12 week old mice). Flow plots show that FF treatment at doses of 0.1 / 0.3 / 0.5 mg / kg did not increase granulocyte monocytic progenitors (GMPs). LS-K: Lineage-Sca1-cKit+ BM cells. **p<0.01, ***p<0.001, "ns" = not significant, ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO-treated) controls. [Figure 69A]Figure 62 shows that FF treatment significantly increases erythroid differentiation in the bone marrow (BM) of naive mice (10-12 week old mice). FF treatment at doses of 0.1 / 0.3 / 0.5 mg / kg significantly increases RI in the BM of wild-type mice. *p<0.05, **p<0.01, ***P<0.001, ****P<0.0001, "ns"=not significant, ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO)-treated controls. [Figure 69B] Figure 62 shows that FF treatment significantly increases erythroid differentiation in the bone marrow (BM) of naive mice (10-12 week old mice). FF treatment at doses of 0.1 / 0.3 / 0.5 mg / kg significantly increases RII in the BM of wild-type mice. *p<0.05, **p<0.01, ***P<0.001, ****P<0.0001, "ns" = not significant, ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO) treated controls. [Figure 69C] Figure 62 shows that FF treatment significantly increases erythroid differentiation in the bone marrow (BM) of naive mice (10-12 week old mice). FF treatment at doses of 0.1 / 0.3 / 0.5 mg / kg significantly increases RIII in the BM of wild-type mice. *p<0.05, **p<0.01, ***P<0.001, ****P<0.0001, "ns"=not significant, ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO)-treated controls. [Figure 69D]Figure 62 shows that FF treatment significantly increases erythroid differentiation in the bone marrow (BM) of naive mice (10-12 week old mice). FF treatment at doses of 0.1 / 0.3 / 0.5 mg / kg significantly increases RIV erythroid progenitors in the BM of wild-type mice. Quantification of data showing absolute numbers of RI, RII, RIII, and RIV erythroid progenitors per million BM cells in naive mice. *p<0.05, **p<0.01, ***P<0.001, ****P<0.0001, "ns" = not significant, ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO)-treated controls. [Figure 70A] Figure 63 shows that FF treatment significantly increases erythroid gene expression in sorted erythroid progenitor cells isolated from the bone marrow (BM) of naive mice (10-12 week old mice). FF treatment at doses of 0.1 / 0.3 / 0.5 mg / kg significantly increases the expression of erythroid genes, such as EPOR, in sorted erythroid progenitor cells isolated from the BM of wild-type mice. *p<0.05, **p<0.01, ***P<0.001, ****P<0.0001, "ns" = not significant, ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO)-treated controls. [Figure 70B] Figure 63 shows that FF treatment significantly increases erythroid gene expression in sorted erythroid progenitor cells isolated from the bone marrow (BM) of naive mice (10-12 week old mice). FF treatment at doses of 0.1 / 0.3 / 0.5 mg / kg significantly increases the expression of erythroid genes, such as EPOR and ASXL1 (Figure 70B), in sorted erythroid progenitor cells isolated from the BM of wild-type mice. *p<0.05, **p<0.01, ***P<0.001, ****P<0.0001, "ns" = not significant, ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO)-treated controls. [Figure 70C]Figure 63 shows that FF treatment significantly increases erythroid gene expression in sorted erythroid progenitor cells isolated from the bone marrow (BM) of naive mice (10-12 week old mice). FF treatment at doses of 0.1 / 0.3 / 0.5 mg / kg significantly increases the expression of erythroid genes, such as NFE2, in sorted erythroid progenitor cells isolated from the BM of wild-type mice. *p<0.05, **p<0.01, ***P<0.001, ****P<0.0001, "ns" = not significant, ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO)-treated controls. [Figure 70D] Figure 63 shows that FF treatment significantly increases erythroid gene expression in sorted erythroid progenitor cells isolated from the bone marrow (BM) of naive mice (10-12 week old mice). FF treatment at doses of 0.1 / 0.3 / 0.5 mg / kg does not affect the expression of ADRB2, the gene encoding the β2-AR, in sorted erythroid progenitor cells isolated from the BM of wild-type mice. *p<0.05, **p<0.01, ***P<0.001, ****P<0.0001, "ns" = not significant, ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO)-treated controls. Because FF treatment in naive mice increases erythropoietin receptor (EPOR) expression on erythroid progenitor cells, FF may be administered in combination with erythropoiesis-stimulating agents (ESAs), such as epoetin alfa and other erythropoietin (EPO) biosimilars, in patients with MDS and other anemias who do not respond to EPO treatment alone. The increase in EPOR on erythroid progenitor cells by FF does not preclude its use in combination with FDA-approved drugs, such as luspatercept, lenalidomide, and / or hypomethylating agents, such as azacitidine and / or decitabine, in patients with MDS and other anemias who do not respond to EPO treatment alone. [Figure 71A]This shows that FF treatment increases RBC parameters in MDS / AML mice (NUP98-HOXD13 transgenic mice). This shows that FF treatment at a low dose of 0.3 mg / kg, three times a week (intraperitoneally), reduces RBC parameters, such as RBCs in the PB of MDS / AML mice. *p<0.05, **p<0.01, ****p<0.0001, ANOVA. n=5-7 mice per group. Veh: vehicle, LC: littermate control. [Figure 71B] This shows that FF treatment increases RBC parameters in MDS / AML mice (NUP98-HOXD13 transgenic mice). This shows that FF treatment at a low dose of 0.3 mg / kg, three times a week (intraperitoneally), reduces RBC parameters such as hemoglobin (Hb) in the PB of MDS / AML mice. *p<0.05, **p<0.01, ****p<0.0001, ANOVA. n=5-7 mice per group. Veh: vehicle, LC: littermate control. [Figure 71C] FF treatment increases RBC parameters in MDS / AML mice (NUP98-HOXD13 transgenic mice). Figures 71A, 71B, and 71C show that FF treatment at a low dose of 0.3 mg / kg, three times a week (intraperitoneally), reduces RBC parameters such as hematocrit (HCT%) in the PB of MDS / AML mice. *p<0.05, **p<0.01, ****p<0.0001, ANOVA. n=5-7 mice per group. Veh: vehicle, LC: littermate control. [Figure 72A] This shows that FF treatment does not affect WBC parameters in MDS / AML mice. FF treatment at a low dose of 0.3 mg / kg three times a week (intraperitoneally) does not affect platelets in the peripheral blood of MDS / AML mice. ***p<0.001, ****p<0.0001, ANOVA. n=5-7 mice per group. Veh: vehicle, LC: littermate control. ns: not significant. [Figure 72B]This shows that FF treatment does not affect WBC parameters in MDS / AML mice. FF treatment at a low dose of 0.3 mg / kg, three times a week (intraperitoneally), does not affect WBC in the peripheral blood of MDS / AML mice. ***p<0.001, ****p<0.0001, ANOVA. n=5-7 mice per group. Veh: vehicle, LC: littermate control. ns: not significant. [Figure 72C] This shows that FF treatment does not affect WBC parameters in MDS / AML mice. FF treatment at a low dose of 0.3 mg / kg three times a week (intraperitoneally) does not affect apoptosis in the bone marrow (measured by Annexin V staining) in the peripheral blood of MDS / AML mice. ***p<0.001, ****p<0.0001, ANOVA. n=5-7 mice per group. Veh: vehicle, LC: littermate control. ns: not significant. [Figure 73A] This shows that FF treatment increases erythroid progenitor cells in the bone marrow of MDS / AML mice. FF treatment at a low dose of 0.3 mg / kg, three times a week (intraperitoneally), increases erythroid progenitor cells. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ANOVA. n=5-7 mice per group. Veh: vehicle, LC: littermate control. ns=not significant. [Figure 73B] This shows that FF treatment increases erythroid progenitor cells in the bone marrow of MDS / AML mice. FF treatment at a low dose of 0.3 mg / kg three times a week (intraperitoneally) does not affect granulocyte-monocyte progenitor cells (GMP) in the BM of MDS / AML mice (Figure 73B). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ANOVA. n=5-7 mice per group. Veh: vehicle, LC: littermate control. ns=not significant. [Figure 73C]FF treatment increases erythroid progenitor cells in the bone marrow of MDS / AML mice. FF treatment at a low dose of 0.3 mg / kg three times a week (intraperitoneally) does not affect megakaryocytic progenitor cells (MkPs). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ANOVA. n=5-7 mice per group. Veh: vehicle, LC: littermate control. ns=not significant. [Figure 74A] This study demonstrates that intraperitoneal FF treatment increases RBC parameters in Apcmin mice, a mouse model of intestinal adenoma that develops anemia. Daily intraperitoneal FF treatment at a low dose of 0.3 mg / kg reduces RBC parameters, including RBC counts, in Apcmin mice, which spontaneously develop anemia at approximately 3 months of age. *p<0.05, ***p<0.001, two-way ANOVA. n=5-6 mice per group. Comparisons are shown between Apcmin + FF and Apcmin + Veh. Veh: vehicle; LC: littermate control mice. [Figure 74B] This study demonstrates that intraperitoneal FF treatment increases RBC parameters in Apcmin mice, a mouse model of intestinal adenoma that develops anemia. Daily intraperitoneal FF treatment at a low dose of 0.3 mg / kg reduces RBC parameters, such as hematocrit % (HCT%), in Apcmin mice, which spontaneously develop anemia at approximately 3 months of age. *p<0.05, ***p<0.001, two-way ANOVA. n=5-6 mice per group. Comparisons are shown between Apcmin + FF and Apcmin + Veh. Veh: vehicle; LC: littermate control mice. [Figure 74C]This study demonstrates that intraperitoneal FF treatment increases RBC parameters in Apcmin mice, a mouse model of intestinal adenoma that develops anemia. Daily intraperitoneal FF treatment at a low dose of 0.3 mg / kg reduces RBC parameters, such as hemoglobin (Hb), in Apcmin mice, which spontaneously develop anemia at approximately 3 months of age. *p<0.05, ***p<0.001, two-way ANOVA. n=5-6 mice per group. Comparisons are shown between Apcmin + FF and Apcmin + Veh. Veh: vehicle; LC: littermate control mice. [Figure 75] FF treatment moderately increased body weight in Apcmin mice. n = 5-6 mice per group. Veh: vehicle, Ctrl (control): littermate control mice. [Figure 76] Figure 1 shows that FF (intraperitoneal) treatment significantly restores (increases) body weight in cisplatin-treated / driven chronic kidney disease (CKD) mice compared to mice treated with cisplatin + vehicle. ****p<0.0001, two-way ANOVA. n=4-5 mice per group. Comparisons are shown between mice treated with cisplatin + FF compared to mice treated with cisplatin + vehicle. [Figure 77A] We demonstrate that cisplatin treatment induces chronic kidney disease (CKD) in wild-type mice. Serum blood urea nitrogen (BUN) measurements show that cisplatin induces kidney damage characteristic of CKD in mice, and that FF treatment does not affect serum BUN or creatinine levels. [Figure 77B] We demonstrate that cisplatin treatment induces chronic kidney disease (CKD) in wild-type mice. Serum creatinine levels indicate that cisplatin induces kidney damage characteristic of CKD in mice, and that FF treatment does not affect serum BUN or creatinine levels. [Figure 78A]This shows that FF treatment reverses anemia in cisplatin-treated CKD mice. FF treatment at a low dose of 0.3 mg / kg, three times per week (intraperitoneally), alleviates RBC parameters such as the % change in hematocrit (HCT%) in cisplatin-induced CKD mice. *p<0.05, two-way ANOVA. n=4-5 mice per group. Comparisons are shown between mice treated with cisplatin + FF compared to mice treated with cisplatin + vehicle. [Figure 78B] This shows that FF treatment reverses anemia in cisplatin-treated CKD mice. FF treatment at a low dose of 0.3 mg / kg, three times a week (intraperitoneally), alleviates RBC parameters, such as the % change in hemoglobin (Hb), in cisplatin-induced CKD mice. *p<0.05, two-way ANOVA. n=4-5 mice per group. Comparisons are shown between mice treated with cisplatin + FF compared to mice treated with cisplatin + vehicle. [Figure 78C] This shows that FF treatment reverses anemia in cisplatin-treated CKD mice. FF treatment at a low dose of 0.3 mg / kg three times a week (intraperitoneally) alleviates RBC parameters, such as % RBC change, in cisplatin-induced CKD mice. *p<0.05, two-way ANOVA. n=4-5 mice per group. Comparisons are shown between mice treated with cisplatin + FF compared to mice treated with cisplatin + vehicle. [Figure 79A] This shows that FF treatment increases RBC parameters in cisplatin-treated CKD mice. FF treatment at a low dose of 0.3 mg / kg, three times a week (intraperitoneally), reduces RBC parameters, such as absolute hemoglobin (Hb) levels, in cisplatin-induced CKD mice. *p<0.05, **p<0.01, ***p<0.001, ***p<0.001, two-way ANOVA. n=4-5 mice per group. ns: not significant. [Figure 79B]This shows that FF treatment increases RBC parameters in cisplatin-treated CKD mice. FF treatment at a low dose of 0.3 mg / kg, three times a week (intraperitoneally), reduces RBC parameters, such as absolute hematocrit % (HCT%), in cisplatin-driven CKD mice. *p<0.05, **p<0.01, ***p<0.001, ***p<0.001, two-way ANOVA. n=4-5 mice per group. ns: not significant. [Figure 79C] This shows that FF treatment increases RBC parameters in cisplatin-treated CKD mice. FF treatment at a low dose of 0.3 mg / kg, three times a week (intraperitoneally), reduces RBC parameters, including absolute RBC counts, in cisplatin-driven CKD mice. *p<0.05, **p<0.01, ***p<0.001, ***p<0.001, two-way ANOVA. n=4-5 mice per group. ns: not significant. [Figure 80A] Figure 1 shows that FF treatment does not affect WBC parameters in cisplatin-treated CKD mice. FF treatment does not affect WBC in cisplatin-driven CKD mice. Two-way ANOVA. n=4-5 mice per group. Comparisons are shown between mice treated with cisplatin + FF compared to mice treated with cisplatin + vehicle. ns: not significant. [Figure 80B] Figure 1 shows that FF treatment does not affect WBC parameters in cisplatin-treated CKD mice. FF treatment does not affect platelets in cisplatin-driven CKD mice. Two-way ANOVA. n=4-5 mice per group. Comparisons are shown between mice treated with cisplatin + FF compared to mice treated with cisplatin + vehicle. ns: not significant. [Figure 81A]Figure 1 shows that FF treatment increases erythroid progenitor cells in cisplatin-treated CKD mice. FF treatment at a low dose of 0.3 mg / kg, three times per week (intraperitoneally), increases peripheral erythroid progenitor cells in the BM of cisplatin-driven CKD mice. *p<0.05, ***p<0.001, two-way ANOVA. n=4-5 mice per group. Comparisons are shown between mice treated with cisplatin + FF and mice treated with cisplatin + vehicle. ns: not significant. [Figure 81B] This shows that FF treatment increases erythroid progenitor cells in cisplatin-treated CKD mice. FF treatment at a low dose of 0.3 mg / kg three times a week (intraperitoneally) does not increase granulocyte-monocyte progenitor cells (GMPs) in the BM of cisplatin-driven CKD mice. *p<0.05, ***p<0.001, two-way ANOVA. n=4-5 mice per group. Comparisons are shown between mice treated with cisplatin + FF and mice treated with cisplatin + vehicle. ns: not significant. [Figure 81C] This shows that FF treatment increases erythroid progenitor cells in cisplatin-treated CKD mice. FF treatment at a low dose of 0.3 mg / kg three times a week (intraperitoneally) does not increase megakaryocytic progenitor cells (MkPs) in the BM of cisplatin-driven CKD mice. *p<0.05, ***p<0.001, two-way ANOVA. n=4-5 mice per group. Comparisons are shown between mice treated with cisplatin + FF and mice treated with cisplatin + vehicle. ns: not significant. [Figure 82A] Figure 1 shows that ADRB2 surface expression on erythroid progenitor cells (EP) is higher than that on non-erythroid progenitor cells (non-EP) in WT mice. Figure 2 shows a flow plot showing ADRB2 expression on the surface of erythroid progenitor cells (EP). ADRB2 is expressed on the surface of erythroid progenitor cells in the bone marrow of wild-type mice. Ms-1-5: Mouse 1 to Mouse 5. Ab: antibody against ADRB2. [Figure 82B]Figure 1 shows that ADRB2 surface expression on erythroid progenitor cells (EP) is higher than that on non-erythroid progenitor cells (non-EP) in WT mice. ADRB2 is expressed on the surface of erythroid progenitor cells in the bone marrow of wild-type mice. Flow plots showing minimal expression of ADRB2 on the surface of non-erythroid bone marrow (BM) progenitor cells (non-EP). Ms-1-5: Mouse 1 to Mouse 5. Ab: antibody against ADRB2. [Figure 83] Figure 1 shows that FF treatment of naive mice increases body weight in a dose-dependent manner. The graph shows the % change in body weight of mice treated with vehicle or FF at doses of 0.1 / 0.3 / 0.5 / 1.0 mg / kg. **p<0.01, ****p<0.0001, "ns"=not significant. [Figure 84A] This study demonstrates that FF treatment of naive mice has a dose-dependent effect on red blood cell (RBC) parameters in wild-type mice (10-12 week-old male mice). FF treatment at doses of 0.3 and 0.5 mg / kg enhanced and maintained RBC parameters, but this effect was not observed at the highest dose of 1.0 mg / kg for each of the following RBC parameters in peripheral blood: hemoglobin (Hb). *p<0.05, **p<0.01, ***p<0.001, ****P<0.0001, "ns" = not significant. One-way ANOVA and two-way ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO)-treated controls. [Figure 84B] This study demonstrates that FF treatment of naive mice has a dose-dependent effect on red blood cell (RBC) parameters in wild-type mice (10-12 week-old male mice). FF treatment at doses of 0.3 and 0.5 mg / kg enhanced and maintained RBC parameters, but this effect was not observed at the highest dose of 1.0 mg / kg for each of the following RBC parameters in peripheral blood: hematocrit (HCT) %. *p<0.05, **p<0.01, ***p<0.001, ****P<0.0001, "ns" = not significant. One-way ANOVA and two-way ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO)-treated controls. [Figure 84C]This study demonstrates that FF treatment of naive mice has a dose-dependent effect on red blood cell (RBC) parameters in wild-type mice (10-12 week-old male mice). FF treatment at doses of 0.3 and 0.5 mg / kg enhanced and maintained RBC parameters, but this effect was not observed at the highest dose of 1.0 mg / kg for each of the following RBC parameters in peripheral blood: RBC count. *p<0.05, **p<0.01, ***p<0.001, ****P<0.0001, "ns" = not significant. One-way ANOVA and two-way ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO)-treated controls. [Figure 85A] FF treatment of naive mice shows a moderate dose-dependent effect on total white blood cell (non-RBC) parameters in wild-type mice (10-12 week-old male mice). FF administration had no effect on white blood cell (WBC) counts at the lowest dose and moderately affected WBC counts at the highest dose of 1.0 mg / kg. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, "ns" = not significant. One-way ANOVA and two-way ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO)-treated controls. [Figure 85B] FF treatment of naive mice showed a moderate dose-dependent effect on total white blood cell (non-RBC) parameters in wild-type mice (10-12 week-old male mice). Conversely, FF treatment moderately affected monocyte counts in the peripheral blood of naive mice at doses of 0.3 and 0.5 mg / kg, but not at the lowest (0.1 mg / kg) or highest (1.0 mg / kg) doses. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, "ns" = not significant. One-way ANOVA and two-way ANOVA. n=5 male mice per group. All comparisons were made relative to vehicle (DMSO)-treated controls. [Figure 85C]FF treatment of naive mice shows a modest dose-dependent effect on total white blood cell (non-RBC) parameters in wild-type mice (10-12 week old male mice). No significant effects on platelets were observed at any dose tested. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, "ns" = not significant, one-way ANOVA and two-way ANOVA. n=5 male mice per group. All comparisons were made against vehicle (DMSO)-treated controls. [Figure 86] An exemplary workflow for bulk RNA sequencing of mice treated with formoterol fumarate (FF) is shown. [Figure 87A] Figure 1 shows differential gene expression in erythroid progenitor cells isolated from the bone marrow of FF-treated and vehicle-treated mice, with a table showing the changes in expression of various genes listed in the last column. [Figure 87B] Figure 1 shows differential gene expression in erythroid progenitor cells isolated from the bone marrow of FF-treated and vehicle-treated mice, and shows a table depicting a heat map of changes in gene expression. [Figure 88A] Figure 1 shows that intraperitoneal treatment with FF at 0.5 mg / kg three times a week enhances HIF-1α expression in bone marrow cells compared to vehicle-treated mice. *p<0.05, **p<0.01, non-parametric Mann-Whitney test. Data are presented as mean ± SEM. All comparisons were made relative to vehicle (DMSO treatment). [Figure 88B] Figure 1 shows that intraperitoneal treatment with FF 0.5 mg / kg three times a week enhances VEGFA expression compared to vehicle-treated mice. *p<0.05, **p<0.01, non-parametric Mann-Whitney test. Data are presented as mean ± SEM. All comparisons were made against vehicle (DMSO treatment). DETAILED DESCRIPTION OF THE INVENTION

[0019] In some aspects, provided herein are methods of treating anemia in a patient in need thereof, comprising administering to the patient an effective amount of formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate). In some aspects, provided herein are methods of promoting differentiation of erythroid progenitor cells into mature erythrocytes in a patient in need thereof, comprising administering an effective amount of formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate). In some embodiments, the method further comprises administering to the patient in need thereof an effective amount of an erythropoiesis-stimulating factor preparation. In some aspects, 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 formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) in combination with an erythropoiesis-stimulating agent, wherein the anemia is refractory to an erythropoiesis-stimulating agent (such as erythropoietin, epoetin alfa, epoetin beta, epoetin omega, epoetin zeta, or darbepoetin alfa). In some embodiments, formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) can be administered in combination with other FDA-approved agents, such as luspatercept, lenalidomide, and / or hypomethylating agents, such as azacitidine or decitabine.

[0020] In some embodiments, provided herein is formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) for use in treating anemia in a patient. In some embodiments, provided herein is the use of formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) in the manufacture of a medicament for treating anemia in a patient. In some embodiments, provided herein is a pharmaceutical composition comprising formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) for use in treating anemia in a patient.

[0021] In some embodiments, the anemia is selected from the group consisting of macrocytic anemia, hemolytic anemia, anemia caused by ribosomal diseases, anemia caused by dysfunction of the 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 its homologous genes, anemia caused by chromosomal translocations in the NUP98 gene or its homologous genes (such as anemia caused by a fusion of NUP98 with an Abd-B group HOX gene (e.g., HOXD13)), stress-induced anemia, anemia secondary to intestinal cancer, anemia associated with inflammatory diseases such as Diamond-Blackfan anemia, aplastic anemia, Shwachman-Diamond syndrome, rheumatoid arthritis, or multiple sclerosis, anemia associated with bone marrow failure syndromes, and anemia secondary to chemotherapy in cancer patients. In some embodiments, the anemia is associated with cancer, and optionally the cancer is a hematological malignancy, such as myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), or any hematological malignancy disclosed herein.

[0022] In some aspects, the methods and uses disclosed herein enhance erythropoiesis in bone marrow cells from MDS patients, thus ameliorating erythroid differentiation defects 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.

[0023] In some embodiments, the methods and uses disclosed herein further have the effect of increasing the patient's weight. In some embodiments, the methods and uses disclosed herein are useful for treating patients suffering from anemia associated with weight loss (e.g., cancer-associated anemia as disclosed herein). In some embodiments, the methods and uses disclosed herein are useful for treating patients suffering from anemia associated with decreased bone density (e.g., cancer-associated anemia as disclosed herein). In some embodiments, the methods and uses disclosed herein are useful for treating patients suffering from anemia associated with muscle wasting (e.g., cancer-associated anemia as disclosed herein).

[0024] In some aspects, the present disclosure provides herein new uses of orally administered formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) for the treatment of anemia, as further described herein.

[0025] I. Definition The articles "a" and "an" are used herein to refer to one or to more than one (i.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.

[0026] The term "cancer" or "tumor" or "hyperproliferative" refers to the presence of cells that have characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features.

[0027] Cancer cells often take the form of tumors, but such cells may exist alone in an animal or may be non-tumorigenic cancer cells, such as leukemia cells. As used herein, the term "cancer" includes pre-cancerous conditions and malignant cancers. Cancers include, but are not limited to, B-cell cancers, e.g., myelomas such as multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain diseases such as alpha chain disease, gamma chain disease, and mu chain disease, benign monoclonal gammopathy, and immunocytic amyloidosis, melanoma, breast cancer, lung cancer, bronchial cancer, colon cancer, prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, hematological tissue cancer, and the like. Other non-limiting examples of cancer types amenable to methods encompassed by the present disclosure include human sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial tumor, lymphangiosarcoma, lymphangioendothelial tumor, synovial tumor, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, 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 adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, liver carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, bone cancer, brain tumor, testicular cancer, Cancers include round cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemias, such as acute lymphocytic leukemia and acute myeloid leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia), chronic leukemias (chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia), and polycythemia vera, lymphomas (Hodgkin's disease and non-Hodgkin's disease), myeloma, multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease. In some embodiments, the cancer is epithelial in nature, including, but not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecological cancer, renal cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer, hi other embodiments, the cancer is breast cancer, prostate cancer, lung cancer, or colon cancer.In yet other embodiments, the epithelial cancer is non-small cell lung cancer, non-papillary renal cell carcinoma, cervical cancer, ovarian cancer (e.g., serous ovarian cancer), or breast cancer. Epithelial cancers may be characterized in a variety of other ways, including, but not limited to, serous, endometrioid, mucinous, clear cell, Brenner, or anaplastic.

[0028] The term "erythroid progenitor cells" refers to precursor cells derived from hematopoietic stem cells that give rise to erythrocytes (red blood cells) after terminal differentiation.

[0029] 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 diseases (e.g., rheumatoid arthritis or multiple sclerosis), anemia caused by dysfunction of the serine / threonine protein kinase RIOK2, anemia caused by one or more mutations and / or deletions in human chromosome 5 or its homologous genes, anemia caused by chromosomal translocations in the NUP98 gene or its homologous genes (such as anemia caused by a fusion of NUP98 with an Abd-B group HOX gene (e.g., HOXD13)), stress-induced anemia, aplastic anemia, anemia secondary to intestinal cancer, Diamond-Blackfan anemia, Shwachman-Diamond syndrome, or anemia secondary to chemotherapy in cancer patients. The anemia may be cancer-associated anemia, and optionally the cancer is a hematological malignancy (e.g., myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), or multiple myeloma (MM)). The anemia may be anemia associated with intestinal cancer, such as colorectal cancer. For example, the anemia may be the result of intestinal adenomas caused by familial adenomatous polyposis coli (FAP). Adenomatous polyposis coli (APC) is a tumor suppressor gene that is mutated in colorectal cancer. Alterations in the APC gene produce a truncated gene product, leading to activation of the Wnt signaling pathway and deregulation of multiple other cellular processes that contribute to tumorigenesis. For further details, see Su et al. Multiple intestinal neoplasia caused by a mutation in the murine homolog of the APC gene. Science. 1992 May 1;256(5057):668-70 and Moser et al. A dominant mutation that predisposes to multiple intestinal neoplasia in the mouse. Science. 1990 January 19;247(4940):322-4. The anemia can be anemia associated with a bone marrow failure disorder.Anemia may be caused by or associated with a ribosomal disease. As used herein, "ribosomal disease" refers to a disease caused by a defect in a ribosomal component or a factor that plays a role in ribosomal assembly, resulting in a defect in ribosomal biogenesis. Congenital ribosomal diseases exhibit a paradoxical transition from early symptoms of reduced cell proliferation to increased cancer risk later in life. Further details regarding ribosomal diseases can be found in Kim R Kampen et al. (2020). Nucleic Acids Res. 48(3):1013-1028. Mutations that disrupt ribosomal biogenesis often affect tissues that depend on cell division for their function. Because blood cell production from the bone marrow is highly dependent on cell division, many ribosomal diseases have an anemic component. Examples of ribosomal disorders include, but are not limited to, Diamond-Blackfan anemia (DBA), 5q-syndrome, Shwachman-Diamond syndrome (SDS), X-linked dyskeratosis congenita (DC), cartilage hair loss (CHH), Treacher Collins syndrome (TCS), Bowen-Conradi syndrome, and North American Indian childhood cirrhosis.

[0030] As used herein, myelodysplastic syndrome (MDS) includes, but is not limited to, a heterogeneous group of myeloid tumors commonly characterized by abnormal cellular morphology and, in some cases, signs of bone marrow failure with a propensity for acute myeloid leukemia (AML). In some cases, MDS is caused by mutations or deletions of human chromosome 5 or chromosomal translocations of the NUP98 gene (e.g., translocations leading to fusion of NUP98 with Abd-B group HOX genes (e.g., HOXD13)). For further details regarding chromosomal translocations of the NUP98 gene, see Lin et al. NUP98-HOXD13 transgenic mice develop a highly penetrant, severe myelodysplastic syndrome that progresses to acute leukemia. Blood. 2005 July 1;106(1):287-95.

[0031] The term "patient" refers to a human suffering from anemia.

[0032] As used herein, the phrase "co-administration" refers to any form of administration of two or more different therapeutic agents such that a second agent is administered while a previously administered therapeutic agent is still effective in the body (e.g., the two agents are effective in a subject simultaneously, which may involve a synergistic effect of the two agents). For example, the different therapeutic agents can be administered either simultaneously or sequentially, in either the same formulation or separate formulations. In certain embodiments, the different therapeutic agents can be administered within about 1 hour, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, or about 1 week of each other. Thus, a subject receiving such treatment can benefit from the combined effect of the different therapeutic agents. As used herein, any two agents and / or additional agents can be co-administered in accordance with the methods provided herein.

[0033] The term "therapeutic effect" refers to a local or systemic effect in humans caused by a pharmacologically active substance. Thus, the term refers to any substance intended for use in the diagnosis, cure, mitigation, or treatment of disease, or the enhancement of desirable physical or mental development and conditions in a patient.

[0034] The terms "therapeutically effective amount" and "effective amount" refer to the amount (e.g., dosage and duration, and enteral or oral administration means) necessary to achieve the desired therapeutic result of treating anemia. The effective amount of a β2-adrenergic receptor agonist may vary depending on factors such as the individual's condition, age, sex, and weight, as well as the ability of the monoclonal antibody to induce a desired response in the individual. An effective amount is also an amount determined by a medical provider, such as a primary care physician, in which the therapeutically beneficial effects outweigh any toxic or adverse effects of the agonist.

[0035] II.Patient The patient can be any human suffering from MDS or anemia (e.g., cancer-associated anemia as disclosed herein). The patient can be any elderly patient or any patient suffering from an age-related disease that presents with anemia.

[0036] In some embodiments, the patient is an adult patient. In some embodiments, the patient is 18 years of age or older. In some embodiments, the patient is an elderly patient. In some embodiments, the patient is 65 years of age or older.

[0037] In some embodiments, the patient is a child at least 5 years old (i.e., 5 years or older). In some embodiments, the patient is 5-18 years old.

[0038] In some embodiments, the patient is one who would benefit from weight gain. In some embodiments, the patient is one who would benefit from increased bone density. In some embodiments, the patient is suffering from anemia associated with weight loss and / or decreased bone density (e.g., cancer-associated anemia as disclosed herein).

[0039] The methods, uses, and compositions encompassed by the present invention can be used in treating anemias such as myelodysplastic syndromes (MDS), and anemia caused by dysfunction of the serine / threonine protein kinase RIOK2, anemia caused by mutations or deletions in human chromosome 5, anemia caused by chromosomal translocations in the NUP98 gene or its homologous genes (such as anemia caused by fusion of NUP98 with an Abd-B group HOX gene (e.g., HOXD13)), macrocytic anemia, anemia associated with inflammatory diseases 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 intestinal cancer, Diamond-Blackfan anemia, and Shwachman-Diamond syndrome. Similarly, the methods and compositions encompassed by the present invention can be used for bone marrow failure syndromes in general, as RIOK2 regulates blood cell development and agonists of RIOK2 activity have been shown to reverse anemia associated with bone marrow failure syndromes such as aplastic anemia, Diamond-Blackfan anemia, dyskeratosis congenita (DC), Fanconi anemia (FA), Pearson's disease, severe congenital neutropenia (SCN), and Shwachman-Diamond syndrome (SDS).

[0040] III. Medication Administration To enhance its effectiveness, formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) can be administered to a patient as disclosed herein in a biologically compatible form suitable for in vivo pharmaceutical administration according to methods encompassed by the present invention. By "biologically compatible form suitable for in vivo administration" is meant an administered form in which the therapeutic effects outweigh any toxic effects. Administration of formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) can be in any pharmacological form, including a therapeutically effective amount of the drug alone or in combination with a pharmaceutically acceptable carrier.

[0041] An exemplary formoterol (molecular formula C 19 H24 N2O4) include, but are not limited to, formoterol fumarate, arformoterol tartrate, 73573-87-2, n-[2-hydroxy-5-(1-hydroxy-2-{[1-(4-methoxyphenyl)propan-2-yl]amino}ethyl)phenyl]formamide, 128954-45-0, CHEBI:63082, or formoterolum [INN-Latin]. Commercially available forms of formoterol include, but are not limited to, AR00CJ9Q from Aaron Chemicals LLC, B1210424 from BenchChem, and AT22502 from AstaTech, Inc.

[0042] Formoterol fumarate is the fumarate form of formoterol. An exemplary molecular form of formoterol fumarate disclosed herein is formoterol fumarate (C 42 H 52 N4O 12 ), formoterol fumarate hydrate (molecular formula C 42 H 54 N4O 13 ), and formoterol fumarate dihydrate (molecular formula C 42 H 56 N4O 14Additional identifiers for formoterol fumarate include, but are not limited to, (±)-2-hydroxy-5-[(1RS)-1-hydroxy-2-[[(1RS)-2-(4-methoxyphenyl)-1-methylethyl]amino]ethyl]formoterol fumarate, (E)-but-2-enedioic acid, N-[2-hydroxy-5-[(1R)-1-hydroxy-2-[[(2R)-1-(4-methoxyphenyl)propan-2-yl]amino]ethyl]phenyl]formamide (IUPAC name), 43229-80-7 (CAS), CHEBI:31633, D01373 (ATC code), and 7848436 / 53477580 (PubChem). Commercially available forms of formoterol fumarate include, but are not limited to, Foradil® from Astellas Pharma Inc., Performomist® from Mylan Specialty LP (Viatris), generic formoterol fumarate solutions from Mylan Pharmaceuticals Inc., Alembic Pharmaceuticals Inc. (i.e., formoterol fumarate), Bryant Ranch Prepack, Lupin Pharmaceuticals, Inc., and Teva Pharmaceuticals USA, Inc., A826230 from Amadis Chemical, 33055 from AstaTech, Inc., and 141492 from ChemShuttle.

[0043] Formoterol is well known in the art, such as U.S. Pat. No. 3,994,974 A and U.S. Pat. No. 6,268,533 B1. Other formulation methods described in Remington's Pharmaceutical Sciences, 21st ed., University of the Sciences, Philadelphia, Pennsylvania, USA (2006) can also be employed in the practice of the present invention. As used herein, formoterol 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).

[0044] Arformoterol (molecular formula C 19 H 24 N2O4) is a stereoisomer of formoterol. Additional identifiers for arformoterol include, but are not limited to, N-[2-hydroxy-5-[(1R)-1-hydroxy-2-[[(2R)-1-(4-methoxyphenyl)propan-2-yl]amino]ethyl]phenyl]formamide (IUPAC), (R,R)-formoterol, 67346-49-0 (CAS), CHEBI:408174, DB01274 (DrugBank), and 3083544 (PubChem).

[0045] As used herein, "arformoterol" includes salt forms of arformoterol. Arformoterol tartrate is the tartrate salt form of arformoterol. Exemplary molecular forms of arformoterol tartrate disclosed herein include arformoterol tartrate (C 23 H 30 N2O 10 or C 19 H 24 Additional identifiers for arformoterol tartrate include, but are not limited to, N-[2-hydroxy-5-[(1R)-1-hydroxy-2-[[(1R)-2-(4-methoxyphenyl)-1-methylethyl]amino]ethyl]phenyl]-formamide, (2R,3R)-2,3-dihydroxybutanedioic acid (1:1 salt) (IUPAC), (R,R)-arformoterol tartrate, 200815-49-2 (CAS), and 9827062 (PubChem). Commercially available arformoterol tartrate salts include, but are not limited to, Brovana® (arformoterol tartrate) from Sunovion Pharmaceuticals, Inc., SML1667 from Sigma-Aldrich, S5217 from Selleckchem, 6219 from Tocris Bioscience, and A12795 from AdooQ Bioscience.

[0046] Further details regarding arformoterol are described in U.S. Pat. No. 9,499,475 B2 and U.S. Pat. No. 9,029,421 B2, which are incorporated herein by reference.

[0047] A therapeutically effective amount of a therapeutic composition of the present invention is defined as an amount effective to achieve the desired result at the required dosage and for the required period of time. For example, a therapeutically effective amount of a drug may vary depending on factors such as the individual's medical condition, age, sex, and weight, as well as the ability of the peptide to induce the desired response in the individual. Dosage regimens can be adjusted to provide the optimal therapeutic response. For example, several divided doses can be administered once daily, or the dose can be proportionally reduced as indicated by the exigencies of the therapeutic situation.

[0048] The agents included in the present invention can be administered alone or in combination with additional therapies. In combination therapy, the agents included in the present invention 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 agents (e.g., luspatercept, lenalidomide, and / or hypomethylating agents (azacitidine or decitabine)), can be delivered to the same cell or different cells, and at the same time or different times. The agents included in the present invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions can include 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.

[0049] The therapeutic agents described herein (e.g., formoterol or a pharmaceutically acceptable salt thereof, such as formoterol fumarate or arformoterol tartrate) can be administered in a convenient manner, such as orally, by injection (subcutaneous, intravenous, intraperitoneal (ip), etc.), inhalation, transdermal application, or rectal administration. Depending on the route of administration, the active compound may be coated with a material to protect the compound from the action of enzymes, acids, and other natural conditions that may inactivate the compound. For example, when administering an agent by a route other than parenteral administration, it may be desirable to coat the agent with, or co-administer the agent with, a material to prevent its inactivation.

[0050] As described in more detail below, pharmaceutical compositions encompassed by the present invention (e.g., compositions comprising formoterol or a pharmaceutically acceptable salt thereof, such as formoterol fumarate or arformoterol tartrate) can be specially formulated for administration in solid or liquid form, including those suitable for: (1) oral administration, e.g., drench (aqueous or non-aqueous solution or suspension), tablet, bolus, powder, granule, paste, or (2) parenteral administration, e.g., as a sterile solution or suspension, e.g., by subcutaneous, intramuscular, intraperitoneal (ip) or intravenous injection.

[0051] The phrase "pharmaceutically acceptable" is used herein to refer to agents, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with human tissues without undue toxicity, irritation, allergic response, or other problem or complication, and commensurate with a reasonable benefit / risk ratio.

[0052] As used herein, the phrase "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, involved in carrying or transporting a chemical substance from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient. Those skilled in the art will recognize or be able to use suitable pharmaceutically acceptable carriers for the therapeutic agents disclosed herein (e.g., formoterol or a pharmaceutically acceptable salt thereof, such as formoterol fumarate or arformoterol tartrate) from pharmaceutically acceptable carriers known in pharmacology (see, e.g., Adejare, Adeboye, ed. Remington: The Science and Practice of Pharmacy. Academic Press, 2020).

[0053] The term "pharmaceutically acceptable salts" refers to the relatively non-toxic inorganic and organic acid addition salts of the therapeutic agents disclosed herein (i.e., formoterol, whose pharmaceutically acceptable salts include, for example, formoterol fumarate and arformoterol tartrate). These salts can be prepared in situ during the final isolation and purification of the therapeutic agent, or by separately reacting the purified therapeutic agent in its free base form with a suitable organic or inorganic acid and isolating the salt so formed. Those skilled in the art will recognize or be able to use suitable pharmaceutically acceptable salts for the therapeutic agents disclosed herein (e.g., formoterol, whose pharmaceutically acceptable salts include, for example, formoterol fumarate and arformoterol tartrate) from pharmaceutically acceptable salts known in pharmacology (see, for example, Berge et al. (1977) J. Pharm. Sci. 66:1-19).

[0054] Wetting agents, emulsifying agents, and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring, and perfuming agents, preservatives, and antioxidants can also be present in the compositions.

[0055] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, and sodium sulfite; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, and alpha-tocopherol; and (3) metal chelators such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0056] Formulations useful in the methods encompassed by the present invention include those suitable for oral administration, intravenous administration, and / or administration by injection (e.g., intraperitoneal (ip) injection). The formulations may be conveniently presented in unit dosage form and may be prepared by any method well known in the art of pharmacy. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the host being treated and the particular mode of administration. In some embodiments, formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) is in a form formulated for oral administration. Exemplary oral forms of formoterol can be found in the art, such as Yokoi et al. (1983) Life Sciences 33:1665-1672. In some embodiments, the medicament provided herein is an oral formulation of formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate).

[0057] In general, the formulations are prepared by uniformly and intimately bringing into association the therapeutic agent with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0058] Formulations suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored base, usually sucrose and acacia or tragacanth), powder, granules, or as a solution or suspension in an aqueous liquid or a non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as a troche (using an inert base such as gelatin and glycerin, or sucrose and acacia), and / or as a mouthwash, etc., each containing a predetermined amount of the therapeutic agent as an active ingredient. The compound may also be administered as a bolus, electuary, or paste.

[0059] In solid dosage forms for oral administration (e.g., capsules, tablets, pills, dragees, powders, granules, etc.), 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 starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia; (3) humectants, such as glycerol; (4) chilling agents, such as hydroxybenzoates, ... Disintegrating agents such as starch, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarders such as paraffin; (6) absorption accelerators such as quaternary ammonium compounds; (7) wetting agents such as acetyl alcohol and glycerol monostearate; (8) absorbents such as kaolin and bentonite clay; (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets, and pills, pharmaceutical compositions may also contain buffering agents. Solid compositions of a similar type may also be used as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or milk sugar, as well as high molecular weight polyethylene glycols.

[0060] Tablets can be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared using binders (e.g., gelatin or hydroxypropylmethylcellulose), lubricants, inert diluents, preservatives, disintegrants (e.g., sodium starch glycolate or cross-linked sodium carboxymethylcellulose), surface-active agents, or dispersing agents. Molded tablets can be made by molding in a suitable machine a mixture of powdered peptide or peptidomimetic moistened with an inert liquid diluent.

[0061] Tablets and other solid dosage forms, such as dragees, capsules, pills, and granules, can optionally be perforated or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulation art. They can also be formulated to provide sustained or controlled release of the active ingredient therein, for example, using hydroxypropylmethylcellulose, other polymer matrices, liposomes, and / or microspheres in various proportions to provide the desired release profile. They can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that 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 optionally be of a composition that releases the active ingredient(s) only, or preferentially, in a delayed manner in a certain part of the gastrointestinal tract. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.

[0062] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.In addition to active ingredients, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan, and mixtures thereof.

[0063] 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.

[0064] Suspensions may contain, in addition to the active agent, suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.

[0065] Pharmaceutical compositions encompassed by the present invention suitable for parenteral administration comprise one or more therapeutic agents in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted into sterile injectable solutions or dispersions immediately before use, which may contain antioxidants, buffers, bacteriostats, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.

[0066] These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.Prevention of microbial activity can be ensured by including various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol sorbic acid, etc.It may also be desirable to include isotonic agents, such as sugars and sodium chloride, in the composition.In addition, the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin, can result in prolonged absorption of injectable pharmaceutical forms.

[0067] In some cases, in order to prolong the effect of a drug, it is desirable to delay the absorption of the drug from subcutaneous or intramuscular injection.This can be achieved by using a liquid suspension of crystalline or amorphous material with poor water solubility.The absorption rate of the drug then depends on its dissolution rate, which may depend on crystal size and crystalline form.Alternatively, delayed absorption of parenterally administered drug forms can be achieved by dissolving or suspending the drug in an oil vehicle.

[0068] Injectable depot dosage forms are made by forming microencapsule matrices of formoterol or its pharmaceutically acceptable salts (e.g., formoterol fumarate or arformoterol tartrate) in biodegradable polymers such as polylactide-polyglycolide. The rate of drug release can be controlled depending on the ratio of drug to polymer and the nature of the particular polymer employed. 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 that are compatible with body tissues.

[0069] When the therapeutic agent included in the present invention is administered to humans as a pharmaceutical, the therapeutic agent may be given as is or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably 0.5 to 90%) of the active ingredient in combination with a pharmaceutically acceptable carrier.

[0070] It will be understood that the total daily dosage of the compounds of the present invention will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for a particular subject will depend on a variety of factors, including the anemia being treated and the severity of the anemia, the specific composition employed, the subject's age, weight, general health, sex, and diet, the time and route of administration, and the plasma half-life of the particular compound employed, the duration of treatment, any medications used in combination or concomitantly with the particular agonist employed, as well as factors well known in the medical art. The daily dosage of the active ingredient may comprise, but is not limited to, about 0.1 to 100 μg per adult per day. Typically, pharmaceutical compositions contain 0.1, 1.0, 5.0, 10.0, 12.0, 15.0, 20.0, 50, 75, or 100 μg of formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate), with 1 to 60 μg being preferred. In some embodiments, the effective amount of formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) is administered at a dose of 100 μg / day or less. In some embodiments, the effective amount of formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) is administered at a dose of 0.1 μg / day to 100 μg / day. In some embodiments, the effective amount of formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) is administered at a dose of 1 μg / day to 60 μg / day. In some embodiments, methods are provided for treating anemia by administering to a patient a tablet or capsule containing formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) at a dose of 0.1 μg to 100 μg. In some embodiments, the tablet or capsule is administered once daily. In some embodiments, a patient is administered a tablet or capsule containing formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) at a dose of 1 μg to 60 μg once daily.

[0071] In some embodiments, methods are provided for treating anemia by administering to a patient one or more tablets or capsules comprising formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) so that the subject receives a total daily dose of 0.1 μg to 100 μg, preferably a total daily dose of 1 μg to 60 μg.

[0072] An effective amount of agent is typically provided in an amount of formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) effective to treat anemia, at a dose and via a route of administration to achieve a plasma concentration of 0.03 to 150 pg / mL, preferably 0.3 to 30 pg / mL.

[0073] Actual dosage levels of the active ingredients in the pharmaceutical compositions encompassed by the present invention can be determined by methods encompassed by the present invention to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and method of administration without being toxic to the patient. [Example]

[0074] Example 1: Formoterol fumarate (FF) enhances erythroid differentiation and mitochondrial function Herein, we describe a novel therapeutic approach targeting anemia in MDS and other human diseases by utilizing formoterol fumarate (FF), an FDA-approved β2-adrenergic receptor agonist for the treatment of COPD and asthma (Hanania et al. (2019) Int J Chron Obstruct Pulmon Dis. 14:117-127; Sharafkhaneh et al. (2010) Int J Chron Obstruct Pulmon Dis. 5:357-366). Herein, we demonstrate that formoterol fumarate (FF) simultaneously enhances mitochondrial biogenesis and erythroid differentiation in primary human hematopoietic stem and progenitor cells (HSPCs). In support of this, depletion of ADRB2, which encodes the β2-adrenergic receptor (β2-AR), significantly impairs not only mitochondrial biogenesis but also erythroid differentiation. FF treatment significantly enhanced erythropoiesis in bone marrow cells derived from MDS patients. Administration of FF significantly enhanced erythroid differentiation and RBC parameters in wild-type mice under both steady-state and stress-induced hemolytic anemia conditions. FF treatment provided a significant survival advantage to mice in response to lethal hemolytic anemia. Therefore, novel uses of existing FDA-approved drugs have been identified to reverse anemia and provide therapeutic benefits for a variety of human diseases, including, but not limited to, hematologic malignancies, aplastic anemia, anemia in chronic kidney disease, ribosomal disorders, anemia secondary to chemotherapy in cancer patients, and bone marrow failure (BMF) disorders.

[0075] Hematological disorders and mitochondrial dysfunction. Mitochondrial dysfunction is often associated with the development of hematological disorders such as MDS and AML (Fontenay et al. (2006) Oncogene 25:4757-4767). For example, alterations in mitochondrial transcription (Schildgen et al. (2011) Exp. Hematol. 39:666-675), deregulated HIF1α expression (Liu et al. (2019) Oncol. Lett. 17:5395-5402; Stergiouet et al. (2021) Int. J. Mol. Sci. 22), the presence of isocitrate dehydrogenase (IDH) mutations that express the oncogenic metabolite 2-hydroxyglutarate (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), and elevated mitochondrial oxidative stress markers (Saigo et al. (2011) Exp. Hematol. 39:666-675). al. (2011) J. Int. Med. Res. 39:1941-1945), and the presence of abnormal oxidation and mutations in mitochondrial DNA (mtDNA) (Coelho-Silva et al. (2021) Sci. 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 function as a key player in the pathogenesis of MDS. MtDNA, together with the nuclear-encoded transcription and translation machinery, encodes 13 polypeptides that form essential components of the electron transport chain (ETC) responsible for oxidative phosphorylation (OXPHOS) (Itoh et al. (2021) Science 371:846-849; Kummer and Ban (2021) Nat. Rev. Mol. Cell Biol. 22:307-325).Thus, the mitochondrial and nuclear genomes remain interwoven in a tightly balanced relationship to determine cellular metabolism and prevent deregulation that would inevitably alter metabolic profiles (Alston et al. (2021) J. Pathol.; Ito and Ito (2018) Exp Hematol 64:1-11; Schildgen et al. (2011) Exp. Hematol. 39:666-675; Zheng et al. (2017) Chin. J. Physiol. 60:338-344). Consistently, elevated tryptophan catabolites in the serum of MDS patients (Berthon et al. (2013) Leuk. Res. 37:573-579) and increased reactive oxygen metabolites in patients with karyotypic abnormalities (Cano et al. (2011) J. Proteome Res. 10:2873-2881; Fracchiolla et al. (2003) Haematologica 88:594-597; Poulaki et al. (2020) Cancers (Basel) 12; Zhong et al. (2015) Genet. Mol. Res. 14:13709-13718) further highlight the involvement of dysregulated metabolites and metabolic pathways in MDS pathogenesis. Dysregulated metabolite levels have also been reported in bone marrow failure diseases (Zhong et al. (2015) Genet. Mol. Res. 14:13709-13718). The present disclosure includes the recognition that mitochondrial defects contribute to the development of hematological disorders.

[0076] β2-adrenergic receptor (β2-AR) agonists and mitochondrial biogenesis The β2-AR is a transmembrane receptor for adrenaline (epinephrine) that mediates 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). The β2-AR is encoded by the ADRB2 gene. Formoterol fumarate (FF), a long-acting, selective β2-AR agonist, is FDA-approved for the treatment of COPD and asthma (Hanania et al. (2019) Int J Chron Obstruct Pulmon Dis 14:117-127; Ni et al. (2018) Cochrane Database Syst Rev 12, CD011594; Sharafkhaneh et al. (2010) Int J Chron Obstruct Pulmon Dis 5:357-366). FF is highly specific for β2-AR, as examined in ADRB2-deficient mice (Cameron et al. (2017) Sci Rep 7:10578). Interestingly, FF has been reported to enhance mitochondrial biogenesis in several in vitro and in vivo models (Peterson et al. (2013) Bioorg Med Chem Lett 23:5376-5381; Wills et al. (2012) J Pharmacol Exp Ther 342:106-118), such as ischemia-reperfusion-mediated kidney injury (Jesinkey et al. (2014) J Am Soc Nephrol 25:1157-1162), spinal cord injury (Scholpa et al. (2019) Exp Neurol 322:113064), and traumatic brain injury (Vekaria et al. (2020) Neurobiol Dis 140:104866.0).FF not only upregulates the expression of mtDNA-encoded genes but also promotes mitochondrial oxygen consumption rate (OCR) in vitro and ex vivo (Arif et al. (2019) Kidney Int 96:656-673). However, the underlying mechanisms of FF-mediated enhancement of mitochondrial biogenesis have yet to be fully elucidated.

[0077] result Formoterol fumarate (FF) enhances erythroid differentiation and mitochondrial function in primary human hematopoietic stem and progenitor cells (HSPCs). Given the involvement of β2-AR in erythropoiesis, we hypothesized that administration of the long-acting, selective β2-AR agonist FF might promote erythroid differentiation. To address this, we treated primary human HSPCs with various doses of FF (Selleckchem). Notably, FF administration dose-dependently increased erythroid differentiation of primary human HSPCs (Figure 1A) without affecting viability, megakaryopoiesis, or myelopoiesis (Figure 1B and Figure 1D). Confirmatory studies showed that formoterol fumarate (FF) from two different vendors (Selleckchem and Sigma) similarly enhanced erythroid differentiation in primary human HSPCs (Figure 3A and Figure 3B). To confirm whether FF administration plays a role further upstream in the erythroid differentiation pathway, we performed a methylcellulose colony formation assay. Indeed, FF treatment significantly increased the formation of burst-forming units-erythroid (BFU-E) and colony-forming units-erythroid (CFU-E) (Figure 4). Consistent with existing literature, treatment with FF enhanced the expression of mtDNA-encoded genes required for OXPHOS (Figure 4A) and increased mitochondrial mass and membrane potential in primary human HSPCs (Figures 5B and 5C). These data demonstrate the therapeutic potential of FF in alleviating anemia.

[0078] FF treatment in in vitro and ex vivo models of MDS Because RIOK2-deficient primary human HSPCs effectively model the impaired erythropoiesis that leads to anemia (Ghosh et al. (2022) Nat Immunol 23:109-121), we next investigated whether treatment with formoterol fumarate (FF) would alleviate anemia in this in vitro model. To address this, RIOK2-deficient and -deficient primary human HSPCs were generated via CRISPR-Cas9-based genome editing as previously described (Figure 6A) (Ghosh et al. (2022) Nat Immunol 23:109-121). Formoterol fumarate consistently enhanced erythroid differentiation of control HSPCs (Figure 6B). Notably, administration of FF stimulated erythropoiesis in RIOK2-knockdown (KD) HSPCs (Figure 6B). Notably, FF treatment enhanced erythroid differentiation of RIOK2-deficient HSPCs to a level comparable to the basal erythropoiesis of control cells (Figure 6B). Because deletion of RPS14 has been reported as a MDS gene (Ebert et al. (2008) Nature 451:335-339; Schneider et al. (2016) Nat Med 22:288-297), RPS14 was similarly knocked down in primary human HSPCs (Figure 6C). FF administration significantly increased erythroid differentiation in RPS14-deficient HSPCs (Figure 6D). Next, to verify the specificity of FF for the β2-adrenergic receptor, ADRB2 was knocked down in primary human HSPCs and treated with FF. FF treatment failed to induce erythropoiesis in ADRB2-deficient HSPCs due to the deletion of the ADRB2 gene encoding the β2-AR.

[0079] These findings prompted further testing to determine whether administering FF to bone marrow (BM) samples from MDS patients would improve erythroid differentiation in an ex vivo environment. To investigate this, cells isolated from BM aspirates of deidentified MDS patients were obtained. Next, BM cells from MDS patients were placed in liquid erythroid differentiation cultures with or without FF treatment, as previously described (Khajuria et al., (2018) Cell 173:90-103). Indeed, treatment with FF (from Selleckchem) significantly enhanced erythroid differentiation of BM cells in the majority of MDS patients, whereas BM cells from a minority of MDS patients were unresponsive (Figure 8A). To further validate this observation, the effect of FF from another vendor (Sigma) was tested on MDS patient-derived cells. Similarly, FF (from Sigma) promoted erythropoiesis in the majority of MDS patients (Figure 8B). Next, CD34+ progenitor cells were FACS-sorted from BM aspirates from MDS patients and seeded in semi-solid methylcellulose culture medium with vehicle (DMSO) or FF treatment. FF significantly promoted the formation of BFU-E and CFU-E in BM progenitor cells from MDS patients (Figures 11A-11C). However, FF administration had no significant effect on the viability, myeloid, or megakaryocytic differentiation of BM cells from MDS patients (Figure 11D). Therefore, we concluded that formoterol fumarate (FF) potently enhances erythroid differentiation not only in primary hematopoietic stem cells from healthy individuals but also in BM cells from MDS patients. Therefore, formoterol fumarate, an FDA-approved drug, can be repurposed to reverse anemia in various hematological disorders.

[0080] FF treatment reduces anemia in vivo The mouse β2-adrenergic receptor shares 87.08% similarity in protein composition with the human analog. This prompted further testing to determine whether formoterol fumarate (FF) treatment in wild-type mice with steady-state or hemolytic anemia would have any mitigating effects in vivo. To address this, 10-12 week-old wild-type C57BL / 6J mice were administered various doses of FF (0.1-0.3-0.5-1.0 mg / kg) or vehicle (0.3% DMSO in saline) daily via intraperitoneal (ip) injection for 3 weeks, followed by weekly submandibular cheek bleeds for complete blood count (CBC). Daily i.p. injections of FF were well tolerated by the mice, as seen in their weight (Figure 14A), motility, activity, and regular breathing patterns (as advised by the animal research facility's veterinarian). At FF doses of 0.3-0.5 and 1.0 mg / kg, mice's body weight increased slightly, but the increase did not reach significance (Figure 14A). However, FF treatment at all doses significantly enhanced mitochondrial biogenesis in mouse peripheral blood mononuclear cells (PBMCs), as evidenced by increased MitoTracker® staining (Figure 14B). Notably, red blood cell (RBC) parameters such as RBC count, hematocrit (HCT) % and hemoglobin (Hb) were slightly elevated in the FF-treated group (Figures 14C-14E), whereas white blood cell (WBC) and monocyte counts remained slightly affected (Figures 14F and 14G).

[0081] These results prompted further investigation to determine the potential effects of FF in mice undergoing non-lethal hemolytic anemia. To address this, we first administered a sublethal dose of phenylhydrazine (PHZ: 50 mg / kg) to 10- to 12-week-old female mice, followed by daily intraperitoneal injections of FF at 0.1 / 0.3 mg / kg or vehicle (0.3% DMSO in saline). PHZ is a potent oxidant that readily oxidizes hemoglobin in RBCs, inducing their immediate lysis and thus causing hemolytic anemia. FF treatment at a low dose of 0.1 / 0.3 mg / kg was sufficient to restore body weight in mice undergoing PHZ-induced hemolytic anemia (Figure 18A). FF treatment significantly enhanced Hb production compared to the vehicle-treated group (Figure 18B). FF administration also provided long-term benefits in increasing the HCT% and RBC count in mice compared to the control group (Figure 18B). FF treatment immediately increased WBC and monocyte counts after PHZ administration, but in contrast to RBC parameters, no long-term benefits were observed in the FF-treated group (Figure 18C). Platelet counts were not statistically significant in the FF-treated group (Figure 18C). These findings are consistent with in vitro and ex vivo experiments, in which FF treatment specifically enhanced erythroid differentiation, with no significant changes in the myeloid and megakaryocytic compartments (Figures 1A-1D and 11A-11D). To exclude the possibility that the FF-mediated increase in RBC parameters was a spurious effect, mice were sacrificed after the study endpoint (day 14) and their bone marrow (BM) progenitor cells were analyzed. Interestingly, FF treatment at low doses of 0.1-0.3 mg / kg significantly enhanced erythroid differentiation, as evidenced by a significant increase in RI, RII, RIII, and RIV erythroid progenitors in the BM of mice (Figures 19A and 19B). This clearly demonstrates that FF acts directly on BM progenitor cells to induce erythroid differentiation. To substantiate these findings, this experiment was repeated in 10-12 week-old male C57BL / 6J mice. In response to sublethal PHZ-induced hemolytic anemia (PHZ dose: 60 mg / kg), FF (0.1 / 0.3 mg / kg)-treated mice exhibited a similarly significant recovery in body weight compared to the vehicle-treated group (Figure 22A).FF treatment consistently improved RBC parameters, such as Hb, RBC count, and HCT%, in the PB of anemic mice compared with vehicle-treated controls (Figure 22B). Again, except for the immediate boost after PHZ treatment, no long-term effects were observed on WBC parameters or platelets in the FF-treated group (Figure 22C). Surprisingly, BM cells from the FF-treated group showed a significant increase in viability, partially due to mitochondrial biogenesis, as evidenced by increased mitoTracker® staining (Figure 23B) and decreased mitochondrial superoxide production (mitoSOX staining) (Figure 23C) (Figure 23A). Thorough analysis of viable lineage-negative BM progenitors showed that FF treatment specifically increased megakaryocytic erythroid progenitors (MEPs), but not common myeloid progenitors (CMPs) or granulocytic monocytic progenitors (GMPs) (Figures 26A-26D). Because MEPs give rise to either megakaryocyte or erythroid progenitors, both BM cell compartments were examined. However, no significant differences were observed in megakaryocyte progenitors (Figure 27), consistent with the significant effect of FF treatment on PB platelets (Figure 22C). FF treatment significantly enhanced RI, RII, RIII, and RIV erythroid progenitors in the BM of male mice (Figures 30A-30B), consistent with the FF-mediated improvement of erythroid differentiation in the BM of female mice (Figures 19A-19C). These findings demonstrate that formoterol fumarate (FF) directly acts on early BM progenitors to stimulate erythroid differentiation, resulting in enhanced RBC parameters in the peripheral blood (PB) in vivo.

[0082] The significant reduction in RBC parameters and weight recovery in anemic mice treated with FF encouraged further testing of the survival benefit of FF against phenylhydrazine (PHZ)-mediated lethal hemolytic anemia. To this end, 10- to 12-week-old male C57BL / 6J mice were first treated with a single lethal dose of 150 mg / kg PHZ, followed by daily intraperitoneal injections of vehicle or 0.1 / 0.3 mg / kg FF. FF treatment at the low dose of 0.1 / 0.3 mg / kg conferred a significant survival benefit compared to the vehicle-treated group (Figure 42A). All control mice (n = 5) became moribund and died by day 3 of the lethal PHZ dose, whereas 3-4 mice in the 0.1 / 0.3 mg / kg FF-treated group were still alive 2 weeks after the lethal dose (Figure 42A). FF treatment significantly restored mouse body weight within the first 7 days of the lethal PHZ dose (Figure 42B). FF injections were stopped 7 days after the lethal PHZ treatment, and it was observed that the mouse weights remained stable (Figure 42B). The weights and survival of these mice are being monitored daily. This unexpected finding prompted us to repeat this lethal hemolytic anemia model in female mice. Consistently, FF treatment at a dose of 0.1 / 0.3 mg / kg conferred a significant survival benefit to female mice, whereas all vehicle-treated mice (n = 5) were moribund or dead by day 3 of the lethal PHZ dose (Figure 42C). FF treatment similarly restored mouse body weight after the lethal anemic stress (Figure 42D). FF treatment continued for 7 days after the lethal PHZ administration, and the weights and survival of these mice are being monitored daily. These data establish that FF produces a significant survival benefit in vivo for hemolytic anemia, thus demonstrating the therapeutic potential of FF in the treatment of anemia.

[0083] Example 2: Materials and Methods from Example 1 Mouse experiments C57BL / 6J mice were obtained from The Jackson Laboratory and housed at the Dana-Farber Cancer Institute (DFCI) Animal Resources Facility for at least 2 weeks before starting the experiments. All drug treatments are mentioned in detail in the text.

[0084] Primary and secondary cell cultures CD34+ primary human hematopoietic stem and progenitor cells (HSPCs) were obtained and cultured from the Fred Hutchinson Cancer Research Center, Seattle, USA, as previously described. Formoterol fumarate was purchased from Selleckchem (#S2020) and Sigma (#F9552), dissolved (in DMSO) according to the manufacturer's instructions, stored, and used at the final working concentration indicated. Frozen BM aspirates from de-identified MDS patients were obtained from the Pasquarello Tissue Bank at DFCI using an IRB-approved protocol (#21-632).

[0085] Mitochondrial fitness test TMRE (T669, Invitrogen) and Mitotracker® (M22426, Life Technologies) and MitoSox® (M36008, Life Technologies) staining was performed according to the manufacturers' instructions.

[0086] CRISPR / Cas9 gene editing Primary human HSPCs were genome-edited as previously described (Ghosh, S., Raundhal, M., Myers, SA, Carr, SA, Chen, X., Petsko, GA, and Glimcher, LH (2022). Identification of RIOK2 as a master regulator of human blood cell development. Nat Immunol 23, 109-121).

[0087] Quantitative RT-PCR As previously described herein.

[0088] Flow cytometry and sorting FACS staining and sorting were performed as previously described (Ghosh et al. (2022) Nat Immunol 23:109-121).

[0089] Methylcellulose assay After genome editing, primary human HSPCs were washed twice with 1x PBS and mixed with semi-solid methylcellulose medium (H4034 StemCell Technologies) by brief vortexing. Cells were seeded into 6-well plates at a density of 2000 cells per well and then incubated in a humidified chamber at 37°C for 14 days. Colony imaging was performed using an EVOS M5000 Imaging System® (ThermoFisher Scientific). Colony-forming cells were then collected by triturating the wells with staining buffer, and multicolor flow cytometry was performed as previously described (Li et al. (2014) Blood 124:3636-3645).

[0090] statistical tests Data are presented as mean ± SEM. An unpaired, two-tailed t-test was used to compare two groups. Analysis of variance (ANOVA) with Tukey's correction or the Kruskal-Wallis test with Dunn's correction was used for comparisons between multiple groups, when applicable according to the data and quantification requirements. GraphPad Prism v8.0 / 9.0 (GraphPad Software Inc., San Diego, CA) was used to perform statistical analysis. Sample size was not predetermined.

[0091] Example 3: Nonlinear dose-specific effects of formoterol fumarate (FF) treatment in wild-type mice As described in Example 1 above, formoterol fumarate (FF) treatment in wild-type mice was well tolerated (Figures 14A-14E). The effects of various doses of FF, ranging from 0.1 to 1.0 mg / kg, were evaluated in naive mice without the external stress of phenylhydrazine (PHZ). Specifically, various doses of FF (0.1-0.3-0.5-1.0 mg / kg) or vehicle (0.3% DMSO in saline) were administered via intraperitoneal (ip) injection daily for 30 days to 10-12 week-old wild-type C57BL / 6J mice.

[0092] Treatment with FF at 0.1 to 1.0 mg / kg induced a dose-dependent increase in body weight, and doses of 0.1 to 0.5 mg / kg moderately increased the body weight of naive mice. In particular, a significant increase in body weight was observed at the highest dose of FF, 1.0 mg / kg (Figure 83).

[0093] Interestingly, a dose-dependent effect was observed on red blood cell (RBC) parameters, with effects observed at low to mid-dose but not at the highest dose. As shown in Figure 84A, hemoglobin (Hb) levels increased with dose from 0.1 mg / kg FF to 0.5 mg / kg FF, but no effect was observed at the highest dose of 1.0 mg / kg FF. Similarly, hematocrit (HCT) % and RBC counts increased in mice treated with FF at doses of 0.3 mg / kg and 0.5 mg / kg, but not at the lowest dose (0.1 mg / kg FF) or the highest dose (1.0 mg / kg FF) (Figures 84B-84C).

[0094] White blood cell (WBC) counts were only slightly affected at the highest dose of 1.0 mg / kg FF (Figure 85A), whereas monocyte counts were only slightly affected at 0.3 mg / kg and 0.5 mg / kg FF doses but not at the highest or lowest doses (1.0 mg / kg and 0.1 mg / kg FF, respectively) (Figure 85B). No effect was observed on platelets following administration of FF at any dose (Figure 85C).

[0095] These results support that FF has a nonlinear dose-specific effect on blood cell parameters, in contrast to its effect on body weight.

[0096] Example 4: Bone mass in mice treated with formoterol fumarate (FF) As described in Example 1 above, formoterol fumarate (FF) treatment is associated with increased body weight, and FF treatment also restored body weight in mice with phenylhydrazine (PHZ)-mediated anemia. FF has been shown to have anabolic effects on bone in an ovariectomized rat model, which may be due, at least in part, to FF's agonistic effect on beta-adrenergic receptors expressed in bone cells. See Kellenberger et al., (1998) Bone 22(5):471-478, incorporated by reference. FF has also been demonstrated to increase muscle mass in mice and improve muscle structure and function after injury. See Gehrig et al., (2010) The American Journal of Pathology 176(1):29-33 and Ryall et al. (2008) J. Appl. Physiol. 105:165-172. To determine whether increased body weight is associated with increased bone and / or muscle mass, dexascans are performed. Specifically, C57BL / 6J mice (2-3 month old, wild-type, steady-state mice (not receiving phenylhydrazine treatment) are treated with FF at a dose of 0.3 mg / kg or vehicle (0.3% DMSO in saline) by intraperitoneal (i.p.) injection five times a week for one month, after which the mice's bone mineral density, muscle mass, and fat mass, as well as free and total water content, are measured by Dexascan. Mice treated with other β2-adrenergic receptor agonists not associated with weight gain are also evaluated.

[0097] Example 5: RNA sequencing of mice treated with formoterol fumarate (FF) In this example, Klf9 (Ren et al., (2018) Yi Chuan.40(11):998-1006 and Zhang et al., (2017) Blood 130(20):2161-2170), Klf13, Klf11 (Emery (2007) J.Cell.Biochem 100(4):1045-55), Hif1a (Wellmann, et al., (2004) J.Cell Sci.117:175-94; Feng et al., (2022) Nature 610(7933):783-790; and Chen et al., (2019) N Engl J Med381:1011-1022),Epas1(Scortegagna et al.(2003)Blood The effects of formoterol fumarate treatment on the expression of genes related to erythropoiesis, including FF (102:1634-40), Ddit4 (Yoshikawa (2023) Cancer Biomark. 37(4):217-225), Cirbp, and Rora (Kim et al. (2008) Arterioscler Thromb Vasc Biol 28(10):1796-802 and Chauvet et al., (2004) Biochem. J. 384:79-85), are described. Differential gene expression in naive wild-type mice was analyzed by bulk RNA sequencing, following the exemplary workflow shown in Figure 86. Exemplary differential gene expression in erythroid progenitor cells isolated from the bone marrow of FF-treated and vehicle-treated mice is shown in Figures 87A-B. In FF-treated mice, the expression of numerous genes involved in erythropoiesis, including Klf9, Klf13, Klf11, Hif1a, Epas1, Ddit4, Cirbp, and Rora, was increased. Notably, Figures 8A-B show that the expression of both HIF-1α and VEGFA was significantly increased during FF treatment in wild-type mice, as verified by qPCR analysis. These data support the multitarget effect of formoterol in increasing erythropoiesis and treating anemia.

[0098] Incorporation by Reference All publications, patents, and patent applications mentioned herein are herein 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.

[0099] equivalent 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 invention described herein which equivalents are intended to be encompassed by the following claims.

[0100] Other abbreviations CFU-GM: Colony-forming unit-granulocyte macrophage CFU-MK: Colony forming unit-megakaryocyte COPD: chronic obstructive pulmonary disease RIOK2: Right open reading frame kinase 2 TMRE: tetramethylrhodamine ethyl ester

Claims

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

2. 2. The method of claim 1, wherein the anemia is selected from the group consisting of macrocytic anemia, hemolytic anemia, anemia caused by ribosomal diseases, anemia caused by dysfunction of the 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 its homologous genes, stress-induced anemia, Diamond-Blackfan anemia, aplastic anemia, Shwachman-Diamond syndrome, anemia associated with inflammatory diseases such as rheumatoid arthritis or multiple sclerosis, anemia secondary to chemotherapy in cancer patients, and anemia associated with bone marrow failure syndromes.

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

4. 3. The method of claim 1 or claim 2, wherein the anemia is associated with cancer, and optionally the cancer is a bowel cancer, such as colon cancer.

5. 1. A method for promoting differentiation of erythroid progenitor cells into mature erythrocytes in a patient in need thereof, comprising administering to said patient in need thereof an effective amount of formoterol or a pharmaceutically acceptable salt thereof.

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

7. 7. The method of claim 6, wherein the anemia is selected from the group consisting of macrocytic anemia, hemolytic anemia, anemia caused by ribosomal diseases, anemia caused by dysfunction of the 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 its homologous genes, stress-induced anemia, Diamond-Blackfan anemia, aplastic anemia, Shwachman-Diamond syndrome, anemia associated with inflammatory diseases such as rheumatoid arthritis or multiple sclerosis, anemia secondary to chemotherapy in cancer patients, and anemia associated with bone marrow failure syndromes.

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

9. 8. The method of claim 6 or claim 7, wherein the anemia is associated with cancer, and optionally the cancer is intestinal cancer, such as colon cancer.

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

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

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

13. 13. The method of claim 12, wherein the anemia is selected from the group consisting of macrocytic anemia, hemolytic anemia, anemia caused by ribosomal diseases, anemia caused by dysfunction of the 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 its homologous genes, stress-induced anemia, Diamond-Blackfan anemia, aplastic anemia, Shwachman-Diamond syndrome, anemia associated with inflammatory diseases such as rheumatoid arthritis or multiple sclerosis, anemia secondary to chemotherapy in cancer patients, and anemia associated with bone marrow failure syndromes.

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

15. 14. The method of claim 12 or claim 13, wherein the anemia is associated with cancer, and optionally the cancer is a bowel cancer, such as colon cancer.

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

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

18. 18. The method of any one of claims 1 to 17, wherein the formoterol or a pharmaceutically acceptable salt thereof is formoterol fumarate.

19. 18. The method of any one of claims 1 to 17, wherein the formoterol or a pharmaceutically acceptable salt thereof is arformoterol.

20. 20. The method of claim 19, wherein the arformoterol is arformoterol tartrate.

21. 21. The method of any one of claims 1 to 20, wherein formoterol or a pharmaceutically acceptable salt thereof is administered in combination with an erythropoiesis-stimulating agent (ESA), such as luspatercept, lenalidomide, epoetin alfa or darbepoetin alfa, and / or a hypomethylating agent, optionally azacitidine and / or decitabine.

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