Methods of treating anemia using formoterol or pharmaceutically acceptable salt thereof

Abstract

In one embodiment, the present invention provides a method 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 some embodiments, the formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or afmoterol tartrate) may be administered in combination with an erythropoiesis stimulating agent, optionally wherein the anemia is refractory to the erythropoiesis stimulating agent. The invention further provides a method of promoting differentiation of erythroid progenitor cells into mature erythrocytes in a patient in need thereof, the method comprising administering an effective amount of formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or afformoterol tartrate). In particular, the formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or afformoterol tartrate) may be administered in combination with other FDA-approved drugs, such as lutetium, lenalidomide, an erythropoiesis stimulating agent (ESA), and / or a demethylating agent, such as azacitidine, and / or decitabine, and may be administered in combination with other FDA-approved drugs, such as lutetium, lenalidomide, an erythropoiesis stimulating agent (ESA), and / or a pharmaceutically acceptable salt thereof. The ESA comprises, but is not limited to, epotin alpha (epotin alpha) or darbepotin alpha (darbepotin alpha), and the ESA comprises, but is not limited to, epotin alpha (epotin alpha) or darbepotin alpha (darbepotin alpha).

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 422,210, filed on November 3, 2022, U.S. Provisional Application No. 63 / 455,540, filed on March 29, 2023, and U.S. Provisional Application No. 63 / 537,307, filed on September 8, 2023, the entire disclosures of each of which are hereby incorporated by reference in their entirety. Background Art

[0003] 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 of multiple hematological disorders, such as anemia in myelodysplastic syndromes (MDS), bone marrow failure (BMF), inflammatory diseases such as chronic kidney disease, ribosomopathies, 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 major goal in designing new 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 low-risk MDS patients (Schiavon et al. (2018) Med. Oncol. 35:76; Park et al. (2019) Br. J. Haematol. 184:134-160).Thus, most MDS patients ultimately become transfusion-dependent and unresponsive to several existing FDA-approved drugs: demethylating agents (Cheng et al. (2021) Hematol. 26:261-270; Kordella et al. (2021) Front. Oncol. 11:650473; Schiffer et al. (2021) Expert Rev. Anticancer Ther. 21:989-1002), lenalidomide (Hecht et al. (2021) Ann. Hematol. 100:1463-1471;), or luspatercept (Chan et al. (2021) Fut. Oncol. 17:1473-1481; Cheng et al. (2021) Hematol. 26:261-270; Hecht et al. (2021) Ann. Hematol. 100:1463-1471; Kordella et al. (2021) Front. Oncol. 11:650473; Kubasch et al. (2021) Blood Adv. 5:1565-1575; List et al. (2021) J. Clin. Oncol. 39:1001-1009), or progress to AML (in approximately 25-30% of patients) in cases where they are unable to undergo allogeneic bone marrow transplantation, the only curative treatment. Thus, there is an urgent need to identify additional novel therapies for promoting erythroid differentiation to alleviate anemia in these conditions. SUMMARY OF THE INVENTION

[0004] The present invention described herein is based in part on the discovery of a novel therapeutic method that targets anemia (e.g., anemia in hematological malignancies such as MDS, cancer patients undergoing chemotherapy treatment, and other conditions 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 in primary human hematopoietic stem and progenitor cells (HSPCs). FF / Ar treatment significantly enhances erythropoiesis in bone marrow cells derived from MDS patients and thus provides a potential therapeutic strategy for improving 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. Repurposing formoterol or a pharmaceutically acceptable salt thereof that stimulates erythroid differentiation (e.g., formoterol fumarate or arformoterol tartrate) has significant therapeutic effects in treating various hematological conditions associated with anemia, such as aplastic anemia, Diamond-Blackfan anemia, Schwachman-Diamond syndrome, MDS, inflammatory diseases such as chronic kidney disease, ribosomopathies, anemia in hematological malignancies, anemia secondary to chemotherapy in cancer patients, anemia secondary to bowel cancer, and also general anemia and aging-related anemia, the hematological malignancies including but not limited to acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and multiple myeloma (MM).

[0005] In some aspects, the present invention provides a method 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).

[0006] In some embodiments, an effective amount of formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) is a dose of ≤100 μg. In some embodiments, a dose of formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) is administered daily (such as once a day). In some embodiments, an 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 preferred embodiments, an 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.

[0007] In some embodiments, an effective amount of formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) is a total daily dose of ≤100 μg. In some embodiments, an effective amount of formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) is a total daily dose of 0.1 μg to 100 μg. In some preferred embodiments, an effective amount of formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) is a total daily dose of 1 μg to 60 μg.

[0008] In some embodiments, the anemia is selected from the group consisting of: megaloblastic anemia, hemolytic anemia, anemia caused by ribosomopathies, anemia caused by serine / threonine-protein kinase RIOK2 deficiency, anemia associated with chronic kidney disease (CKD), anemia caused by one or more mutations and / or deletions in human chromosome 5 or its orthologs, anemia caused by chromosomal translocation in the NUP98 gene or its orthologs, such as anemia caused by the infusion of the NUP98 gene with an Abd-B group HOX gene (e.g., HOXD13), stress-induced anemia, anemia secondary to bowel cancer, 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. 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 bowel cancer (such as colorectal cancer).

[0009] In some aspects, provided herein are methods for promoting the differentiation of erythroid progenitor cells of a patient into mature red blood cells 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 a dose of ≤100 μg. In some embodiments, a dose of formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) is administered daily (such as once a 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 0.1 μg / day to 100 μg / day. In some preferred 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.

[0010] In some embodiments, the effective amount of formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) is a total daily dose of ≤100 μg. In some embodiments, the effective amount of formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) is 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 a total daily dose of 1 μg to 60 μg.

[0011] In some embodiments, formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) can be orally administered to the patient. In some embodiments, the patient is a human suffering from anemia. In some embodiments, the method can further comprise administering (e.g., co-administering) an effective amount of an erythropoiesis-stimulating agent (ESA) or other FDA-approved drugs, such as luspatercept, lenalidomide, and / or demethylating agents, including but not limited to epoetin alfa or darbepoetin alfa, azacitidine, or decitabine, to the patient in need thereof. In some embodiments, the erythropoiesis-stimulating agent comprises erythropoietin, epoetin alfa, epoetin beta, epoetin omega, epoetin zeta, or darbepoetin alfa.

[0012] In some aspects, provided herein are methods of treating anemia in a patient in need thereof, the methods 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 drugs such as roxadustat, lenalidomide, and / or demethylating agents (such as azacitidine or decitabine). The anemia can be selected from the group consisting of megaloblastic anemia, hemolytic anemia, anemia caused by ribosomopathies, anemia caused by serine / threonine-protein kinase RIOK2 deficiency, anemia associated with chronic kidney disease (CKD), anemia caused by one or more mutations and / or deletions in human chromosome 5 or its orthologs, anemia caused by chromosomal translocations in the NUP98 gene or its orthologs, such as anemia caused by the infusion of the NUP98 gene with an Abd-B class HOX gene (e.g., HOXD13), stress-induced anemia, anemia secondary to bowel cancer, 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.

[0013] 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 has anemia associated with weight loss, decreased bone density, and / or muscle atrophy. In some embodiments, the anemia associated with weight loss, decreased bone density, and / or muscle atrophy is anemia associated with cancer as disclosed herein.

[0014] In some embodiments, provided is a method of treating anemia and weight gain in a subject in need thereof. In some embodiments, the patient has anemia associated with weight loss (e.g., anemia associated with cancer as disclosed herein).

[0015] In some embodiments, provided is a method of treating anemia and increased bone density in a subject in need thereof. In some embodiments, the patient has anemia associated with decreased bone density (e.g., anemia associated with cancer as disclosed herein).

[0016] In some embodiments, a method for treating anemia and increasing muscle mass in a subject in need thereof is provided. In some embodiments, the patient has anemia associated with reduced muscle mass (e.g., anemia associated with cancer as disclosed herein).

[0017] 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 bowel cancer (such as colorectal cancer). BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings, which are incorporated herein and form a part of this specification, are provided by way of illustration only and not as a limitation.

[0019] Figures 1A - 1D Illustrates formoterol fumarate-induced erythropoiesis. Treatment of primary human HSPCs with formoterol fumarate (FF) ( Figure 1A ) enhanced erythroid differentiation (CD235a) in a dose-dependent manner, but did not affect viability ( Figure 1B ), megakaryocyte (CD41-61) ( Figure 1C ), or myeloid (CD11b) differentiation ( Figure 1D ). n = 3 healthy donor-derived HSPCs. **p < 0.01, ***p < 0.001, one-way analysis of variance (ANOVA). "ns" (not significant) applies to cases where no statistical significance was shown. Data are represented as mean ± SEM. All comparisons were made relative to control (0 μM: vehicle (DMSO)-treated).

[0020] Figure 2 Shows that formoterol fumarate (FF) and arformoterol tartrate (Arf) induce erythropoiesis. Treatment of primary human HSPCs with 1 / 5 μM formoterol fumarate (FF) or arformoterol tartrate (Arf) induced erythroid differentiation (CD235a) in a dose-dependent manner. Representative of n = 5 healthy donor-derived HSPCs; n = 4 technical replicates within each donor. **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way analysis of variance (ANOVA). Data are represented as mean ± SEM. All comparisons were made relative to control (DMSO-treated). ns = not significant.

[0021] Figures 3A - 3B Illustrates that formoterol fumarate increases erythropoiesis. Treatment with formoterol fumarate from Selleckchem ( Figure 3A ) and Sigma (Figure 3B ) Formoterol fumarate (FF) 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). "ns" (not significant) applies to cases where no statistical significance is shown. Data are represented as mean ± SEM. All comparisons are relative to the control (0 μM: vehicle (DMSO)-treated).

[0022] Figure 4 Indicates that administration of formoterol fumarate (FF) in primary human HSPCs enhances the formation of burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E), which are precursors of red blood cells (RBC). N = 4 healthy donor-derived HSPCs. *p < 0.05, Student T test. Data are represented as mean ± SEM. All comparisons are relative to the control, i.e., the vehicle (DMSO-treated).

[0023] Figures 5A - 5C Indicates that formoterol fumarate (FF) enhances mitochondrial function. Figure 5A Indicates that treatment of primary human HSPCs with formoterol fumarate (FF) enhances the expression of the mitochondrial DNA-encoded genes MT-ND1, MT-CytB, MT-CO1, and MT-ATP6. Figure 5B Indicates that treatment of primary human HSPCs with formoterol fumarate (FF) enhances mitochondrial membrane potential (TMRE staining). Figure 5C Indicates that FF treatment increases mitochondrial mass in HSPCs ( staining). *p < 0.05, **p < 0.01, one-way analysis of variance (ANOVA) / Student t test. "ns" (not significant) applies to cases where no statistical significance is shown. Data are represented as mean ± SEM. All comparisons are relative to the control (0 μM / vehicle (DMSO)-treated).

[0024] Figures 6A - 6F Indicates that formoterol fumarate (FF) stimulates erythroid differentiation in RIOK2 knockdown (KD) HSPCs and RPS14 KD HSPCs, but not in ADRB2-deficient HSPCs. Figure 6A and Figure 6B Indicates that intraperitoneal (i.p.) injection of formoterol fumarate (FF) increases erythroid differentiation (CD235a) in RIOK2 KD HSPCs compared to vehicle (DMSO) treatment. Figure 6C and Figure 6DIndicates that, compared to vehicle (DMSO) treatment, intraperitoneal injection of FF increases erythroid differentiation (CD235a) in RPS14 KD HSPCs. Figure 6E and Figure 6F Depicts that FF is unable to induce erythroid differentiation due to the lack of the ADRB2 gene, which encodes β2-AR. *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA. ns: not significant. Data are represented as mean ± SEM. All comparisons were made relative to vehicle (DMSO)-treated.

[0025] Figure 7 Shows an exemplary workflow for using liquid cultures to study the effects of formoterol fumarate (FF) on MDS patient-derived cells.

[0026] Figures 8A - 8B Indicates that formoterol fumarate (FF) enhances erythroid differentiation in MDS patient-derived bone marrow cells. Erythroid differentiation (CD235a) in vehicle (DMSO) relative to FF-treated ( Figure 8A : Selleck; Figure 8B : Sigma) MDS patient-derived bone marrow cells; n = 40 uncharacterized MDS patients. Vehicle: DMSO; FF: formoterol fumarate. ****p < 0.0001, non-parametric Wilcoxon paired signed rank test. All comparisons were made relative to vehicle (DMSO)-treated controls.

[0027] Figure 9 Shows the results of liquid culture (ex vivo) experiments, which indicate that formoterol fumarate (FF) does not affect myelopoiesis / megakaryopoiesis in 40 MDS patient-derived cells.

[0028] Figure 10 Shows an exemplary workflow for studying the effects of formoterol fumarate (FF) on hematopoietic progenitor cells isolated from MDS patients.

[0029] Figures 11A - 11D Indicates that formoterol fumarate (FF) enhances the differentiation of erythroid progenitors in MDS patient-derived bone marrow cells. Figure 11A Shows pictures depicting an increase in burst-forming unit-erythroid (BFU-E) in MDS bone marrow (BM) cells treated with FF from Selleckchem and FF from Sigma compared to vehicle-treated BM cells. Figure 11B and Figure 11CShows the number of erythroid progenitors BFU-E and CFU-E (erythroid colony-forming units) in DMSO-treated MDS bone marrow cells relative to FF-treated MDS bone marrow cells; n = 23 MDS patients. Agent: DMSO; FF: Formoterol fumarate. Figure 11D Indicates that FF treatment does not affect viability, myelopoiesis (CFU-GM), or megakaryopoiesis (CFU-Mk) in bone marrow cells from MDS patients. **p < 0.01, ****p < 0.0001, non-parametric Wilcoxon paired signed-rank test. ns: not significant. All comparisons were made relative to controls treated with the agent (DMSO).

[0030] Figure 12 Indicates that agonists and antagonists of the β2-adrenergic receptor do not affect megakaryopoiesis. Administration of 1 / 5 μM formoterol fumarate (FF) or arformoterol tartrate (Arf) to primary human HSPCs does not affect megakaryocyte differentiation (CD41 / 61). Representative of HSPCs from n = 5 healthy donors; n = 4 technical replicates within each donor. One-way ANOVA. Data are presented as mean ± SEM. All comparisons were made relative to controls (treated with DMSO). ns = not significant.

[0031] Figure 13 Shows an exemplary workflow of a preclinical study with formoterol fumarate (FF) treatment in vivo.

[0032] Figures 14A - 14G Indicates that formoterol fumarate (FF) treatment moderately increases RBC parameters in wild-type mice at steady state. Figure 14A Shows a graph that shows the percentage change in body weight of mice in the case of treatment with the agent or FF at doses of 0.1 / 0.3 / 0.5 / 1.0 mg / kg. Figure 14B Indicates that FF administration enhances mitochondrial biogenesis in peripheral blood mononuclear cells (PBMCs) as observed by staining 14 days after daily intraperitoneal (i.p.) injection. Figures 14C - 14E Indicates that FF treatment moderately increases RBC parameters in peripheral blood, such as RBC count, hematocrit (HCT) %, and hemoglobin (Hb), 14 days after daily intraperitoneal injection. Figure 14F and Figure 14G Indicates that 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. "ns" (not significant) applies to cases where no statistical significance was shown. n = 3 mice / group. All comparisons were made relative to controls treated with the agent (DMSO).

[0033] Figure 15 Shows the complete blood count (CBC) results on day 8 of formoterol fumarate (FF) treatment by intraperitoneal injection in wild-type C57BL / 6J mice. "ns" (not significant) applies to cases where no statistical significance was shown.

[0034] Figure 16 Shows the reticulocyte count on day 8 after daily injection of formoterol fumarate (FF) treatment by intraperitoneal injection in wild-type C57BL / 6J mice. *p < 0.05, ANOVA. "ns" (not significant) applies to cases where no statistical significance was shown.

[0035] Figure 17 Shows an exemplary workflow of in vivo formoterol fumarate (FF) treatment in the case of phenylhydrazine (PHZ) study and sub-lethal dose (50 mg / kg).

[0036] Figures 18A - 18C Indicates that formoterol fumarate (FF) treatment significantly improves the RBC parameters in phenylhydrazine (PHZ)-mediated stress-induced anemic mice (10 - 12-week-old mice). Figure 18A Indicates that FF treatment at 0.1 / 0.3 mg / kg doses increases the absolute body weight and the % change in body weight in mice after sub-lethal PHZ-induced hemolytic anemia. PHZ dose: 50 mg / kg. Figure 18B Indicates that FF administration enhances and maintains RBC parameters in peripheral blood after PHZ-induced hemolytic anemia, such as hemoglobin (Hb), hematocrit (HCT) %, and RBC count. Figure 18C Indicates that FF treatment increases the WBC and monocyte counts in peripheral blood after 4 days of daily intraperitoneal injection, but does not maintain the effect beyond 14 days. Platelet count was not statistically significant. *p < 0.05, **p < 0.01, ****P < 0.0001, one-way and two-way ANOVA. "ns" (not significant) applies to cases where no statistical significance was shown. N = 4 female mice / group. All comparisons were made relative to vehicle (DMSO)-treated controls.

[0037] Figures 19A - 19B Indicates that formoterol fumarate (FF) treatment significantly elevates erythroid differentiation in the bone marrow (BM) of phenylhydrazine (PHZ)-mediated stress-induced anemic mice (10 - 12-week-old mice). Figure 19AA flow chart is shown, which depicts that treatment with FF at a dose of 0.1 / 0.3 mg / kg significantly increases RI, RII, RIII, and RIV erythroid progenitors in the BM of mice after sub-lethal PHZ-induced hemolytic anemia. PHZ dose: 50 mg / kg. Figure 19B Shows the quantification of the data presented in Figure 19A , which shows the absolute numbers of RI, RII, RIII, and RIV erythroid progenitors per million BM cells in mice after sub-lethal PHZ-induced hemolytic anemia. *p < 0.05, **p < 0.01, ***P < 0.001, ANOVA. N = 4 female mice / group. All comparisons were made relative to the vehicle (DMSO) control.

[0038] Figure 20 Shows an exemplary workflow of in vivo formoterol fumarate (FF) treatment in the case of phenylhydrazine (PHZ) study and sub-lethal dose (60 mg / kg).

[0039] Figure 21 Indicates that treatment with formoterol fumarate (FF) increases staining, thus showing the mitochondrial mass in peripheral blood mononuclear cells (PBMCs) on day 4 after PHZ treatment.

[0040] Figures 22A - 22C Indicates that treatment with formoterol fumarate (FF) significantly improves the RBC parameters in phenylhydrazine (PHZ)-mediated stress-induced anemic mice (10 - 12-week-old mice). Figure 22A Indicates that treatment with FF at a dose of 0.1 / 0.3 mg / kg increases the absolute body weight and the % change in body weight of male mice after sub-lethal PHZ-induced hemolytic anemia. PHZ dose: 60 mg / kg. Figure 22B Indicates that FF administration significantly enhances and maintains RBC parameters in peripheral blood, such as hemoglobin (Hb), hematocrit (HCT)%, and RBC count, after sub-lethal PHZ-induced hemolytic anemia. Figure 22C Indicates that FF treatment increases the WBC and monocyte counts in peripheral blood after 4 days of daily intraperitoneal injection, but does not maintain the effect for more than 14 days. Platelet counts were not statistically significant. *p < 0.05, **p < 0.01, ***P < 0.001, ****P < 0.0001, two-way ANOVA. "ns" (not significant) applies to cases where no statistical significance was shown. N = 5 male mice / group. All comparisons were made relative to the vehicle (DMSO)-treated control.

[0041] Figures 23A - 23CIt was shown that formoterol fumarate (FF) treatment significantly enhanced the viability and mitochondrial biogenesis in BM progenitors of phenylhydrazine (PHZ)-mediated stress-induced anemic mice (10 - 12-week-old mice). Figure 23A It was shown that FF treatment at 0.1 / 0.3 mg / kg dose significantly increased the viability in BM cells of mice after sub-lethal PHZ-induced hemolytic anemia; PHZ dose: 60 mg / kg. Figure 23B It was shown that FF administration enhanced mitochondrial biogenesis in BM cells after PHZ-induced hemolytic anemia as observed by staining. Figure 23C It was shown that FF administration significantly reduced mitochondrial superoxide production indicating mitochondrial fitness in BM cells after PHZ-induced hemolytic anemia as observed by staining. *p < 0.05, ****P < 0.0001, ANOVA. N = 5 male mice / group. All comparisons were made relative to vehicle (DMSO)-treated controls.

[0042] Figure 24 Flow cytometry plots are shown for the study of bone marrow progenitors (BMP).

[0043] Figures 25A - 25B It was shown that formoterol fumarate (FF) enhanced LS-K cells in the bone marrow (BM). *p < 0.05, **p < 0.01, ANOVA. ns: not significant.

[0044] Figures 26A - 26D It was shown that formoterol fumarate (FF) treatment significantly elevated megakaryocyte-erythroid progenitors (MEP) in the BM of phenylhydrazine (PHZ)-mediated stress-induced anemic mice (10 - 12-week-old mice), but did not significantly elevate common myeloid progenitors (CMP) or granulocyte-monocyte progenitors (GMP). Figure 26A A flow chart is shown that depicts that FF treatment at 0.1 / 0.3 mg / kg dose significantly increased MEP in the BM of mice after PHZ-induced sub-lethal hemolytic anemia. CMP and GMP were not affected. LS-K: lineage-Sca1-cKit+ BM cells. PHZ dose: 60 mg / kg. Figures 26B - 26D A quantification of the data presented in Figure 26A is shown, which shows the absolute numbers of CMP, GMP, and MEP progenitors per million BM cells in mice after PHZ-induced hemolytic anemia. **p < 0.01, ***P < 0.001, ANOVA. ns: not significant. N = 5 male mice / group. All comparisons were made relative to vehicle (DMSO-treated) controls.

[0045] Figure 27 It was shown that formoterol fumarate (FF) treatment did not affect megakaryocyte progenitors in the BM of phenylhydrazine (PHZ)-mediated stress-induced anemic mice (mice aged 10 - 12 weeks). The flow chart depicts that FF treatment at a dose of 0.1 / 0.3 mg / kg did not affect megakaryocyte progenitors (MkP) in the BM of mice after PHZ-induced sub-lethal hemolytic anemia. LS-K: lineage-Sca1-cKit+ BM cells. PHZ dose: 60 mg / kg. N = 5 male mice / group.

[0046] Figures 28A - 28B It was shown that formoterol fumarate (FF) selectively enhanced CFU-E progenitor cells in the BM. *p < 0.05, ***p < 0.001, ANOVA.

[0047] Figures 29A - 29B It was shown that formoterol fumarate (FF) selectively enhanced proerythroblasts in the BM.

[0048] Figures 30A - 30B It was shown that formoterol fumarate (FF) treatment significantly increased erythroid differentiation in the BM of phenylhydrazine (PHZ)-mediated stress-induced anemic mice (mice aged 10 - 12 weeks). Figure 30A The flow chart is shown, which depicts that FF treatment at a dose of 0.1 / 0.3 mg / kg significantly increased RI, RII, RIII, and RIV erythroid progenitors in the BM of mice after PHZ-induced sub-lethal hemolytic anemia. PHZ dose: 60 mg / kg. Figure 30B The quantification of the data presented in Figure 30A is shown, which 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 / group. All comparisons were made relative to the vehicle (DMSO)-treated control.

[0049] Figure 31 The schematic diagram of FF / Arf treatment by intraperitoneal injection in PHZ-treated mice at a sub-lethal dose of 60 mg / kg is shown.

[0050] Figure 32 It was shown that FF / Arf treatment by intraperitoneal injection enhanced body weight in PHZ-treated mice at a sub-lethal dose (60 mg / kg). ns = not significant.

[0051] Figures 33A - 33BIndicates that intraperitoneal FF / Arf treatment at a sub-lethal dose (60 mg / kg) does not affect the WBC parameters in phenylhydrazine (PHZ)-treated mice. FF / Arf treatment does not affect platelets and WBC in peripheral blood after intraperitoneal administration. ***p < 0.001, ANOVA. N = 5 male mice / group. All comparisons were made relative to vehicle (DMSO)-treated controls. ns = not significant.

[0052] Figures 34A - 34B Indicates that intraperitoneal FF / Arf treatment at a sub-lethal dose (60 mg / kg) moderately increases the RBC parameters in phenylhydrazine (PHZ)-treated mice. FF / Arf treatment increases RBC parameters in peripheral blood, such as hematocrit (HCT) % and RBC, 7 days after intraperitoneal administration. *p < 0.05, ANOVA. N = 5 male mice / group. All comparisons were made relative to vehicle (DMSO)-treated controls. ns = not significant.

[0053] Figures 35A - 35B Indicates that intraperitoneal FF / Arf treatment at a sub-lethal dose (60 mg / kg) increases the RBC parameters in phenylhydrazine (PHZ)-treated mice. FF and Arf treatment increase hemoglobin (Hb) in peripheral blood ( Figure 35A ) 7 days and ( Figure 35B ) 14 days after intraperitoneal administration. *p < 0.05, ANOVA. N = 5 male mice / group. All comparisons were made relative to vehicle (DMSO)-treated controls. ns = not significant.

[0054] Figures 36A - 36C Indicates that intraperitoneal FF / Arf treatment at a sub-lethal dose (60 mg / kg) increases LSK, LS-K, and mitochondrial activity in the BM of phenylhydrazine (PHZ)-treated mice. FF and Arf treatment increase ( Figure 36A ) LS-K, ( Figure 36B ) LSK, and ( Figure 36C ) mitochondrial membrane potential (TMRE staining) in bone marrow progenitors (BMP) 14 days after intraperitoneal administration. *p < 0.05, **p < 0.01, ***p < 0.001, ANOVA. N = 5 male mice / group. All comparisons were made relative to vehicle (DMSO)-treated controls. ns = not significant.

[0055] Figures 37A - 37BIt was shown that intraperitoneal treatment with FF / Arf increased megakaryocyte-erythroid progenitors (MEP) in the bone marrow (BM) of phenylhydrazine (PHZ)-treated mice at a sub-lethal dose (60 mg / kg), but did not increase granulocyte-monocyte progenitors (GMP). FF and Arf treatment increased ( Figure 37A ) MEP in bone marrow progenitors (BMP) 14 days after intraperitoneal administration, but did not increase ( Figure 37B ) GMP. *p < 0.05, ANOVA. N = 5 male mice / group. All comparisons were made relative to vehicle (DMSO)-treated controls. ns = not significant.

[0056] Figures 38A - 38B It was shown that intraperitoneal FF / Arf treatment consistently enhanced erythroid progenitors in the bone marrow (BM) of phenylhydrazine (PHZ)-treated mice at a sub-lethal dose (60 mg / kg). Figure 38A A flow chart is shown that depicts the effect of FF / treatment at a dose of 0.3 mg / kg on RI, RII, RIII, and RIV erythroid progenitors in the BM of mice. Figure 38B A quantification of the data presented in Figure 38A is shown, which 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 / group. All comparisons were made relative to vehicle (DMSO)-treated controls. ns = not significant.

[0057] Figures 39A - 39B It was shown that intraperitoneal FF treatment enhanced erythroid colony-forming units (CFU-e) in the bone marrow (BM) of phenylhydrazine (PHZ)-treated mice at a sub-lethal dose (60 mg / kg). Figure 39A A figure is shown that depicts the long-term effect of FF treatment at 0.3 mg / kg on CFU-e production in the BM of mice. Figure 39B A figure is shown that depicts enhanced transferrin receptor expression in CFU-e derived from BMP of FF / Arf-treated mice. *p < 0.05, ***P < 0.001, ANOVA. N = 5 male mice / group. All comparisons were made relative to vehicle (DMSO)-treated controls. ns = not significant.

[0058] Figure 40 A flow cytometry plot is shown for studying erythroid progenitors (EryP) in the BM of mice.

[0059] Figure 41It was shown that formoterol fumarate (FF) did not affect granulocyte myeloid progenitors (GMP) in the bone marrow (BM).

[0060] Figures 42A - 42D It was shown that formoterol fumarate (FF) treatment conferred a remarkable survival benefit in phenylhydrazine (PHZ)-mediated lethal hemolytic anemia in mice (10 - 12-week-old mice). Figure 42A The Kaplan - Meier survival plot is shown, which shows 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 / group. In the vehicle-treated group (gray circles), 1 mouse died on day 1 and 4 mice died on day 2 after the lethal PHZ dose. In the FF 0.1 mg / kg group (orange circles), 2 mice died on day 2 and 3 mice survived after the lethal PHZ dose. In the FF 0.3 mg / kg group (maroon circles), 1 mouse died on day 2 and 4 mice survived after the lethal PHZ dose. Figure 42B It is a figure that shows that FF treatment at 0.1 / 0.3 mg / kg doses led to a steady increase in the body weight of male mice after PHZ-induced lethal hemolytic anemia. Figure 42C The Kaplan - Meier survival plot is shown, which shows 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 / group. In the vehicle-treated group (gray circles), 3 mice died on day 2 and 2 mice died on day 3 after the lethal PHZ dose. In the FF 0.1 mg / kg group (orange circles), 1 mouse died on day 2 and 4 mice survived after the lethal PHZ dose. In the FF 0.3 mg / kg group (maroon circles), 2 mice died on day 2 and 3 mice survived after the lethal PHZ dose. Figure 42D It is a figure that shows that FF treatment at 0.1 / 0.3 mg / kg doses led to a steady increase in the body weight of female mice after PHZ-induced lethal hemolytic anemia. *p < 0.05, log-rank (Mantel - Cox) test. n = 5 mice / group. All comparisons were made relative to the vehicle (DMSO)-treated control.

[0061] Figures 43 - 44 The flow cytometry plots of formoterol fumarate (FF) using semi-solid methylcellulose cultures on MDS patient-derived cells are shown.

[0062] Figure 45 Indicates that formoterol fumarate (FF) significantly rescues erythropoiesis in RIOK2 knockdown human hematopoietic stem and progenitor cells (RIOK2 KD HSPC) in vitro. *p<0.05, **p<0.01, ANOVA.

[0063] Figure 46 Indicates that formoterol fumarate (FF) moderately rescues erythropoiesis in RPS14- and APC-knockdown human hematopoietic stem and progenitor cells (RPS14 and APC KD HSPC) in vitro. *p<0.05, **p<0.01, ANOVA. ns: not significant.

[0064] Figure 47 Shows a schematic of FF treatment by oral gavage in wild-type mice at steady state.

[0065] Figures 48A - 48C Indicates that FF treatment by oral gavage increases RBC parameters in wild-type mice at steady state. FF treatment increases RBC parameters in peripheral blood, such as hematocrit (HCT)%, RBC count, and hemoglobin (Hb), after 14 days of daily oral gavage (o.g.) administration. *p<0.05, ANOVA. N = 5 mice / group. All comparisons were made relative to vehicle-treated (DMSO) controls. ns = not significant.

[0066] Figures 49A - 49C Indicates 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 in peripheral blood, such as WBC, monocytes, and platelets, after 14 days of daily oral gavage (o.g.) administration. ANOVA. N = 5 mice / group. All comparisons were made relative to vehicle-treated (DMSO) controls. ns = not significant.

[0067] Figure 50 Insets A-B indicate that FF treatment by oral gavage enhances the viability and mitochondrial activity of bone marrow progenitors (BMP) in wild-type mice at steady state. FF treatment increases viability and mitochondrial membrane potential (measured by TMRE staining) in BMP after 14 days of daily oral gavage (o.g.) administration. *p<0.05, **p<0.01, ****p<0.0001, ANOVA. n = 5 mice / group. All comparisons were made relative to vehicle-treated (DMSO) controls. ns = not significant.

[0068] Figure 51Panels A - C show that FF treatment by oral gavage enhances MEP and MkP, but not GMP, in the bone marrow (BM) of wild - type mice at steady - state. FF treatment does not affect granulocyte - monocyte progenitors (GMP) in the BM after 14 days of daily oral gavage (o.g.) administration, but increases megakaryocyte - erythroid progenitors (MEP) and megakaryocyte progenitors (MkP). ***p < 0.001, ****p < 0.0001, ANOVA. n = 5 mice / group). All comparisons were made relative to vehicle (DMSO) - treated controls. ns = not significant.

[0069] Figures 52A - 52B show that FF treatment by oral gavage enhances erythroid progenitors in the bone marrow (BM) of wild - type mice at steady - state. Figure 52A Shows a flow chart depicting that FF treatment at 0.1 / 0.5 / 1.0 mg / kg doses significantly increases RI, RII, RIII, and RIV erythroid progenitors in the BM of mice. Figure 52B Shows the Figure 52A quantification of the data presented in, which 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 / group. All comparisons were made relative to vehicle (DMSO) - treated controls. ns = not significant.

[0070] Figure 53 show that FF treatment moderately increases the body weight of steady - state mice after FF treatment by oral gavage (o.g.). Graph showing the % change in body weight of mice moderately increased by FF treatment by oral gavage. *p < 0.05, **p < 0.01, two - way ANOVA. n = 5 male mice / group. All comparisons were made relative to vehicle (DMSO) - treated controls. ns = not significant.

[0071] Figure 54 show that FF treatment by oral gavage confers a remarkable survival benefit to mice (10 - 12 - week - old mice) treated with a lethal dose of phenylhydrazine (PHZ) at 135 mg / kg. Kaplan - Meier survival plots showing the survival benefit in mice after PHZ - induced lethal hemolytic anemia (PHZ - 135 mg / kg) for 0.1 / 0.5 mg / kg doses of FF treatment by oral gavage. Log - rank (Mantel - Cox) test. n = 5 mice / group. All comparisons were made relative to vehicle (DMSO) - treated controls.

[0072] Figure 55Shows a schematic diagram of FF treatment by oral gavage at a sub-lethal dose of 60 mg / kg in mice treated with phenylhydrazine (PHZ).

[0073] Figure 56 Indicates that FF treatment by oral gavage (o.g.) does not affect body weight in phenylhydrazine (PHZ)-treated mice at a sub-lethal dose (60 mg / kg). ns = not significant.

[0074] Figures 57A - 57B Indicates that FF treatment by oral gavage increases RBC parameters in phenylhydrazine (PHZ)-treated mice at a sub-lethal dose (60 mg / kg). FF treatment increases RBC parameters in peripheral blood such as hematocrit (HCT) % and hemoglobin (Hb) after oral gavage (o.g.) administration. *p < 0.05, **p < 0.01, ***P < 0.001, ANOVA. n = 5 male mice / group. All comparisons were made relative to vehicle (DMSO)-treated controls. ns = not significant.

[0075] Figures 58A - 58B Indicates that FF treatment by oral gavage does not affect WBC parameters in phenylhydrazine (PHZ)-treated mice at a sub-lethal dose (60 mg / kg). FF treatment does not affect WBC and platelets in peripheral blood after oral gavage (o.g.) administration. ANOVA. N = 5 male mice / group. All comparisons were made relative to vehicle (DMSO)-treated controls. ns = not significant.

[0076] Figures 59A - 59B Indicates that FF treatment by oral gavage enhances the viability and mitochondrial activity of bone marrow progenitors (BMP) in phenylhydrazine (PHZ)-treated mice at a sub-lethal dose (60 mg / kg). FF treatment increases viability and mitochondrial membrane potential (measured by TMRE staining) in BMP after 14 days of daily oral gavage (o.g.) administration. *p < 0.05, **p < 0.01, ***p < 0.001, ANOVA. n = 5 mice / group. All comparisons were made relative to vehicle-treated (DMSO) controls. ns = not significant.

[0077] Figures 60A - 60BIndicates that FF treatment by oral gavage enhances MkP in the bone marrow (BM) of phenylhydrazine (PHZ)-treated mice at a sub-lethal dose (60 mg / kg), but not GMP. FF treatment does not affect granulocyte-monocyte progenitors (GMP) in BMP but increases megakaryocyte progenitors (MkP) after 14 days of daily oral gavage (o.g.) administration. ***p < 0.001, ****p < 0.0001, ANOVA. n = 5 mice / group). All comparisons were made relative to a vehicle (DMSO)-treated control. ns = not significant.

[0078] Figures 61A - 61B Indicates that FF treatment by oral gavage enhances erythroid progenitors in the bone marrow (BM) of phenylhydrazine (PHZ)-treated mice at a sub-lethal dose (60 mg / kg). Figure 61A Shows a flow chart depicting that FF treatment at 0.1 / 0.3 / 0.5 / 1.0 mg / kg doses significantly increases RI, RII, RIII, and RIV erythroid progenitors in the BM of mice. Figure 61B Shows the Figure 61A Quantification of the data presented in, which 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 / group. All comparisons were made relative to a vehicle (DMSO)-treated control. ns = not significant.

[0079] Figure 62 Shows a schematic diagram of the experimental setup for analyzing the effects of formoterol fumarate (FF) in naïve mice without external stress of phenylhydrazine (PHZ).

[0080] Figure 63 Indicates that Figure 62 The FF treatment shown in increases the body weight of wild-type mice at steady state. Figure, which shows the percentage change in body weight of mice in the case of vehicle treatment or FF treatment at 0.1 / 0.3 / 0.5 mg / kg doses. **p < 0.01, “ns” = not significant. Although the 0.1 mg / kg group and 0.3 mg / kg group did not show statistical significance in Graphpad PRISM, the body weight increased significantly compared to the vehicle-treated control.

[0081] Figures 64A - 64C Indicates that Figure 62 The FF treatment shown in significantly increases red blood cell (RBC) parameters in wild-type mice (10 - 12-week-old male mice). FF administration enhances and maintains RBC parameters in peripheral blood, such as ( Figure 64A) RBC count, ( Figure 64B ) hemoglobin (Hb), and ( Figure 64C ) hematocrit (HCT). According to Graphpad PRISM, the 0.1 mg / kg and 0.5 mg / kg groups did not show statistical significance in terms of HCT%. *p < 0.05, **p < 0.01, ***p < 0.001, ****P < 0.0001, "ns" = not significant, one-way and two-way ANOVA. N = 5 male mice / group. All comparisons were made relative to the vehicle (DMSO)-treated control.

[0082] Figures 65A - 65C Indicates that Figure 62 the FF treatment shown in did not affect the total white blood cell (non-RBC) parameters of wild-type mice (10 - 12-week-old male mice). FF administration did not affect ( Figure 65A ) white blood cell count (WBC) and ( Figure 65C ) platelet count, but moderately affected the monocyte count in the peripheral blood of naive mice ( Figure 65B ). *p < 0.05, **p < 0.01, ***p < 0.001, ****P < 0.0001, "ns" = not significant, one-way and two-way ANOVA. N = 5 male mice / group. All comparisons were made relative to the vehicle (DMSO)-treated control.

[0083] Figures 66A - 66C Indicates that Figure 62 the FF treatment shown in significantly enhanced mitochondrial biogenesis in bone marrow progenitors (BMP) of wild-type mice (10 - 12-week-old mice). Figure 66A Indicates that FF administration enhanced mitochondrial biogenesis in BMP of naive mice as observed by MitoTracker staining. Figure 66B Indicates that FF administration enhanced the mitochondrial membrane potential in BMP of naive mice as observed by TMRE staining. Figure 66C Indicates that FF administration significantly reduced mitochondrial superoxide production indicating mitochondrial fitness in BMP of naive mice as observed by MitoSox staining. *p < 0.05, **p < 0.01, ***p < 0.001, ****P < 0.0001, "ns" = not significant. N = 5 male mice / group. All comparisons were made relative to the vehicle (DMSO)-treated control.

[0084] Figures 67A - 67B Indicates that Figure 62The FF treatment shown in significantly enhances the viability in bone marrow (BM) progenitors and LS-K cells (Lineage-Sca1-cKit+) in wild-type mice (10 - 12-week-old mice). The FF treatment at 0.1 / 0.3 / 0.5 mg / kg doses significantly increases the ( Figure 67A ) viability and ( Figure 67B ) LS-K cells in the BM of naive mice. *p < 0.05, ****P < 0.0001, ANOVA. N = 5 male mice / group. All comparisons were made relative to vehicle (DMSO)-treated controls.

[0085] Figures 68A - 68C Indicates that Figure 62 the FF treatment shown in significantly elevates megakaryocyte-erythroid progenitors (MEP) and megakaryocyte progenitors (MkP) in the bone marrow (BM) of naive mice (10 - 12-week-old mice), but not granulocyte-monocyte progenitors (GMP). Flow chart depicting that the FF treatment at 0.1 / 0.3 / 0.5 mg / kg doses significantly increases the ( Figure 68A ) megakaryocyte-erythroid progenitors (MEP) and ( Figure 68B ) megakaryocyte progenitors (MkP) in the BM of wild-type mice, but does not increase the ( Figure 68C ) granulocyte-monocyte progenitors (GMP). LS-K: Lineage-Sca1-cKit+ BM cells. **p < 0.01, ***P < 0.001, “ns” = not significant, ANOVA. N = 5 male mice / group. All comparisons were made relative to vehicle (DMSO-treated) controls.

[0086] Figures 69A - 69D Indicates that Figure 62 the FF treatment shown in significantly elevates erythroid differentiation in the bone marrow (BM) of naive mice (10 - 12-week-old mice). The FF treatment at 0.1 / 0.3 / 0.5 mg / kg doses significantly increases the ( Figure 69A ) RI, ( Figure 69B ) RII, ( Figure 69C ) RIII and (D) RIV erythroid progenitors in the BM of wild-type mice. Quantification of data showing the 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 / group. All comparisons were made relative to vehicle (DMSO)-treated controls.

[0087] Figures 70A - 70D Indicates that Figure 63The FF treatment shown in the figure significantly increased erythroid gene expression in sorted erythroid progenitor cells isolated from the bone marrow (BM) of naive mice (10 - 12-week-old mice). The FF treatment at doses of 0.1 / 0.3 / 0.5 mg / kg significantly increased the expression of erythroid genes such as ( Figure 70A ) EPOR, ( Figure 70B ) ASXL1, and ( Figure 70C ) NFE2 in sorted erythroid progenitor cells isolated from the BM of wild-type mice, but did not affect the expression of the β2-AR-encoding gene ADRB2 ( Figure 70D ) in the sorted erythroid progenitor cells. *p < 0.05, **p < 0.01, ***P < 0.001, ****P < 0.0001, "ns" = not significant, ANOVA. N = 5 male mice / group. All comparisons were made relative to the vehicle (DMSO)-treated control. Since the FF treatment in naive mice increased the expression of the erythropoietin receptor EPOR in erythroid progenitor cells, FF could potentially be co-administered with erythropoiesis-stimulating agents (ESAs) such as epoetin alfa and other biosimilars of erythropoietin (EPO) to patients with MDS and other anemias who are unresponsive to treatment with EPO alone. This increase in EPOR in erythroid progenitor cells by FF also does not rule out the co-administration of FF with other FDA-approved drugs such as luspatercept, lenalidomide, and / or demethylating agents such as azacitidine and / or decitabine in patients with MDS and other anemias who are unresponsive to treatment with EPO alone.

[0088] Figures 71A - 71C showed that the FF treatment increased RBC parameters in MDS / AML mice (NUP98-HOXD13 transgenic mice). Figure 71A 、 Figure 71B and Figure 71C showed that a low dose of FF treatment at 0.3 mg / kg three times a week (intraperitoneally) alleviated RBC parameters in the PB of MDS / AML mice, such as RBC, hemoglobin (Hb), and hematocrit (HCT%). *p < 0.05, **p < 0.01, ****p < 0.0001, ANOVA. n = 5 - 7 mice / group. Veh: vehicle; LC: littermate control.

[0089] Figures 72A - 72C showed that the FF treatment did not affect the WBC parameters in MDS / AML mice. The low dose of FF treatment at 0.3 mg / kg three times a week (intraperitoneally) had no effect on ( Figure 72A ) platelets and ( Figure 72B ) WBC in the peripheral blood of MDS / AML mice, as well as ( Figure 72C) Apoptosis (measured by annexin V staining) was not affected. ***p < 0.001, ****p < 0.0001, ANOVA. n = 5 - 7 mice / group. Veh: vehicle; LC: littermate control. ns: not significant.

[0090] Figures 73A - 73C showed that FF treatment increased erythroid progenitors in the bone marrow of MDS / AML mice. Low-dose FF treatment at 0.3 mg / kg three times a week (intraperitoneally) Figure 73A ) elevated erythroid progenitors in the BM of MDS / AML mice but did not affect Figure 73B ) granulocyte-monocyte progenitors (GMP) or Figure 73C ) megakaryocyte progenitors (MkP). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ANOVA. n = 5 - 7 mice / group. Veh: vehicle; LC: littermate control. ns = not significant.

[0091] Figures 74A - 74C showed that FF (intraperitoneal) treatment elevated RBC parameters in Apcmin mice. This is a mouse model of intestinal adenomas with anemia. Low-dose FF treatment at 0.3 mg / kg per day (intraperitoneally) alleviated the RBC parameters of Apcmin mice that spontaneously developed anemia at about 3 months of age, such as Figure 74A ) RBC Figure 74B ) hematocrit % (HCT%) and Figure 74C ) hemoglobin (Hb). *p < 0.05, ***p < 0.001, two-way ANOVA. n = 5 - 6 mice / group. Comparison was shown between Apcmin + FF and Apcmin + vehicle. Veh: vehicle; LC: littermate control mice.

[0092] Figure 75 showed that FF treatment moderately increased the body weight of Apcmin mice. n = 5 - 6 mice / group. Veh: vehicle; Ctrl (control): littermate control mice.

[0093] Figure 76 showed that FF (intraperitoneal) treatment significantly rescued (elevated) the body weight of 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 / group. Comparison was shown between mice treated with cisplatin + FF and mice treated with cisplatin + vehicle.

[0094] Figures 77A - 77B showed that cisplatin treatment induced chronic kidney injury (CKD) in wild-type mice. Figure 77A) Serum blood urea nitrogen (BUN) and ( Figure 77B ) serum creatinine levels, indicating that cisplatin-induced kidney injury in mice is characterized by CKD and that FF treatment does not affect serum BUN and creatinine levels.

[0095] Figures 78A - 78C Indicating that FF treatment rescues anemia in cisplatin-treated CKD mice. Low-dose FF treatment at 0.3 mg / kg three times a week (intraperitoneally) alleviates RBC parameters in mice with cisplatin-driven CKD, such as ( Figure 78A ) hematocrit (HCT%) ( Figure 78B ) hemoglobin (Hb) and ( Figure 78C ) % change in RBC. *p < 0.05, two-way ANOVA. n = 4 - 5 mice / group. Comparison is shown between cisplatin + FF-treated mice and cisplatin + vehicle-treated mice.

[0096] Figures 79A - 79C Indicating that FF treatment elevates RBC parameters in cisplatin-treated CKD mice. Low-dose FF treatment at 0.3 mg / kg three times a week (intraperitoneally) alleviates RBC parameters in mice with cisplatin-driven CKD, such as ( Figure 79A ) hemoglobin (Hb) ( Figure 79B ) hematocrit % (HCT%) and ( Figure 79C ) absolute value of RBC. *p < 0.05, **p < 0.01, ***p < 0.001, ***p < 0.001, two-way ANOVA. n = 4 - 5 mice / group. ns: not significant.

[0097] Figures 80A - 80B Indicating that FF treatment does not affect WBC parameters in cisplatin-treated CKD mice. FF treatment does not affect ( Figure 80A ) WBC and ( Figure 80B ) platelets in mice with cisplatin-driven CKD. Two-way ANOVA. n = 4 - 5 mice / group. Comparison is shown between cisplatin + FF-treated mice and cisplatin + vehicle-treated mice. ns: not significant.

[0098] Figures 81A - 81C Indicating that FF treatment elevates erythroid progenitors in cisplatin-treated CKD mice. Low-dose FF treatment at 0.3 mg / kg three times a week (intraperitoneally) elevates ( Figure 81A ) terminal erythroid progenitors in the BM of mice with cisplatin-driven CKD, but does not elevate ( Figure 81B ) granulocyte-macrophage progenitors (GMP) or ( Figure 81C)Megakaryocyte progenitor cells (MkP). *p < 0.05, ***p < 0.001, two-way ANOVA. n = 4 - 5 mice / group. Comparisons are shown between cisplatin + FF-treated mice and cisplatin + vehicle-treated mice. ns: not significant.

[0099] Figures 82A - 82B It is shown that in WT mice, the surface expression of ADRB2 on erythroid progenitor cells (EP) is higher than that on non-erythroid progenitor cells (non-EP). ADRB2 is expressed on the surface of erythroid progenitor cells in the bone marrow of wild-type mice. Figure 82A A flow chart is shown, which depicts the expression of ADRB2 on the surface of erythroid progenitor cells (EP). Figure 82B A flow chart is shown, which depicts that the expression of ADRB2 on the surface of non-erythroid bone marrow (BM) progenitor cells (non-EP) is the least. Ms-1 - 5: Mouse 1 - Mouse 5; Ab: antibody against ADRB2.

[0100] Figure 83 It is shown that FF treatment in naive mice increases body weight in a dose-dependent manner. Figure, which shows the percentage change in body weight of mice in the case of vehicle treatment or FF treatment at doses of 0.1 / 0.3 / 0.5 / 1.0 mg / kg. **p < 0.01, ****p < 0.0001, "ns" = not significant.

[0101] Figures 84A - 84C It is shown that FF treatment in 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 mg / kg and 0.5 mg / kg enhances and maintains 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: ( Figure 84A ) Hemoglobin (Hb), ( Figure 84B ) Hematocrit (HCT) %, and ( Figure 84C ) RBC count. *p < 0.05, **p < 0.01, ***p < 0.001, ****P < 0.0001, "ns" = not significant, one-way and two-way ANOVA. N = 5 male mice / group. All comparisons were made relative to vehicle (DMSO)-treated controls.

[0102] Figures 85A - 85C It is shown that FF treatment in naive mice has 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 does not affect at the lowest dose ( Figure 85A)The white blood cell count (WBC) was counted, and at the highest dose of 1.0 mg / kg, it moderately affected the WBC. In contrast, FF treatment moderately affected the monocyte count in the peripheral blood of naive mice at doses of 0.3 mg / kg and 0.5 mg / kg ( Figure 85B ), but did not affect the monocyte count at the lowest dose (0.1 mg / kg) or the highest dose (1.0 mg / kg). No significant effects were observed on platelets at any of the tested doses. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, "ns" = not significant, one-way and two-way ANOVA. N = 5 male mice / group. All comparisons were made relative to vehicle (DMSO)-treated controls.

[0103] Figure 86 An example workflow of bulk RNA sequencing of formoterol fumarate (FF)-treated mice is shown.

[0104] Figure 87A -B shows the differential gene expression in erythroid progenitor cells isolated from the bone marrow of FF-treated mice relative to that in erythroid progenitor cells isolated from the bone marrow of vehicle-treated mice, where the changes in the expression of various genes listed in the last column are shown ( Figure 87A ) and a heatmap of the changes in gene expression ( Figure 87B ).

[0105] Figure 88A -B shows that compared with vehicle-treated mice, intraperitoneal treatment with 0.5 mg / kg of FF three times a week enhanced the expression of HIF-1α ( Figure 88A ) and VEGFA ( Figure 88B ) in bone marrow cells. *p < 0.05, **p < 0.01, non-parametric Mann-Whitney test. Data are represented as mean ± SEM. All comparisons were made relative to vehicle (DMSO-treated). Detailed Description

[0106] In some aspects, provided herein are methods of treating anemia in a patient in need thereof, the methods 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 some aspects, provided herein are methods of promoting the differentiation of erythroid progenitor cells into mature red blood cells in a patient in need thereof, the methods comprising administering an effective amount of formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate). In some embodiments, the methods may further comprise administering to the patient in need thereof an effective amount of an erythropoiesis-stimulating agent. In some aspects, provided herein are methods of treating anemia in a patient in need thereof, the methods 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 (such as erythropoietin, epoetin α, epoetin β, epoetin ω, epoetin ζ, or darbepoetin α). In some embodiments, formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) may be administered in combination with other FDA-approved drugs (such as roxadustat, lenalidomide, and / or demethylating agents (such as azacitidine or decitabine)).

[0107] In some embodiments, provided herein is formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) for 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), the formoterol or pharmaceutically acceptable salt thereof for treating anemia in a patient.

[0108] In some embodiments, the anemia is selected from the group consisting of: megaloblastic anemia, hemolytic anemia, anemia caused by ribosomopathies, anemia caused by serine / threonine-protein kinase RIOK2 deficiency, anemia associated with chronic kidney disease (CKD), anemia caused by one or more mutations and / or deletions in human chromosome 5 or its orthologs, anemia caused by chromosomal translocations in the NUP98 gene or its orthologs, such as anemia caused by the infusion of the NUP98 gene with an Abd-B group HOX gene (e.g., HOXD13), stress-induced anemia, anemia secondary to bowel cancer, Diamond-Blackfan anemia, aplastic anemia, Shwachman-Diamond syndrome, anemia associated with inflammatory diseases such as 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, optionally wherein the cancer is a hematological malignancy such as myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), or any hematological malignancy disclosed herein.

[0109] In some aspects, the methods and uses disclosed herein enhance erythropoiesis in bone marrow cells derived from MDS patients and thus improve 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.

[0110] In some embodiments, the methods and uses disclosed herein also have the effect of increasing the weight of a patient. In some embodiments, the methods and uses disclosed herein can be used to treat a patient suffering from anemia associated with weight loss (e.g., anemia associated with cancer disclosed herein). In some embodiments, the methods and uses disclosed herein can be used to treat a patient suffering from anemia associated with decreased bone density (e.g., anemia associated with cancer disclosed herein). In some embodiments, the methods and uses disclosed herein can be used to treat a patient suffering from anemia associated with muscle wasting (e.g., anemia associated with cancer disclosed herein).

[0111] In some aspects, the present disclosure provides herein a new use of formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) for oral administration to treat anemia as further described herein.

[0112] I. Definitions

[0113] As used herein, the articles "a" and "an" refer to one or more than one (i.e., at least one) of the grammatical objects of said articles. For example, "an element" means one element or more than one element.

[0114] The term "cancer" or "tumor" or "hyperplastic" refers to the presence of cells having the typical characteristics of cancer cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features.

[0115] Cancer cells typically occur in the form of tumors, but such cells can exist alone in an animal body or can be non-tumorigenic cancer cells, such as leukemia cells. As used herein, the term "cancer" includes pre-cancerous as well as malignant cancers. Cancers include, but are not limited to, B-cell cancers, such as myelomas, for example multiple myeloma, Waldenström macroglobulinemia ( macroglobulinemia), heavy chain diseases (e.g., alpha chain disease, gamma chain disease, and mu chain disease), benign monoclonal gammopathy, and immunocytic amyloidosis, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, bladder cancer, brain cancer or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine cancer or endometrial cancer, oral cancer 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, cancers of the hematopoietic tissues, etc. Other non-limiting examples of cancer types applicable to the methods covered by the present disclosure include human sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, 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, cholangiocarcinoma, liver cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, bone cancer, brain cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, meningioma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, such as acute lymphocytic leukemia and acute myelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia); chronic leukemias (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphomas (Hodgkin's disease and non-Hodgkin's disease), myeloma, multiple myeloma, Waldenström macroglobulinemia, and heavy chain disease. In some embodiments, the cancer is epithelial in nature and includes, but is not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecological cancers, kidney cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer. In other embodiments, the cancer is breast cancer, prostate cancer, lung cancer, or colon cancer. In still other embodiments, the epithelial cancer is non-small cell lung cancer, non-papillary renal cell carcinoma, cervical cancer, ovarian cancer (e.g., serous ovarian cancer), or breast cancer. Epithelial cancers can be characterized in various other ways, including but not limited to serous, endometrioid, mucinous, clear cell, Brenner, or undifferentiated.

[0116] The term "erythroid progenitor cell" refers to a progenitor cell derived from hematopoietic stem cells that gives rise to erythrocytes (red blood cells) upon terminal differentiation.

[0117] As used herein, the term "anemia" includes megaloblastic anemia, hemolytic anemia, anemia associated with inflammation such as chronic kidney disease (CKD) and other inflammatory diseases such as autoimmune disorders (e.g., rheumatoid arthritis or multiple sclerosis), anemia caused by deficiency of serine / threonine-protein kinase RIOK2, anemia caused by one or more mutations and / or deletions in human chromosome 5 or its orthologs, anemia caused by chromosomal translocation in the NUP98 gene or its orthologs, such as anemia caused by the fusion of NUP98 with Abd-B class HOX genes (e.g., HOXD13), stress-induced anemia, aplastic anemia, anemia secondary to bowel cancer, Diamond-Blackfan anemia, Shwachman-Diamond syndrome or anemia secondary to chemotherapy treatment of cancer patients. The anemia can be cancer-related anemia, optionally wherein 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 can be anemia associated with bowel cancer (such as colorectal cancer). For example, the anemia can be caused by intestinal adenomas resulting from familial adenomatous polyposis (FAP). Adenomatous polyposis coli (APC) is a tumor suppressor gene that is mutated in colorectal cancer. Alterations in the APC gene produce truncated gene products, leading to activation of the Wnt signaling pathway and dysregulation of multiple other cellular processes, thereby resulting in tumorigenesis. For more details, see Su et al., Multiple intestinal neoplasia caused by a mutation in the murine homolog of the APC gene. Science. May 1, 1992; 256(5057):668-70 and Moser et al., A dominant mutation that predisposes to multiple intestinal neoplasia in the mouse. Science. Jan 19, 1990; 247(4940):322-4. The anemia can be anemia associated with bone marrow failure disorders. The anemia can be anemia caused by or associated with ribosomopathies. As used herein, "ribosomopathy" is a disease caused by a defect in ribosomal components or factors that play a role in ribosome assembly, which results in a defect in ribosome biogenesis. Congenital ribosomopathies exhibit a paradoxical transition from early symptoms due to reduced cell proliferation to an increased risk of cancer in later life.More details regarding ribosomopathies can be found in: Kim R Kampen et al. (2020). Nucleic Acids Res. 48(3):1013–1028. Mutations that disrupt ribosome biogenesis often affect tissues that rely on cell division to function. Many ribosomopathies have an anemia component, as the production of blood cells by the bone marrow is highly dependent on cell division. Examples of ribosomopathies include, but are not limited to, Diamond–Blackfan anemia (DBA), 5q syndrome, Shwachman–Diamond syndrome (SDS), X-linked dyskeratosis congenita (DC), cartilage–hair hypoplasia (CHH), Treacher–Collins syndrome (TCS), Bowen–Conradi syndrome, and North American Indian childhood cirrhosis.

[0118] As used herein, myelodysplastic syndrome (MDS) includes, but is not limited to, a heterogeneous group of myeloid neoplasms that are commonly characterized by bone marrow failure with manifestations of abnormal cell morphology and, in some cases, a propensity for acute myeloid leukemia (AML). In some instances, MDS is caused by mutations or deletions on human chromosome 5 or chromosomal translocations in the NUP98 gene (e.g., translocations that result in the fusion of NUP98 with Abd-B class HOX genes (e.g., HOXD13)). For more details regarding chromosomal translocations in the NUP98 gene, see Lin et al. NUP98-HOXD13 transgenic mice develop a highly penetrant, severe myelodysplastic syndrome that progresses to acute leukemia. Blood. July 1, 2005;106(1):287-95.

[0119] The term "patient" refers to a person suffering from anemia.

[0120] As used herein, the phrase "co-administering" refers to any form of administering two or more different therapeutic agents such that a second agent is administered while a previously administered therapeutic agent is still active in the body (e.g., the two agents are both active in the subject at the same time, which can include a synergistic effect of the two agents). For example, the different therapeutic agents can be administered simultaneously or sequentially in the same formulation or in separate formulations. In certain embodiments, the different therapeutic agents can be administered within about one hour, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, or about one week of each other. Thus, a subject receiving such treatment can benefit from the combined effects of the different therapeutic agents. As used herein, any two agents and / or additional agents can be co-administered according to the methods provided herein.

[0121] The term "therapeutic effect" refers to a local or systemic effect in the human body caused by a pharmacologically active substance. Thus, the term means any substance intended for use in the diagnosis, cure, mitigation, or treatment of a disease or to enhance the desired physical or mental development and condition of a patient (e.g., a person suffering from anemia).

[0122] The terms "therapeutically effective amount" and "effective amount" refer to the amount required to achieve the desired therapeutic effect for treating anemia (e.g., at a certain dosage, for a certain period of time, and for a particular mode of administration, such as enteral or oral). The effective amount of an agonist of the β2-adrenergic receptor can vary depending on factors such as the individual's disease state, age, sex, and weight, and the ability of the monoclonal molecule to elicit the desired response in the individual. The effective amount is also the amount in which the drug provider (e.g., the attending physician) determines that any toxic or harmful effects of the agonist are outweighed by the therapeutically beneficial effects.

[0123] II. Patients

[0124] A patient can be any person suffering from MDS or anemia (e.g., anemia associated with the cancers disclosed herein). A patient can be any elderly patient, or any patient suffering from an age-related condition who exhibits anemia.

[0125] 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.

[0126] In some embodiments, the patient is a child at least 5 years of age (i.e., 5 years of age or older). In some embodiments, the patient is between 5 and 18 years of age.

[0127] 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 has anemia associated with weight loss and / or decreased bone density (e.g., anemia associated with cancer as disclosed herein).

[0128] The methods, uses, and compositions encompassed by the present invention can be used for myelodysplastic syndromes (MDS) and the following anemias: anemia caused by serine / threonine-protein kinase RIOK2 deficiency, anemia caused by mutations or deletions in human chromosome 5, anemia caused by chromosomal translocations in the NUP98 gene or its orthologs, such as anemia caused by the infusion of NUP98 with Abd-B group HOX genes (e.g., HOXD13), megaloblastic anemia, anemia associated with inflammatory disorders such as rheumatoid arthritis or multiple sclerosis, anemia associated with chronic kidney disease (CKD), stress-induced anemia, chemotherapy-induced anemia in cancer patients, aplastic anemia, anemia secondary to bowel cancer, Diamond-Blackfan anemia, and Shwachman-Diamond syndrome. Similarly, the methods and compositions encompassed by the present invention can also be used within various bone marrow failure syndromes, as it has been determined that RIOK2 regulates blood cell development and agonists of RIOK2 activity reverse anemia associated with bone marrow failure syndromes such as aplastic anemia, Diamond-Blackfan anemia, dyskeratosis congenita (DC), Fanconi anemia (FA), Pearson syndrome, severe congenital neutropenia (SCN), Shwachman-Diamond syndrome (SDS), etc.

[0129] III. Administered Agents

[0130] Formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) can be administered to the patients disclosed herein in a biocompatible form suitable for in vivo drug administration to enhance its effect. "Biocompatible form suitable for in vivo administration" means a form in which any toxic effects are outweighed by the therapeutic effects. The administration of formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) can be carried out in any pharmacological form, including a therapeutically active amount of the agent alone or a combination of the agent with a pharmaceutically acceptable carrier.

[0131] Exemplary formoterol (molecular formula C 19 H 24 N 2 O 4) including but 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 formoterol [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.

[0132] Formoterol fumarate is the fumarate salt form of formoterol. Exemplary molecular forms of formoterol fumarate disclosed herein include formoterol fumarate (C 42 H 52 N 4 O 12 ), formoterol fumarate hydrate (molecular formula C 42 H 54 N 4 O 13 ), and formoterol fumarate dihydrate (molecular formula C 42 H 56 N 4 O 14 ). Additional 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]formanilide 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 those from Astellas Pharma Inc. from Mylan Specialty L.P. (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 Est Co., and 141492 from ChemShuttle.

[0133] Formoterol is well known in the art, such as in US 3,994,974 A and US 6,268,533 B1. In practicing the present invention, other formulation methods described in Remington's Pharmaceutical Sciences, 21st Edition, University of the Sciences in Philadelphia, Philadelphia, Pa., USA (2006) can be employed. 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).

[0134] Arformoterol (molecular formula C 19 H 24 N 2 O 4 ) 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).

[0135] As used herein, "arformoterol" includes the 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 N2 O 10 or C 19 H 24 N 2 O 4 ·C 4 H 6 O 6 )。Additional identifiers for formoterol 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-dihydroxybutanedioate (1:1 salt) (IUPAC), (R,R)-formoterol tartrate, 200815-49-2 (CAS), and 9827062 (PubChem). Commercially available forms of formoterol tartrate include, but are not limited to, (formoterol tartrate) from Sunovion Pharmaceuticals, Inc., SML1667 from Sigma-Aldrich, S5217 from Selleckchem, 6219 from Tocris Bioscience, and A12795 from AdooQ Bioscience.

[0136] Additional details regarding formoterol can be found in US 9,499,475 B2 and US 9,029,421 B2, which are hereby incorporated by reference.

[0137] The administration of a therapeutically active amount of the therapeutic composition covered by the present invention is defined as the amount effective to achieve the desired result in the necessary dosage and period of time. For example, the therapeutically active amount of the agent can vary depending on factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the peptide to elicit the desired response in the individual. The dosage regimen can be adjusted to provide the optimal therapeutic response. For example, several separate doses can be administered daily, or the dosage can be proportionally reduced, as indicated by the exigencies of the therapeutic situation.

[0138] The agents covered by the present invention can be administered alone or in combination with additional therapies. In combination therapies, one agent covered by the present invention and another agent, such as an erythropoiesis-stimulating agent (e.g., erythropoietin, epoetin alpha, epoetin beta, epoetin omega, epoetin zeta, or darbepoetin alpha) or other FDA-approved drugs (such as luspatercept, lenalidomide, and / or demethylating agents (such as azacitidine or decitabine)) can be delivered to the same or different cells and can be delivered at the same or different times. The agents covered by the present invention can be incorporated into a pharmaceutical composition suitable for administration. Such compositions can contain one or more agents or one or more molecules that produce such one or more agents and a pharmaceutically acceptable carrier.

[0139] 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 by oral administration, injection (subcutaneous, intravenous, intraperitoneal (i.p.), etc.), inhalation, transdermal application, or rectal administration. Depending on the route of administration, the active compound can be coated in a material to protect the compound from the effects of enzymes, acids, and other natural conditions that can inactivate the compound. For example, for the administration of an agent by a route other than parenteral administration, it may be desirable to coat the agent with a material or co-administer the agent with the material to prevent inactivation of the agent.

[0140] As described in detail below, the pharmaceutical compositions covered by the present invention (e.g., compositions containing formoterol or a pharmaceutically acceptable salt thereof (such as formoterol fumarate or arformoterol tartrate)) can be specifically formulated for administration in solid or liquid form, the forms including those suitable for: (1) oral administration, such as infusions (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes; or (2) parenteral administration, such as by subcutaneous, intramuscular, intraperitoneal (i.p.) or intravenous injection, such as sterile solutions or suspensions.

[0141] The phrase "pharmaceutically acceptable" as used herein refers to agents, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for contact with the tissues of humans without excessive toxicity, irritation, allergic response, or other problems or complications and are commensurate with a reasonable benefit / risk ratio.

[0142] As used herein, the phrase "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, involved in carrying or transporting a chemical from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Those skilled in the art will recognize 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) or will be able to adapt suitable pharmaceutically acceptable carriers for the therapeutic agents (see, e.g., Adejare, Adeboye, eds., Remington: the science and practice of pharmacy. Academic Press, 2020).

[0143] The term "pharmaceutically acceptable salt" refers to relatively non-toxic inorganic and organic acid addition salts of the therapeutic agents disclosed herein (i.e., formoterol, pharmaceutically acceptable salts of which 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 thus formed. Those skilled in the art will recognize suitable pharmaceutically acceptable salts for the therapeutic agents disclosed herein (e.g., formoterol, pharmaceutically acceptable salts of which include, for example, formoterol fumarate and arformoterol tartrate) or will be able to adapt suitable pharmaceutically acceptable salts for the therapeutic agents (see, e.g., Berge et al. (1977) J. Pharm. Sci. 66:1-19).

[0144] Wetting agents, emulsifying agents, and lubricants (such as sodium lauryl sulfate and magnesium stearate), as well as coloring agents, release agents, coating agents, sweetening agents, flavoring agents, and perfumes, preservatives, and antioxidants may also be present in the composition.

[0145] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0146] 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 (i.p.) injection). These formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient that may 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 dosage forms of formoterol can be found in the art, such as Yokoi et al. (1983) Life Sciences 33:1665-1672. In some embodiments, the pharmaceutical agents provided herein are oral formulations of formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate).

[0147] Generally, formulations are prepared by uniformly and intimately bringing into association a therapeutic agent with a liquid carrier or a finely divided solid carrier or both and then, if necessary, shaping the product.

[0148] Formulations suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavoured base, usually sucrose and acacia or tragacanth), powders, granules; or as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil liquid emulsion; or as an elixir or syrup; or as a soft gelatin capsule (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 compounds may also be administered in the form of a bolus, electuary, or paste.

[0149] In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules, etc.), the active ingredient is mixed with one or more pharmaceutically acceptable carriers such as sodium citrate or calcium phosphate dibasic and / or any one of the following: (1) fillers or extenders such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders such as carboxymethyl cellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia; (3) humectants such as glycerol; (4) disintegrants such as agar-agar, 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, for example, cetyl alcohol and glycerol monostearate; (8) absorbents such as kaolin and bentonite; (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, the pharmaceutical composition may also contain buffering agents. Solid compositions of a similar type may also be used as fillers in soft and hard gelatin capsules, the gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols.

[0150] Tablets may be prepared by compressing or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binders (e.g., gelatin or hydroxypropyl methylcellulose), lubricants, inert diluents, preservatives, disintegrants (e.g., sodium starch glycolate or cross-linked sodium carboxymethylcellulose), surfactants, or dispersing agents. Molded tablets may be prepared by molding in a suitable machine a mixture of powdered peptides or peptidomimetics moistened with an inert liquid diluent.

[0151] Tablets and other solid dosage forms, such as dragees, capsules, pills, and granules, may be optionally coated or prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulation art. The dosage forms may also be formulated to provide slow or controlled release of the active ingredient therein using, for example, different ratios of hydroxypropyl methylcellulose, other polymer matrices, liposomes, and / or microspheres, the different ratios being used to provide the desired release profile. The dosage forms may be sterilized, for example, by filtration through a bacteria-retaining filter or by incorporating a sterilizing agent in the form of a sterile solid composition that is soluble in sterile water or some other sterile injectable medium just prior to use. These compositions may also optionally contain emulsifying agents and the composition may be formulated so as to cause the active ingredient to be released only or preferentially in a particular part of the gastrointestinal tract, optionally in a delayed manner. Examples of embedding compositions that may be used include polymeric substances and waxes. The active ingredient may also be in microencapsulated form, if appropriate, with one or more of the above excipients.

[0152] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. Besides the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers, and emulsifying agents, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 - butanediol, oils (specifically, cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan and mixtures thereof.

[0153] Besides inert diluents, oral compositions may also include adjuvants such as wetting agents, emulsifying agents, and suspending agents, sweetening agents, flavoring agents, coloring agents, perfuming agents, and preservatives.

[0154] Besides the active pharmaceutical agent, suspensions may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol, and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, and tragacanth and mixtures thereof.

[0155] The pharmaceutical compositions covered by the present invention suitable for parenteral administration comprise a combination of one or more therapeutic agents 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, bacteriostatic agents, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.

[0156] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. The action of microorganisms can be ensured against by including various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. It may also be necessary to include isotonic agents such as sugars, sodium chloride, etc. in the composition. Additionally, extended absorption of injectable pharmaceutical forms can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0157] In some cases, in order to prolong the action of the drug, it is necessary to slow down the absorption of the drug from subcutaneous or intramuscular injection. This can be achieved by using liquid suspensions of poorly water - soluble crystalline or amorphous materials. The absorption rate of the drug depends on its dissolution rate, which in turn can depend on crystal size and crystal form. Alternatively, delayed absorption of parenterally administered drug forms is achieved by dissolving or suspending the drug in an oily vehicle.

[0158] An injectable depot form is prepared by forming a microcapsule matrix of formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) in a biodegradable polymer such as polylactic acid - polyethylene glycol. The drug release rate can be controlled according to the ratio of the drug to the polymer and the nature of the specific polymer employed. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot - type injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

[0159] When the therapeutic agent covered by the present invention is administered to a human as a medicament, it can be administered per se 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.

[0160] It should be understood that the total daily dose of the compounds of the present invention will be determined by the attending physician within the scope of reasonable medical judgment. The specific therapeutically effective dose level for any 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 age, weight, general health status, gender and diet of the subject; the time of administration, the route of administration and the plasma half-life of the specific compound employed; the duration of the treatment; drugs used in combination with or concomitantly with the specific agonist employed and similar factors well known in the medical arts. The daily dose of the active ingredient can include, but is not limited to, from about 0.1 μg to 100 μg per adult per day. Generally, the pharmaceutical composition contains 0.1 μg, 1.0 μg, 5.0 μg, 10.0 μg, 12.0 μg, 15.0 μg, 20.0 μg, 50 μg, 75 μg, 100 μg formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate), preferably 1 μg to 60 μg. 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 / 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 0.1 μg / day to 100 μg / day. In some preferred 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. In some embodiments, there is provided a method of treating anemia by administering to a patient a tablet or capsule in a dose of 0.1 μg to 100 μg, said tablet or capsule containing formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate). In some embodiments, the tablet or capsule is administered once daily. In some embodiments, a tablet or capsule containing formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) is administered once daily to a patient in a dose of 1 μg to 60 μg.

[0161] In some embodiments, there is provided a method of treating anemia by administering to a patient one or more tablets or capsules such that the subject receives a total daily dose of 0.1 μg to 100 μg, preferably 1 μg to 60 μg, said tablet or capsule containing formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate).

[0162] A therapeutically effective amount of a drug is typically provided at a certain dosage and administration route to achieve a therapeutically effective amount for treating anemia with formoterol or a pharmaceutically acceptable salt thereof (e.g., formoterol fumarate or arformoterol tartrate) and a plasma level of 0.03 - 150 pg / mL (preferably 0.3 - 30 pg / mL).

[0163] The actual dosage level of the active ingredient in the pharmaceutical compositions covered by the present invention can be determined by the methods covered by the present invention to obtain an amount of the active ingredient that effectively achieves the desired therapeutic response for a particular patient, composition, and mode of administration and is non-toxic to the patient.

[0164] Examples

[0165] Example 1: Formoterol fumarate (FF) enhances erythroid differentiation and mitochondrial function

[0166] A novel therapeutic approach for targeting anemia in MDS and other human conditions by utilizing formoterol fumarate (FF), a β2 - adrenergic receptor agonist approved by the FDA 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), is described herein. It is shown herein that formoterol fumarate (FF) simultaneously enhances mitochondrial biogenesis and erythroid differentiation in primary human hematopoietic stem and progenitor cells (HSPCs). Consistent with this, deletion of ADRB2, which encodes the β2 - adrenergic receptor (β2 - AR), significantly impairs erythroid differentiation as well as mitochondrial biogenesis. FF treatment significantly enhances erythropoiesis in bone marrow cells derived from MDS patients. Administration of FF significantly enhances erythroid differentiation and RBC parameters in wild - type mice in both steady - state and stress - induced hemolytic anemia settings. FF treatment confers a remarkable survival benefit to mice responding to lethal hemolytic anemia. Thus, a novel use of an existing FDA - approved drug for reversing anemia is disclosed, and therapeutic benefits are provided for a range of human diseases, including but not limited to anemia in hematological malignancies, aplastic anemia, anemia in chronic kidney disease, ribosomopathies, anemia secondary to chemotherapeutic agents in cancer patients, and bone marrow failure (BMF) disorders.

[0167] Hematological disorders and mitochondrial dysfunction.

[0168] Mitochondrial dysfunction is commonly associated with the pathogenesis of hematological disorders such as MDS and AML (Fontenay et al. (2006) Oncogene 25:4757-4767). For example, mitochondrial transcriptional alterations (Schildgen et al. (2011) Exp Hematol 39:666-675), dysregulation of HIF1α expression (Liu et al. (2019) Oncol Lett 17:5395-5402; Stergiou et al. (2021) Int J Mol Sci 22), the presence of isocitrate dehydrogenase (IDH) mutations that express the oncometabolite 2-hydroxyglutaric acid (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), elevated mitochondrial oxidative stress markers (Saigo et 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 suggest a key role of mitochondrial function defects in the pathogenesis of MDS. MtDNA encodes 13 polypeptides in conjunction with the nuclear-encoded transcriptional and translational machinery, which forms an integral part 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 genome remains intertwined with the nuclear genome in a tightly balanced relationship to govern cellular metabolism and prevent dysregulation that would inevitably alter metabolite 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 metabolite 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 emphasize the involvement of metabolite and metabolic pathway dysregulation in the etiology of MDS. Dysregulation of metabolite levels has also been reported in bone marrow failure diseases (Zhong et al. (2015) Genet. Mol. Res. 14:13709-13718). The present disclosure encompasses the recognition that mitochondrial defects contribute to the pathogenesis of hematological disorders.

[0169] β2-Adrenergic Receptor (β2-AR) Agonists and Mitochondrial Biogenesis

[0170] The β2-AR is a cell membrane-spanning receptor for adrenaline / epinephrine, which mediates smooth muscle relaxation and bronchodilation through adenylate cyclase stimulation (Abosamak and Shahin (2021). StatPearls Database (StatPearls) (Treasure Island (FL)); Johnson (2006) Journal of Allergy and Clinical Immunology (J Allergy Clin Immunol) 117, 18-24; quiz 25; Yang et al. (2021) Life Sciences (Life Sci) 265:118864). The β2-AR is encoded by the ADRB2 gene. The long-acting selective β2-AR agonist formoterol fumarate (FF) is FDA-approved for the treatment of COPD and asthma (Hanania et al. (2019) International Journal of Chronic Obstructive Pulmonary Disease 14:117-127; Ni et al. (2018) Cochrane Database of Systematic Reviews (Cochrane Database Syst Rev) 12, CD011594; Sharafkhaneh et al. (2010) International Journal of Chronic Obstructive Pulmonary Disease 5:357-366). FF is highly specific for the β2-AR as examined in ADRB2-depleted mice (Cameron et al. (2017) Scientific Reports 7:10578). Interestingly, FF has been reported to enhance mitochondrial biogenesis in several in vitro and in vivo models (Peterson et al. (2013) Bioorganic & Medicinal Chemistry Letters (Bioorg Med Chem Lett) 23:5376-5381; Wills et al. (2012) Journal of Pharmacology and Experimental Therapeutics (J Pharmacol Exp Ther) 342:106-118), such as ischemia-reperfusion-mediated kidney injury (Jesinkey et al. (2014) Journal of the American Society of Nephrology (J Am Soc Nephrol) 25:1157-1162), spinal cord injury (Scholpa et al. (2019) Experimental Neurology (Exp Neurol) 322:113064), and traumatic brain injury (Vekaria et al. (2020) Neurobiology of Disease (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 International (Kidney Int) 96:656-673). However, the underlying mechanism of FF-mediated enhancement of mitochondrial biogenesis remains poorly defined.

[0171] Results

[0172] Formoterol fumarate (FF) enhances erythroid differentiation and mitochondrial function in primary human hematopoietic stem and progenitor cells (HSPCs).

[0173] Given that β2-AR is involved in erythropoiesis, it was hypothesized that administration of the long-acting selective β2-AR agonist FF might promote erythroid differentiation. To address this, primary human HSPCs were treated with different doses of FF (Selleckchem). Notably, FF administration elevated erythroid differentiation in primary human HSPCs in a dose-dependent manner ( Figure 1A ), without affecting viability, megakaryopoiesis, or myelopoiesis ( Figure 1B and Figure 1D ). As confirmation, formoterol fumarate (FF) from 2 different suppliers (Selleckchem and Sigma) similarly enhanced erythroid differentiation in primary human HSPCs ( Figure 3A and Figure 3B ). To determine whether FF administration acts more upstream in the erythroid differentiation pathway, methylcellulose colony formation assays were performed. Indeed, FF treatment significantly increased the formation of burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) ( Figure 4 ). Consistent with the existing literature, treatment with FF enhanced the expression of mtDNA-encoded genes required for OXPHOS ( Figure 4 A), and elevated mitochondrial mass and membrane potential in primary human HSPCs ( Figure 5B and Figure 5C ). These data suggest the therapeutic potential of FF in alleviating anemia.

[0174] FF treatment in in vitro and ex vivo MDS models

[0175] Since RIOK2-deficient primary human HSPCs effectively model impaired erythropoiesis leading to anemia (Ghosh et al. (2022) Nat Immunol 23:109–121), it was next investigated whether treatment with formoterol fumarate (FF) alleviates anemia in this in vitro model. To address this, RIOK2-proficient primary human HSPCs and RIOK2-deficient primary human HSPCs were generated by CRISPR-Cas9-based genome editing ( Figure 6A ), as previously described (Ghosh et al. (2022) Nat Immunol 23:109–121). Formoterol fumarate consistently enhanced erythroid differentiation in control HSPCs ( Figure 6B)。Notably, erythropoiesis in FF-stimulated RIOK2-knockdown (KD) HSPCs was administered ( Figure 6B )。Importantly, FF treatment elevated erythroid differentiation of RIOK2-deficient HSPCs to a level comparable to basal erythropoiesis in control cells ( Figure 6B )。Since RPS14 deletion has been reported as an MDS genotype (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 )。Administration of FF significantly increased erythroid differentiation in RPS14 KD 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. Due to the lack of the ADRB2 gene encoding β2-AR, FF treatment failed to induce erythropoiesis in ADRB2-deficient HSPCs.

[0176] These findings prompted additional tests to determine whether administration of FF in MDS patient-derived bone marrow (BM) samples improves erythroid differentiation in an ex vivo setting. To explore this question, cells isolated from BM aspirates of de-identified MDS patients were purchased. Next, MDS patient-derived BM cells 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 in BM cells of most MDS patients, but a few MDS patient bone marrow (BM) cells 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) increased erythropoiesis in most MDS patients ( Figure 8B )。Next, CD34+ progenitors from MDS patient-derived BM aspirates were FACS sorted and seeded in semi-solid methylcellulose medium with vehicle (DMSO) or FF treatment. FF significantly increased the formation of BFU-E and CFU-E in MDS patient-derived BM progenitors ( Figures 11A - 11C )。However, the effect of FF administration on the viability, myeloid, and megakaryocytic differentiation of MDS patient-derived BM cells was not significant ( Figure 11D)。Therefore, the conclusion is that formoterol fumarate (FF) not only effectively enhances erythroid differentiation in primary hematopoietic stem cells from healthy individuals but also promotes erythroid differentiation in BM cells derived from MDS patients. Therefore, the FDA-approved drug formoterol fumarate can be repurposed to reverse anemia in a range of hematological disorders.

[0177] FF treatment alleviates anemia in vivo

[0178] The protein composition of the murine β2 - adrenergic receptor has 87.08% similarity to that of the human analogue. This encouraged further testing to determine whether formoterol fumarate (FF) treatment in wild - type mice under steady - state or hemolytic anemia conditions has any alleviating effect in vivo. To address this question, different doses of FF 0.1 - 0.3 - 0.5 - 1.0 mg / kg or vehicle (saline containing 0.3% DMSO) were administered daily by intraperitoneal (i.p.) injection for 3 weeks in 10 - 12 - week - old wild - type C57BL / 6J mice, while submandibular cheek bleeds were performed weekly to analyze complete blood count (CBC). The mice tolerated daily intraperitoneal injection of FF well, as seen by Figure 14A body weight ( Figure 14A ), mobility, activity, and regular breathing pattern (as recommended by the veterinary staff of the animal research facility). The 0.3 - 0.5 mg / kg and 1.0 mg / kg FF doses moderately increased the body weight of the mice, but the values did not reach significance ( ). However, FF treatment at all doses significantly enhanced mitochondrial biogenesis in the peripheral blood mononuclear cells (PBMC) of the mice, as demonstrated by an increase in Figure 14B staining ( Figures 14C - 14E ). Importantly, red blood cell (RBC) parameters such as RBC count, hematocrit (HCT)% and hemoglobin (Hb) were slightly elevated in the FF - treated groups ( Figure 14F ), while white blood cell (WBC) count and monocyte count were still slightly affected ( Figure 14G ).

[0179] These results prompted further research to determine the possible effects of FF in mice undergoing sub-lethal hemolytic anemia. To address this, a sub-lethal dose of phenylhydrazine (PHZ: 50 mg / kg) was first administered to female mice aged 10 - 12 weeks, followed by daily intraperitoneal injection of 0.1 / 0.3 mg / kg FF or vehicle (saline containing 0.3% DMSO). PHZ is a strong oxidizing agent that readily oxidizes hemoglobin in RBCs, triggering their immediate lysis and thereby causing hemolytic anemia. Treatment with a low dose of 0.1 / 0.3 mg / kg FF was sufficient to restore the body weight of mice undergoing PHZ-induced hemolytic anemia ( Figure 18A ). Compared to the vehicle-treated group, FF treatment significantly enhanced Hb production ( Figure 18B ). Additionally, compared to the control group, FF administration produced long-term benefits in elevating the HCT% and RBC count of mice ( Figure 18B ). Although FF treatment caused an immediate increase in WBC count and monocyte count after PHZ administration, in contrast to RBC parameters, no long-term benefits were observed in the FF-treated group ( Figure 18C ). Platelet count was 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 and produced insignificant changes in the myeloid and megakaryocyte compartments ( Figures 1A - 1D and Figures 11A - 11D ). To eliminate the possibility that the FF-mediated increase in RBC parameters could be a spurious effect, mice were sacrificed after the study endpoint (14 days), and their bone marrow (BM) progenitors were analyzed. Interestingly, treatment with a low dose of 0.1 - 0.3 mg / kg FF significantly enhanced erythroid differentiation, as evidenced by a marked increase in RI, RII, RIII, and RIV erythroid progenitors in the BM of mice ( Figure 19A and 19B ). This clearly indicates that FF acts directly on BM progenitors to induce erythroid differentiation. To confirm these findings, this experiment was repeated in male C57BL / 6J mice aged 10 - 12 weeks, and similarly, a significant recovery in body weight was observed in FF (0.1 / 0.3 mg / kg)-treated mice compared to the vehicle-treated group in response to sub-lethal PHZ-induced hemolytic anemia (PHZ dose: 60 mg / kg) ( Figure 22A ). Compared to vehicle-treated controls, FF treatment consistently improved RBC parameters in the PB of anemic mice, such as Hb, RBC count, and HCT% ( Figure 22B ). Similarly, in the FF-treated group, no long-term effects were observed in WBC parameters or platelets, except for an immediate increase after PHZ treatment ( Figure 22C)。Surprisingly, BM cells from the FF-treated group showed a significant increase in viability ( Figure 23A ), which was partly due to elevated mitochondrial biogenesis, as demonstrated by staining increase ( Figure 23B ) and decreased mitochondrial superoxide production (mitoSOX staining) ( Figure 23C ). Thorough analysis of live lineage-negative BM progenitors showed that FF treatment specifically increased megakaryocyte-erythroid progenitors (MEPs), but not common myeloid progenitors (CMPs) or granulocyte-macrophage progenitors (GMPs) ( Figures 26A - 26D ). Since MEPs give rise to megakaryocytes or erythroid progenitors, two BM cell compartments were examined. However, no significant differences were observed in megakaryocyte progenitors ( Figure 27 ), which is consistent with the insignificant 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 ), which is consistent with the FF-mediated enhancement of erythroid differentiation in the BM of female mice ( Figure 19A -19C). These findings suggest that formoterol fumarate (FF) acts directly on early BM progenitors to stimulate erythroid differentiation, which ultimately enhances RBC parameters in peripheral blood (PB) in vivo.

[0180] The marked remission of RBC parameters and the recovery of body weight in FF-treated anemic mice encouraged further testing of the survival benefit of FF in response to phenylhydrazine (PHZ)-mediated lethal hemolytic anemia. For this purpose, 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 injection of vehicle or 0.1 / 0.3 mg / kg FF. Compared with the vehicle-treated group, low-dose FF treatment at 0.1 / 0.3 mg / kg produced a remarkable survival benefit ( Figure 42A ). All control mice (n = 5) were moribund and died 3 days after the lethal PHZ dose, while 3-4 mice in the 0.1 / 0.3 mg / kg FF treatment group remained alive 2 weeks after the lethal dose ( Figure 42A ). FF treatment significantly restored the body weight of mice within the first 7 days of the lethal PHZ dose ( Figure 42B ). FF injection was stopped 7 days after the lethal PHZ treatment, and the body weight of the mice was observed to remain stable ( Figure 42B)。The body weights and survival rates of these mice were monitored daily. This unexpected finding prompted the repetition of this lethal hemolytic anemia model in female mice. Consistently, FF treatment at a dose of 0.1 / 0.3 mg / kg provided a significant survival benefit to female mice, while all (n = 5) vehicle-treated mice were moribund or dead by day 3 at the lethal PHZ dose ( Figure 42C )。FF treatment similarly restored the body weight of mice after lethal anemia stress ( Figure 42D )。FF treatment was continued for 7 days after the lethal PHZ dose, and the body weights and survival rates of these mice were monitored daily. These data confirm that FF produces a significant survival benefit in vivo in response to hemolytic anemia, thereby demonstrating the therapeutic potential of FF in treating anemia.

[0181] Example 2: Materials and methods of Example 1

[0182] Mouse experiments

[0183] C57BL / 6J mice were obtained from Jackson laboratories and were housed in the animal resource facility of the Dana-Farber Cancer Institute (DFCI) for at least 2 weeks before the start of the experiment. All drug treatments were described in detail in the text.

[0184] Primary cell culture and subculture

[0185] CD34+ primary human hematopoietic stem and progenitor cells (HSPC) were obtained from the Fred-Hutchinson Cancer Research center, Seattle, USA, and were cultured as previously described. Formoterol fumarate was purchased from Selleckchem (#S2020) and Sigma (#F9552), and was dissolved (in DMSO) and stored according to the manufacturer's instructions, and was used at the indicated final working concentration. Cryopreserved BM aspirates from de-identified MDS patients were obtained from the Pasquarello tissue bank at DFCI using an IRB-approved protocol (#21-632).

[0186] Mitochondrial fitness test

[0187] TMRE (T669, Invitrogen) and (M22426, Life Technologies) and (M36008, Life Technologies) staining were performed according to the manufacturer's instructions.

[0188] CRISPR / Cas9 gene editing

[0189] Genome editing of primary human HSPCs was performed as previously described (Ghosh, S., Raundhal, M., Myers, S.A., Carr, S.A., Chen, X., Petsko, G.A. and Glimcher, L.H. (2022). Identification of RIOK2 as a master regulator of human blood cell development. Nature Immunology 23, 109-121).

[0190] Quantitative RT-PCR

[0191] As previously described.

[0192] Flow cytometry and sorting

[0193] FACS staining and sorting were performed as previously described (Ghosh et al. (2022) Nature Immunology 23:109-121).

[0194] Methylcellulose assay

[0195] After genome editing, primary human HSPCs were rinsed twice with 1x PBS and mixed with semi-solid methylcellulose medium (H4034 StemCell Technologies) by brief vortexing. Cells were plated at a density of 2000 per well in 6-well plates and then incubated for 14 days in a humidified chamber at 37°C. Colonies were imaged using an EVOS M5000 imaging system (ThermoFisher Scientific). Colony-forming cells were then collected by wet-grinding the wells with staining buffer and multicolor flow cytometry was performed as previously described (Li et al. (2014) Blood 124:3636-3645).

[0196] Statistical tests

[0197] Data are presented as mean ± SEM. Unpaired two-tailed t-tests were used to compare two groups. Analysis of variance (ANOVA) with Tukey's correction or the Kruskal-Wallis test with Dunn's correction was used to compare multiple groups as applicable according to the data and quantitative requirements. GraphPad Prism v8.0 / 9.0 (GraphPad Software Inc., San Diego, CA) was used for statistical analysis. The sample size was not predetermined.

[0198] Example 3: Nonlinear dose-specific effects of formoterol fumarate (FF) treatment in wild-type mice

[0199] As described in Example 1 above, formoterol fumarate (FF) treatment of wild-type mice was well tolerated ( Figures 14A - 14E ). The effects of different doses of FF from 0.1 mg / kg to 1.0 mg / kg were evaluated in naïve mice in the absence of any external stress with phenylhydrazine (PHZ). Specifically, different doses of FF 0.1 - 0.3 - 0.5 - 1.0 mg / kg or vehicle (saline containing 0.3% DMSO) were administered daily by intraperitoneal (i.p.) injection to 10 - 12-week-old wild-type C57BL / 6J mice for a 30-day period.

[0200] 0.1 - 1.0 mg / kg FF treatment showed a dose-dependent increase in body weight at doses of 0.1 - 0.5 mg / kg, resulting in a moderate increase in the body weight of naïve mice. Significantly, at the highest dose of 1.0 mg / kg FF, a marked increase in body weight was observed ( Figure 83 ).

[0201] Interestingly, a dose-dependent effect on red blood cell (RBC) parameters was observed, where effects were seen at low to moderate doses but not at the highest dose. As Figure 84A shown, the hemoglobin (Hb) level increased with the 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, the hematocrit (HCT)% and RBC count were elevated in mice treated with 0.3 mg / kg dose and 0.5 mg / kg dose of FF, but not at the lowest dose (0.1 mg / kg FF) or the highest dose (1.0 mg / kg FF) ( Figures 84B - 84C ).

[0202] The white blood cell (WBC) count was only slightly affected at the highest dose of 1.0 mg / kg FF ( Figure 85A ), while the monocyte count was only slightly affected at the FF doses of 0.3 mg / kg and 0.5 mg / kg, but not affected at the highest or lowest doses (1.0 mg / kg FF and 0.1 mg / kg FF, respectively) ( Figure 85B ). No effect was observed on platelets with the administration of any dose of FF ( Figure 85C ).

[0203] These results support that, in contrast to the effect on body weight, FF has a non-linear dose-specific effect on blood cell parameters.

[0204] Example 4: Bone mass in mice treated with formoterol fumarate (FF)

[0205] As described in Example 1 above, treatment with formoterol fumarate (FF) was associated with an increase in body weight, and FF treatment also restored the body weight of phenylhydrazine (PHZ)-mediated anemic mice. FF has been shown to have an anabolic effect on bone in an ovariectomized rat model, which may be at least partly due to the agonistic effect of FF on β-adrenergic receptors expressed on bone cells. See Kellenberger et al. (1998) Bone 22(5):471-478, which is incorporated by reference. FF has also been shown 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 investigate whether the increase in body weight was associated with an increase in bone mass and / or muscle mass, a DEXA scan was performed. Specifically, C57BL / 6J mice (2-3 month-old wild-type steady-state mice (untreated with phenylhydrazine)) were treated with FF at a dose of 0.3 mg / kg or vehicle (saline containing 0.3% DMSO) by intraperitoneal (i.p.) injection 5 times a week for one month, followed by a DEXA scan to measure bone density, muscle and fat mass, and also free and total water content in the mice. Mice treated with other β2-adrenergic receptor agonists not associated with weight gain were also evaluated.

[0206] Example 5: RNA sequencing in mice treated with formoterol fumarate (FF)

[0207] This example describes the effect of formoterol fumarate treatment on the expression of genes related to erythropoiesis, including 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 Med 381:1011 - 1022), Epas1 (Scortegagna et al. (2003) Blood 102:1634 - 40), Ddit4 (Yoshikawa (2023) Cancer Biomark. 37(4):217 - 225), Cirbp, and Rora (Kim et al. (2008) Arterioscler Throm Vasc Biol 28(10):1796 - 802 and Chauvet et al., (2004) Biochem. J. 384:79 - 85). According to Figure 86 the example workflow depicted in Figure 87A -B shows the example differential gene expression of erythroid progenitor cells isolated from the bone marrow of FF - treated mice relative to those isolated from the bone marrow of vehicle - treated mice. The expression of many genes involved in erythropoiesis is increased in FF - treated mice, including Klf9, Klf13, Klf11, Hif1a, Epas1, Ddit4, Cirbp, and Rora. Notably, Figure 88A -B shows that the expression of HIF - 1α and VEGFA is significantly increased during FF treatment in wild - type mice, as verified by qPCR analysis. These data support the multi - target effect of formoterol in increasing erythropoiesis and treating anemia.

[0208] Incorporation by Reference

[0209] All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application (including any definitions herein) will control.

[0210] Equivalent Forms

[0211] 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. Such equivalents are intended to be encompassed by the following claims.

[0212] Other Abbreviations

[0213] CFU-GM: Colony Forming Unit - Granulocyte Macrophage

[0214] CFU-Mk: Colony Forming Unit - Megakaryocyte

[0215] COPD: Chronic Obstructive Pulmonary Disease

[0216] RIOK2: Right Open Reading Frame Kinase 2

[0217] TMRE: Tetramethylrhodamine Ethylester

Claims

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

2. The method according to claim 1, wherein the anemia is selected from the group consisting of: megaloblastic anemia, hemolytic anemia, anemia caused by ribosomopathy, anemia caused by serine / threonine-protein kinase RIOK2 deficiency, anemia associated with chronic kidney disease (CKD), anemia caused by one or more mutations and / or deletions in human chromosome 5 or its orthologs, stress-induced anemia, Diamond-Blackfan anemia, aplastic anemia, Schwachman-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 syndrome.

3. The method according to claim 1 or claim 2, wherein 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).

4. The method according to claim 1 or claim 2, wherein the anemia is associated with cancer, optionally wherein the cancer is intestinal cancer such as colorectal cancer.

5. A method for promoting the differentiation of erythroid progenitor cells into mature red blood cells 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.

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

7. The method according to claim 6, wherein the anemia is selected from the group consisting of: megaloblastic anemia, hemolytic anemia, anemia caused by ribosomopathy, anemia caused by serine / threonine-protein kinase RIOK2 deficiency, anemia associated with chronic kidney disease (CKD), anemia caused by one or more mutations and / or deletions in human chromosome 5 or its orthologs, stress-induced anemia, Diamond-Blackfan anemia, aplastic anemia, Schwachman-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 syndrome.

8. The method according to claim 6 or claim 7, wherein 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).

9. The method according to claim 6 or claim 7, wherein the anemia is cancer-related, optionally wherein the cancer is bowel cancer, such as colorectal cancer.

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

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

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

13. The method according to claim 12, wherein the anemia is selected from the group consisting of megaloblastic anemia, hemolytic anemia, anemia caused by ribosomopathies, anemia caused by serine / threonine-protein kinase RIOK2 deficiency, anemia associated with chronic kidney disease (CKD), anemia caused by one or more mutations and / or deletions in human chromosome 5 or its orthologs, 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. The method according to claim 12 or claim 13, wherein the anemia is cancer-related, 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).

15. The method according to claim 12 or claim 13, wherein the anemia is cancer-related, optionally wherein the cancer is bowel cancer, such as colorectal cancer.

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

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

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

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

20. The method according to claim 19, wherein the formoterol is arformoterol tartrate.

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

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