Amelioration Of Anemia Research Articles

Therapy for Myelodysplastic Syndromes Beyond the Front Line in 2024 in Canada

Management of anemia and/or transfusion dependence (TD) after failure of erythropoietic-stimulating agents (ESA) and therapeutic options after hypomethylating agent (HMA) failures remain the biggest challenges for physicians treating lower and higher-risk myelodysplastic syndromes (MDS), respectively. Fortunately, new therapies are available (or soon to be approved), and innovations in prognostic refinement using next-generation sequencing may also facilitate more precision medicine. This review highlights commercially available (or soon to be) options for the amelioration of anemia and transfusion dependence when ESA’s fail and the management of higher-risk MDS when hypomethylating agents fail or cease working. While not all of these agents are currently funded or approved in Canada, some are available for off-label access or purchase.

TRACK_9: Testosterone replacement assessment: Classical vs. functional hypogonadism-knowledge from a 9-year study.

The longitudinal efficacy and clinical utility of Testosterone Therapy (TTh) in ameliorating functional hypogonadism (FH) remain contentious, with long-term data being scarce. To address this lacuna, a comprehensive long-term registry study, stratifying patients across a spectrum of hypogonadal etiologies, offers a robust investigative paradigm. This 9-year registry, encompassing 650 patients (equivalent to 4,362 cumulative years of treatment), included 188 patients diagnosed with FH (mean age 42.3±11.3 years) and 462 individuals with classical hypogonadism (CH). The cohort segregated into 266 men with primary hypogonadism (PH, mean age 34.0±11.7 years) and 196 with secondary hypogonadism (SH, mean age 31.9±12.0 years). Uniform treatment across the cohort involved intramuscular administration of testosterone undecanoate (1,000mg). A comparative analysis was conducted focusing on anthropometric, metabolic, and safety parameters. Serum testosterone levels increased from 6.6±2.4 to 19.3±2.9nmol/L (p<0.001). TTh was linked with weight reduction and decreased waist circumference (WC) in both CH and FH cohorts (both p<0.001). Cox regression and Kaplan-Meier analyses delineated disparities: men with FH demonstrated a higher propensity for losing>10% body weight and>5% WC compared to CH (hazard ratio [HR] 1.3 [1.1-1.4], p=0.008 and HR 1.4 [1.3-1.5], p=0.001). Increases in hematocrit>50% were uniform across groups, albeit amelioration of anemia was more pronounced in FH versus CH (p=0.002). Increments of prostate-specific antigen (PSA) levels were more likely to occur in FH (HR 1.3 [1.1-1.6], p=0.003). FH patients exhibited pronounced improvements in metabolic parameters and in aging male symptom score (AMS) and IIEF-EF questionnaire scores. These effects were markedly modulated by age and initial weight. Subgroup analysis of age-matched obese patients revealed an accentuated impact of TTh in CH compared to FH. The therapeutic outcomes of TTh across distinct hypogonadal populations demonstrate heterogeneous responses, significantly influenced by diagnostic categorization, age, and baseline risk factor profiles.

Antiplasmodial Activity of β-Ionone and the Effect of the Compound on Amelioration of Anaemia and Oxidative Organ Damage in Mice Infected with Plasmodium berghei.

Owing to evolution of parasite strains that are resistant to existing antimalarial drugs, research for novel antimalarial medicines is progressing on numerous fronts. Herein, we evaluated the in vivo anti-Plasmodium berghei activity of β-ionone including its ameliorative potential towards P. berghei-associated anaemia and oxidative organ damage. Mice were infected with chloroquine-sensitive strain of P. berghei and then treated with β-ionone at doses of 10 and 20mg/kg body weight (BW) for seven days. The parasitemia, packed cell volume and redox sensitive biomarkers in the liver, brain and spleen were estimated. Our result showed that β-ionone, in a dose-dependent fashion, significantly (p < 0.05) repressed the multiplication of P. berghei. More so, the compound, at doses of 10 and 20mg/kg BW, significantly (p < 0.05) mitigated anaemia and organ damage induced by P. berghei. Overall, the findings demonstrated that β-ionone has antiplasmodial actions and plays a mitigative role against P. berghei-induced anaemia and oxidative organ damage.

ALK2 Inhibition and a Modified Activin Receptor Type Iia Ligand Trap Cotherapy Maximized Hematologic Improvements in a Mouse Model of Anemia of Inflammation

Introduction: Anemia of inflammation (AI) is commonly seen in patients with chronic diseases, including chronic kidney disease (CKD). Current therapeutic options to mitigate anemia in AI include treating the underlying inflammatory disease, oral or intravenous iron supplementation, erythropoietin, and blood transfusions. The persistent release of inflammatory cytokines in AI not only induces the iron regulator hepcidin to inhibit iron absorption and recycling, but also directly impacts the marrow to suppress erythropoiesis, further worsening the ineffective, iron-restricted erythropoiesis. Therefore, targeting both iron and erythropoiesis could comprehensively address anemia in AI. We previously showed that ALK2 inhibition was sufficient to reduce serum hepcidin and improve hemoglobin in a CKD mouse model. We also demonstrated that a modified activin receptor IIA ligand trap RKER-050 increased hemoglobin and red blood cell production by promoting erythroid differentiation in mice. Here, we explored the potential of additive improvements in hematologic parameters that could be achieved with a combination of hepcidin suppression by ALK2 inhibition and increased hematopoiesis with RKER-050 in a mouse model of AI. Methods: C57BL/6 mice were fed a control or 0.2% adenine with 40 ppm iron diet (Ade 40) for 5 weeks to induce CKD. After confirming anemia, CKD mice were subcutaneously treated with 3 mg/kg isotype control or KTI-m216, a neutralizing antibody against ALK2, twice a week for 4 weeks while continuing the Ade 40 diet or switching to a 0.2% adenine with 3 ppm low iron diet (Ade 3). Combination therapy was evaluated in CKD mice on Ade 40 diet receiving biweekly isotype or KTI-m216 and also received either weekly PBS or 7.5 mg/kg RKER-050 intraperitoneally for 4 weeks. Results: CKD mice developed impaired kidney function and characteristics of AI, including increased serum IL-6 and hepcidin, hypoferremia, tissue iron retention, and anemia compared to control mice. Serum hepcidin was reduced >95% in KTI-m216 treated mice on both Ade diets compared to mice receiving isotype control (CKD control). Transferrin saturation (TSAT) increased 65% in KTI-m216 treated mice on Ade 40 diet, with a similar trend in mice on Ade 3 diet. When compared to CKD control of the same diet, KTI-m216 treated mice had similar hematologic improvements: hemoglobin (+2.2 g/dL), hematocrit (+5.4%), red blood cells (+1.1 M/μl), and reticulocyte hemoglobin (CHr, +1.8 pg). These data support that increased circulating iron by KTI-m216 mediated hepcidin suppression can increase erythropoiesis in CKD mice. Data also suggest that dietary iron contribution is negligible in supporting erythropoiesis in this context. With combination therapy, mice receiving KTI-m216 alone (KTI-m216 and PBS) and a combined KTI-m216 and RKER-050 therapy had a similar reduction in serum hepcidin (>91%) and a similar increase in TSAT (>1.7-fold), compared to mice receiving vehicle (isotype and PBS) controls. Mice treated with KTI-m216 alone increased hemoglobin to 11.4 g/dL compared to vehicle treated mice with hemoglobin of 8.0 g/dL. Treatment with a combined KTI-m216 and RKER-050 therapy showed a further increase in hemoglobin to 13.5 g/dL. Additionally, red blood cell production was 6.4 M/μl in mice given vehicle controls and increased to 8.3 M/μl in KTI-m216 alone and 9.8 M/μl in combined KTI-m216 and RKER-050; hematocrit was 23.4% in vehicle control, 32.5% in KTI-m216 alone and 38.7% in combined KTI-m216 and RKER-050. Although hematocrit was not fully rescued, hemoglobin and red blood cells in mice receiving a combined KTI-m216 and RKER-050 therapy were not different from the non-CKD controls. Conclusion: These data illustrate that inhibition of ALK2 with KTI-m216 could potentially release sufficient iron from the recycling pathway to ameliorate anemia, but not restore RBC parameters to normal levels. Combined treatment with activin receptor ligand trap, RKER-050 and KTI-m216 normalized the RBC production in this preclinical model. These data support that increasing erythropoiesis with RKER-050 combined with iron mobilization with KTI-m216 has the potential to achieve the maximal amelioration of anemia in AI.

Management of Myelofibrosis-Associated Anemia: Focus on Standard Agents and Novel Therapeutics in Phase 3 Clinical Trials.

The management of myelofibrosis is risk-adapted when considering transplant eligibility and symptom-directed, prioritizing the most burdensome symptoms for the patient. Unfortunately, myelofibrosis-anemia is common, multifactorial in its origin, and impactful regarding prognosis. While clinical trials are advised, not all patients have convenient access, and therefore, hematologists should be aware of the data supporting the use of conventional agents such as erythropoietin-stimulating agents, steroid treatments (danazol and prednisone), and immunomodulatory drugs (thalidomide and lenalidomide). This review summarizes the conventional approach to treating myelofibrosis-anemia and highlights recent data from 3 novel agents that are under phase 3 evaluation. Momelotonib is a JAK1/2 and ACVR1 inhibitor that has demonstrated not only improvements in splenomegaly and symptoms, but also amelioration of anemia on the SIMPLIFY 1 and 2 clinical trial program. This may occur through suppression of hepcidin production. Luspatercept promotes late-stage hematopoiesis, and the phase 2 study has shown promise in ameliorating anemia as a monotherapy, and especially in combination with ruxolitinib. Finally, CP-0160, a BET inhibitor, has shown efficacy as an anemia-directed agent, when used as monotherapy and in combination. This agent reduces cytokine production and promotes erythroid differentiation. Results have been presented for patients previously treated with JAK inhibitors, as well as those who were naïve to JAK inhibitor therapy. Safety and effectiveness are reviewed for both conventional and selected novel agents used in the treatment of MF-anemia. A practical approach to treatment is presented, and data from ASH 2020 are presented.

New drugs for myeloid neoplasms with ring sideroblasts: Luspatercept vs imetelstat.

New drugs for myeloid neoplasms with ring sideroblasts: Luspatercept vs imetelstat.

Stabilizing HIF to Ameliorate Anemia

Erythropoietin (EPO) production in the kidney is regulated by the oxygen-sensing transcription factor HIF-1α, which is degraded under normoxic conditions by HIF-prolyl hydroxylase (HIF-PHD). Inhibition of HIF-PHD by roxadustat leads to increased EPO production, better iron absorption, and amelioration of anemia in chronic kidney disease (CKD).

Iron Restriction Improves Markers of Disease Severity in the Townes Mouse Model of Sickle Cell Anemia

Introduction: Sickle cell anemia (SCA) is caused by mutations in β-globin that result in the production of the abnormal hemoglobin, HbS, with deleterious effects on erythrocyte shape and life span. Because the propensity of erythrocytes to sickle is inversely proportional to the concentration of HbS in the cell, decreasing the mean cellular hemoglobin concentration (MCHC) represents a potential therapeutic approach. Whereas iron restriction in healthy individuals does not alter MCHC, concomitant iron deficiency has been associated with decreased MCHC in SCA patients. Isolated case reports have linked iron restricted erythropoiesis with decreased hemolysis, increased red cell lifespan, and improvement in certain outcomes in SCA patients. We systematically examined the effects of iron restriction on erythropoietic outcomes in SCA utilizing the Townes murine model to investigate the hypothesis that mice with dietary iron deficiency will demonstrate a decreased MCHC, decreased erythrocyte sickling propensity, and improved anemia compared with mice on an iron sufficient diet. Methods: Townes SCA mice were weaned to diets containing either 20 ppm iron (low) or 48 ppm iron (sufficient) and maintained on those diets until sacrifice at 2 months of age. Blood was collected for complete blood count by submandibular or cardiac puncture. Spleen weight was normalized to body weight for calculation of the splenic index. Red cell deformability, defined by the elongation index (EI), and the oxygen pressure at which sickling occurs (point of sickling) during deoxygenation were characterized by oxygenscan ektacytometry using a laser optical rotational red cell analyzer (Mechatronics, The Netherlands). Results: SCA mice fed a 20 ppm low iron diet demonstrate a significant decrease in MCHC compared to SCA mice fed a 48 ppm iron sufficient diet (17.7+1.1 vs 22.7+5.5 g/dL; p &lt;0.05). Mice fed the low iron diet produced more circulating RBCs (5.98+1.03 vs 4.61+0.84 x 106 cells/uL; p &lt;0.05) and a higher hematocrit (39.4+7.1 vs 24.6+4.6%; p &lt;0.05) with a corresponding decrease in splenic index (0.074+0.14 vs 0.087+0.006 g/g body weight; p &lt;0.05) compared to mice fed the iron sufficient diet. RBCs from mice fed the low iron diet showed a significant decrease in the oxygen pressure at the point of sickling compared to RBCs from mice fed the iron sufficient diet (31.9+4.7 vs 40.6+4.6 mmHg; p &lt;0.01). The low iron diet also resulted in an overall improvement in deformability as evidenced by an increase in the EI minimum compared to the iron sufficient diet (0.25+.07 vs 0.12+.03 au; p &lt;0.01). No significant differences between groups were found in maximum deformability (EI max). The improved EI minimum translates to a decreased change in elongation (Delta EI), defined as EI max minus EI min, in RBCs from mice fed the low iron diet compared to mice fed the iron sufficient diet (0.11+.08 vs .23+.02 au; p &lt;0.01). Conclusions: Decreased dietary iron intake is associated with a decrease in the MCHC in Townes SCA mice. The lower MCHC is accompanied by an amelioration of anemia, decreased extramedullary erythropoiesis, improved RBC deformability and a shift in the initiation of sickling to lower oxygen pressures during deoxygenation. We speculate that iron restriction may decrease the clinical complications arising from both hemolysis and sickling in SCA. Future studies will utilize this model system to further characterize the effects of manipulating iron homeostasis on erythropoietic outcomes in SCA. Disclosures Fleming: Protagonist: Membership on an entity's Board of Directors or advisory committees; Silence Therapeutics: Consultancy; Ultragenyx: Consultancy.

Clinical and Molecular Spectrum of Glucose-6-Phosphate Isomerase Deficiency. Report of 12 New Cases.

Glucose-6-phosphate isomerase (GPI, EC 5.3.1.9) is a dimeric enzyme that catalyzes the reversible isomerization of glucose-6-phosphate to fructose-6-phosphate, the second reaction step of glycolysis. GPI deficiency, transmitted as an autosomal recessive trait, is considered the second most common erythro-enzymopathy of anaerobic glycolysis, after pyruvate kinase deficiency. Despite this, this defect may sometimes be misdiagnosed and only about 60 cases of GPI deficiency have been reported. GPI deficient patients are affected by chronic non-spherocytic hemolytic anemia of variable severity; in rare cases, intellectual disability or neuromuscular symptoms have also been reported. The gene locus encoding GPI is located on chromosome 19q13.1 and contains 18 exons. So far, about 40 causative mutations have been identified. We report the clinical, hematological and molecular characteristics of 12 GPI deficient cases (eight males, four females) from 11 families, with a median age at admission of 13 years (ranging from 1 to 51); eight of them were of Italian origin. Patients displayed moderate to severe anemia, that improves with aging. Splenectomy does not always result in the amelioration of anemia but may be considered in transfusion-dependent patients to reduce transfusion intervals. None of the patients described here displayed neurological impairment attributable to the enzyme defect. We identified 13 different mutations in the GPI gene, six of them have never been described before; the new mutations affect highly conserved residues and were not detected in 1000 Genomes and HGMD databases and were considered pathogenic by several mutation algorithms. This is the largest series of GPI deficient patients so far reported in a single study. The study confirms the great heterogeneity of the molecular defect and provides new insights on clinical and molecular aspects of this disease.

Inherited hemolytic anemia: a possessive beginner's guide.

Significant advances have been made in diagnosis and clinical management of inherited red cell membrane disorders that result in hemolytic anemia. Membrane structural defects lead to hereditary spherocytosis (HS) and hereditary elliptocytosis (HE), whereas altered membrane transport function accounts for hereditary xerocytosis (HX) and hereditary overhydrated stomatocytosis (OHS). The degrees of membrane loss and resultant increases in cell sphericity determine the severity of anemia in HS and HE, and splenectomy leads to amelioration of anemia by increasing the circulatory red cell life span. Alterations in cell volume as a result of disordered membrane cation permeability account for reduced life span red cells in HX and OHS. Importantly, splenectomy is not beneficial in these 2 membrane transport disorders and is not recommended because it is ineffective and may lead to an increased risk of life-threatening thrombosis. Rational approaches are now available for the diagnosis and management of these inherited red cell disorders, and these will be discussed in this review.

Lack of GDF11 Does Not Ameliorate Erythropoiesis in β-Thalassemia and Does Not Prevent the Activity of the Trap-Ligand RAP-536

Mutations in the HBB gene causes β-thalassemia (BT). Treatment for BT presents a major clinical challenge in the United States, as patients require chronic and expensive treatment for survival. A new drug in Phase III clinical trials, Luspatercept (ACE-536), has been shown to improve BT symptoms via an erythropoietin (EPO) -independent pathway. ACE-536 is a peptide drug identical to the extracellular domain of activin receptor IIB (ACVR2B). Upon administration, it competes with ACVR2B to bind members of the transforming growth factor (TGF) β superfamily. Growth differentiation factor 11 (GDF11) has been pinpointed as the primary target by which the trap ligand exerts its therapeutic efforts. Studies in murine models of BT using RAP-536 (the mouse analog of ACE-536), have suggested that Gdf11 is overexpressed in erythroblasts and that overexpression functions to inhibit erythroid differentiation. Interestingly, however, ACE-536 and RAP-536 have been shown to stimulate RBC synthesis in healthy humans and mice, where GDF11/Gdf11 overexpression has not been reported. Additionally, the expression data in mice has been questioned because of the unavailability of antibodies that can discriminate between Gdf11 and other TGF-β ligands.Due to the novelty of RAP-536 promoting erythropoiesis through an Epo-independent pathway and the lack of specific antibodies to distinguish between TGF-β ligands, we resorted to genetic tools to investigate the role of Gdf11 in erythropoiesis. For our study, we generated Hbb+/+Gdf11flox/flox and Hbbth3/+Gdf11flox/flox mice and crossed them with EpoRCre and VavCre transgenic lines, resulting in offspring harboring the Gdf11 deletion in erythroid cells and the complete hematopoietic compartment. If Gdf11 is secreted by erythroid cells, and it plays a role in inhibiting erythroid differentiation, then mice lacking Gdf11 in either erythroid cells or all hematopoietic cell lineages should show some increase in red blood cell (RBC) production, hemoglobin (Hb) and hematocrit (Hb). Furthermore, in Hbbth3/+mice, where Gdf11 has been proposed to be overexpressed, improvements in erythroid cell differentiation should be most apparent. Surprisingly, we did not detect any differences in RBC number, Hb or Hct levels of Gdf11 deficient Hbb+/+ or Hbbth3/+ mice compared to their Gdf11 containing controls. The discrepancy between our results and published data could be explained if Gdf11 is produced by non-hematopoietic tissues and indirectly influences erythropoiesis. Since Gdf11-/- are embryonic-lethal, we crossed Hbb+/+ Gdf11flox/floxand Hbbth3/+ Gdf11flox/flox mice with a tamoxifen (TAM) inducible Cre recombinase under the global Rosa26 promoter (RosaCre) to assess the effect of a pancellular deletion of Gdf11. No detectable differences were found in RBC, Hb or Hct levels of fl these animals after TAM treatment either acutely nor up to 5-6 months post deletion of Gdf11. Administration of RAP-536 significantly improved and increased hematopoietic parameters in the peripheral blood in all six models lacking Gdf11. In the RAP-536-treated Hbbth3/+ models, amelioration of anemia was noted by a decrease in spleen size and improved ineffective erythropoiesis indicated by an increased hematological parameters and increased ratio of mature to immature erythroblasts in spleen analyzed by FACS. Therefore, lack of Gdf11 at the erythroid, hematopoietic and pancellular level did not prevent a response to the drug.Next, we investigated the effects of RAP-536 directly on erythroid cells. Since the drug causes increases in RBC and Hb of normal patients, we challenged CD34+ cells with RAP-536 at various concentrations. Results showed no increases in cell numbers, erythroid viability, hemoglobin content nor differentiation. Currently we are investigating the mRNA expression of activin receptors IIA and IIb along with TGF-β ligands in healthy and BT CD34+ cells as well as in erythroid specific progenitors of the Hbbth3/+ mouse model.Our findings suggest that Gdf11 is not the sole target of RAP-536, nor that Gdf11 is required to promote improvement of erythropoiesis. Most importantly, we show that in the absence of Gdf11, RAP-536 is effective at increasing hematological parameters in both Hbb+/+ and Hbbth3/+ mice. The results of this study demonstrate potential alternative target(s) for the action of RAP-536. Future work will focus on identifying the unknown targets of RAP-536. DisclosuresCasu:Aevi Genomic Medicine, Inc: Research Funding; Ionis Pharmaceuticals, Inc.: Research Funding. Martinez:Acceleron Pharma: Employment. Suragani:Acceleron Pharma: Employment. Kumar:Acceleron Pharma: Employment. Rivella:Ionis Pharmaceuticals, Inc: Consultancy; Protagonist: Consultancy; Disc Medicine: Consultancy; MeiraGTx: Other: SAB.

On Demand Recruitment of Macrophages Is Required for Erythroid Niche Formation during Stress Erythropoiesis in the Bone Marrow

Every second more than 2 million new erythrocytes are released from the bone marrow of human adults, highlighting the tremendous turn-over of these cells. In parallel to hematopoietic stem cell niches, the last stages of erythropoiesis take place in specialized bone marrow niches, termed ‘erythroid niches’. Concretely, the erythroid niche is composed of a ‘central macrophage’ which is surrounded by erythroid progenitor cells. Regardless of steady-state or stress erythropoiesis, iron availability is, beside erythropoietin, a key factor determining erythroid output and red blood cell quality, as reflected by hemoglobin content of these cells. It is well established that systemic iron availability for erythropoiesis, in the form of iron saturated transferrin (Tf), is mainly maintained via a recycling process of senescent red blood cells, which takes place in macrophages of the reticuloendothelial system. Yet, it is still a matter of debate if also central macrophages are involved in iron supply for red blood cell development in a more direct way due to their close proximity to developing red blood cells. Using a myeloid-specific knockout mouse strain, lacking the solely known iron exporter ferroportin (Fpn; Fpnfl/flLysMCre+/+ mice) and specific reporter mice (ROSA26tdTomatofl/fl Cx3cr1CreERT2 mice), we examined the connection between iron metabolism, erythropoiesis and central macrophages.Analysis of Fpnfl/flLysMCre+/+ animals at steady state revealed microcytic anemia, higher tissue iron loading, reduced hepatic hepcidin expression and distorted erythroid precursor population distribution in the bone marrow with no significant chances in Tf saturation (Tf-Sat). The latter is giving a first hint, that local bone marrow Fpn expression on macrophages may be important for iron supply for erythropoiesis. Strikingly, further work up via flow cytometry demonstrated that disturbances seen in bone marrow erythropoiesis were accompanied by nearby loss of resident bone marrow macrophages (defined as CD11blo, F4/80pos, MerTKpos). In parallel, a CD11bhi, F4/80pos, MerTKpos population came into existence, suggesting that these cells may compensate for the loss of ‘canonical’ central macrophages.Attempting to explain these intriguing results, we sought to investigate differentiation pathways and turnover of bone marrow central macrophages. First, we used the ROSA26tdTomatofl/fl Cx3cr1CreERT2 monocyte-specific reporter mice and techniques of transient monocyte labelling in utero and in adult phlebotomized animals to determine the origin of central macrophages. We could show that those cells undergo constant replenishment by circulating monocytes. Notably, the rate of this process got markedly increased upon recovery from blood loss and concomitant expansion of the central macrophage population. Second, by administration of a CCR2/CCR5 inhibitor (cenicriviroc), diminishing monocyte egress from the bone marrow and tissue infiltration, we could demonstrate decreased reticulocyte count during stress erythropoiesis, thus strengthening the direct impact of macrophages to support effective erythroid output.Next, effects of stress-induced erythropoiesis were investigated in Fpnfl/flLysMCre+/+ compared to Fpnfl/flLysMCre-/- mice. Amelioration of anemia after phlebotomy was extended, microcytosis was more pronounced and reticulocyte egress was diminished but prolonged. Of interest, Fpnfl/flLysMCre+/+ mice on a diet containing an 8-times higher iron content during phlebotomy, thus transiently increasing Tf-Sat, recovered from anemia wildtype-like. These results indicate that stress erythropoiesis with a high iron demand depends, under normal iron availability, in part on central macrophages and their nursing function to overcome the increased demand of iron.Ongoing experiments aim to identify how recruited bone marrow macrophages, i.e. central macrophages, contribute to erythropoiesis during stress - if central macrophages directly supply developing erythroid cells with iron in a Tf-free fashion or, if they are suppliers of additional growth factors that work synergistically with the Tf-bound iron to drive hemoglobin production. In summary our data clearly show that macrophages need to be recruited to the bone marrow for effective erythroid output during stress erythropoiesis. DisclosuresWeiss:Kymab Ltd.: Consultancy. Theurl:Kymab Ltd.: Consultancy, Research Funding.

Gene Therapy for Sickle Cell Anemia Using a Modified Gamma Globin Lentivirus Vector and Reduced Intensity Conditioning Transplant Shows Promising Correction of the Disease Phenotype

▪Background: Genetic transfer of an anti-sickling β87-globin lentiviral vector (LV) into hematopoietic stem cells (HSC) followed by myeloablative transplant has cured one child with sickle cell anemia (SCA) (NEJM 2017), although it was not successful in 7 subsequent adult SCA patients, and modifications to intensify ablative conditioning, improve HSC dose, gene transfer are underway (Blood 130 Suppl 1: 527, 2017). Based upon our preclinical data (Blood 2009), we embarked upon a Reduced Intensity Conditioning (RIC) Phase I/II Pilot Study on Gene Transfer in Patients with SCA with a modified γ-Globin LV (NCT02186418), hypothesizing this approach will be safe, feasible and efficacious; Moreover, RIC will have significantly less toxicity, costs, and be implementable in many transplant centers, including those in some of the resource-poor countries, where supportive therapies for myeloablative transplants are unavailable, and where majority of SCA patients exist.Methods: Adult patients with severe SCA deemed eligible were transfused/erythrocytapheresed prior to HSC collection and transfused for 6 months post-transplant (PT) to Hb>10g/dl and HbS~30%. CD34+ HSC were collected via bone marrow harvest (BMH) and/or plerixafor mobilized Peripheral Blood Stem Collection (PBSC), selected for CD34+ cells and transduced. Patients received a single dose of IV melphalan (140mg/m2 BSA) 36hr prior to infusion of γ-globin modified (GM)-HSC. Patients were monitored for adverse events (AE), engraftment, vector copy number (VCN), modified HbF (HbF*) expression and clinical features of SCA.Results: Two SCA patients (35yo and 25yo) with HbS-β0 thalassemia genotype were treated. CD34+ HSC were collected via multiple BMH (P1) and BMH+PBSC (P2). Follow up data are available for 6 and 12mo on P1 and P2. P1 received 1x106 CD34+ cells/kgbw [vector copy number (VCN) 0.22], and P2 received 6.9x106 CD34+ cells/kgbw [VCN 0.46]. Time to neutrophil engraftment (ANC ≥ 500) was day 9 and 7 post-transplant (PT) in P1 and P2, respectively, and time to Plt recovery (Plt>50K) was day 14 PT in both. Patients included in this trial had severe disease and continued to have pre-existing chronic pain requiring significant opiates; hence ~80% of the AEs were pain events; other AEs were anticipated transient laboratory AEs associated with melphalan. Following GM-HSC infusion, both patients showed a progressive rise in HbF* (a point mutation in the γ-globin LV allows distinction from endogenous HbF by HPLC) starting from day 30 PT. Since patients had transfused HbA containing RBCs in the initial 6 months, HbF*/(HbF*+HbS) was calculated, and was 20% and 21% in P1 and P2 at day 180 PT and VCN 0.2-0.4, detected in all lineages. Integration site analysis, performed on the infused products (Day 0), at day 30 PT on P1 and P2, and on day 180 PT on P1 demonstrated highly polyclonal pattern of integration. At 1 yr PT, P1 had 20% HbF* (2.1g/dl HbF*, total Hb 10.6) with a stable VCN of 0.2-0.4 in multiple lineages in bone marrow and peripheral blood. The baseline Hb of P1 was 7.5-8.5g/dL prior to transplant. In the preceding 2 years prior to transplant, both patients were admitted for pain crises/acute chest >5-6 times/yr, and had chronic pain requiring chronic opiates. Chronic pain persisted for 4-5 months PT in P1, after which P1 has not required IV opiates, negligible oral opiates and has had no hospital visits/admissions with acute sickle events. P2 has required decreasing amounts of oral opiates for chronic back pain.Conclusions: Early results from 2 SCA adults treated with a modified γ-globin LV modified autologous HSC following RIC transplant showed excellent safety, feasibility, with minimal post-transplant toxicity, rapid count recovery, and sustained stable genetically modified cells in peripheral blood and bone marrow. The first patient shows significant clinical amelioration of the SCA phenotype at 1 year PT, with 20% vector-derived HbF (HbF*) that has caused amelioration of anemia, near elimination of chronic pain and absence of acute sickle events. The second patient, although still early post-transplant shows a similar HbF* trajectory. Additional study data will demonstrate whether this level of HbF* will provide consistent clinical benefit to patients with severe SCA. These early results, especially following a RIC transpant, are extremely promising; and if sustained, will provide a ‘transportable’ safe and feasible gene therapy for SCA. DisclosuresMalik:CSL Behring: Patents & Royalties. Quinn:Global Blood Therapeutics: Research Funding; Silver Lake Research Corporation: Research Funding; Amgen: Research Funding.

Leucine for the Treatment of Transfusion Dependence in Patients with Diamond Blackfan Anemia

▪Diamond Blackfan anemia (DBA) is a rare, inherited bone marrow failure syndrome characterized by anemia, congenital anomalies and a predisposition to cancer. The patients usually present during infancy or early childhood, but can also present in adulthood. In the majority of cases DBA is due to a mutation in a small or large ribosomal protein (RP) subunit leading to RP haploinsufficiency. The only treatments for the anemia of DBA are red cell transfusions (accompanied by iron chelation), oral corticosteroid therapy or stem cell transplantation. Pospisilova et al. (Haematologica 2007; 92(5):e66-67) reported one complete and two partial erythroid responses after the use of the branched chain amino acid L-leucine in 6 select patients. In skeletal muscle, leucine supplementation can upregulate components of the protein synthetic machinery, including ribosomal proteins, promoting protein translation. Mouse and fish models of DBA respond with amelioration of anemia to L-leucine. We therefore proposed to study the effect of L-leucine on transfusion dependence and growth in subjects with DBA.Methods: The primary objectives were to determine the feasibility of administering L-leucine in subjects with DBA who are red cell transfusion-dependent and to determine the efficacy of L-leucine to produce a hematologic and growth response. The secondary objective was to determine the safety profile of L-leucine. Twelve study sites were involved in this multi-center, Phase I/II study with an anticipated accrual of 50 subjects. A dose of 700 mg/M2 orally three times per day for 9 months was used. Inclusion criteria included age > 2 years, the diagnosis of DBA and transfusion dependence with adequate kidney and liver function. Response was evaluated at 9 months with Complete Response (CR) defined as no further transfusions required and Hb >9; Partial Response (PR): Hb < 9 gm/dL with an increase in reticulocyte count and transfusion interval; and No Response (NR): no change in transfusion needs, Hb or reticulocyte count . Growth percentiles were evaluated at baseline and at completion of 9 months of treatment and the growth velocity change was calculated.Results: The study opened July 2014 and closed February 2017; 55 subjects were consented; 12 were screen failures; 43 subjects were evaluable. There were 21 males; the median age was 9 years 1 month (2 years 5 months - 46 years 1 month). There were no untoward side effects experienced by any subject that were attributable to the L-leucine. Two subjects had an erythroid CR and 1 subject had a PR. The CRs occurred at 1 month and 3 months after start of L-leucine. The subject with PR had an elevated reticulocyte count but was not able to maintain a Hb >9 gm/dL without a transfusion and thus was not transfusion independent. Of the 30 subjects with growth potential who received L-leucine 10 experienced a positive growth velocity change at 9 months of therapy compared to baseline. At a median age of 7.5 years, the mean pre-leucine height percentile was 27 +/- 17.9 and the post-leucine height percentile was 35 +/- 19.9 (p <0.01). The mean weight percentile pre-leucine was 35.4 +/- 26.6 versus the post-Leucine weight percentile of 38.4 +/- 28.1.Conclusions: The administration of L-leucine is safe. L-leucine administration resulted in an erythroid response in 7% of subjects and an increased growth velocity in 33% of growing subjects. Based upon extrapolation from animal models, it is likely that this dose was suboptimal. We hypothesize that higher doses of L-leucine will lead to hematologic responses in more subjects who are transfusion dependent. The potential benefit of added growth in children with DBA may improve final adult height. DisclosuresGlader:Agios: Consultancy, Research Funding.

Gene editing gets a head start

Emerging Therapies The development of gene-editing technologies into therapies for human disease is an exciting prospect. A crucial question is whether there are advantages to correcting disease-causing mutations before rather than after birth, and whether this approach is even feasible. In a proof-of-concept study in mice, Ricciardi et al. accomplished successful in utero correction of a hemoglobin gene mutation that causes β-thalassemia, a serious blood disorder. They injected nanoparticles containing gene-editing machinery (triplex-forming peptide nucleic acids and single-stranded donor DNA) intravenously into midgestational mouse fetuses. After birth, the treated mice showed sustained amelioration of anemia and survived longer than untreated mice. Nat. Commun. 10.1038/s41467-018-04894-2 (2018).

Safety and Efficacy of Ferric Carboxymaltose in the Treatment of Iron Deficiency Anaemia in Patients with Inflammatory Bowel Disease, in Routine Daily Practice.

Iron deficiency and iron deficiency anaemia are common complications in inflammatory bowel disease [IBD] patients. Anaemia in IBD is attributable to chronic blood loss and/or impaired iron intake and absorption. International guidelines recommend intravenous iron supplementation in IBD patients, since oral supplements are frequently poorly tolerated and can exacerbate inflammation. Intravenous ferric carboxymaltose [FCM; Ferinject® 50 mg ferric iron[III]/mL suspension] was approved in Europe in 2007 for correction of iron deficiency, and can be administered in single 15-min infusions of up to 1000 mg. A prospective non-interventional post-marketing study was performed in 101 centres in Germany to assess the efficacy, tolerability, and convenience of Ferinject® in clinical practice in a large cohort of IBD patients. Primary endpoints were haemoglobin [Hb] normalisation or increase ≥2 g/dL [responders], and normalisation of serum ferritin [s-ferritin] and transferrin saturation. Adverse events [AEs], clinical signs/symptoms, and disease activity indices were also analysed. In all, 224 subjects (127 Crohn's disease [CD]; 97 ulcerative colitis [UC]) were treated. Mean total iron dose was 1139 mg [range: 100 mg-4800 mg], with 76.7% of doses between 500 mg and 2000 mg; 63.3% of patients responded, and no adverse drug reactions or drug-attributed serious adverse events [SAEs] or deaths occurred. Mean increases of Hb [10.0 to 12.3 g/dL], ferritin [52 μg/L to 103 μg/L], transferrin saturation [TSAT, 15% to 25%], and s-iron [6.1 to 12.4 μmol/L] were significant [p = 0.0001]. Clinical scores and quality of life improved due to the amelioration of anaemia symptoms. Ferinject®-therapy was proven to be effective and safe in a large cohort of patients with IBD-associated anaemia in routine practice. Rapid, high-dose application is convenient for physicians and reduces patients' time lost from work.

Red cell membrane disorders.

Significant advances have been made in our understanding of the structural basis for altered cell function in various inherited red cell membrane disorders with reduced red cell survival and resulting hemolytic anemia. The current review summarizes these advances as they relate to defining the molecular and structural basis for disorders involving altered membrane structural organization (hereditary spherocytosis [HS] and hereditary elliptocytosis [HE]) and altered membrane transport function (hereditary overhydrated stomatocytosis and hereditary xerocytosis). Mutations in genes encoding membrane proteins that account for these distinct red cell phenotypes have been identified. These molecular insights have led to improved understanding of the structural basis for altered membrane function in these disorders. Weakening of vertical linkage between the lipid bilayer and spectrin-based membrane skeleton leads to membrane loss in HS. In contrast, weakening of lateral linkages among different skeletal proteins leads to membrane fragmentation and decreased surface area in HE. The degrees of membrane loss and resultant increases in cell sphericity determine the severity of anemia in these two disorders. Splenectomy leads to amelioration of anemia by increasing the circulatory red cell life span of spherocytic red cells that are normally sequestered by the spleen. Disordered membrane cation permeability and resultant increase or decrease in red cell volume account for altered cellular deformability of hereditary overhydrated stomatocytosis and hereditary xerocytosis, respectively. Importantly, splenectomy is not beneficial in these two membrane transport disorders and in fact contraindicated due to severe postsplenectomy thrombotic complications.

Transdermal delivery of oleanolic acid attenuates pro-inflammatory cytokine release and ameliorates anaemia in P. berghei malaria

Malaria remains a major health problem in many tropical areas. Severe malaria infection is associated with secondary complications including anaemia leading to a need for the search of affordable antimalarial agents that can clear the parasitaemia and ameliorate anaemia during infection. The current study investigated the effects of transdermally delivered OA on malaria parasites, HCT and selected plasma cytokine concentrations in P. berghei-infected male Sprague-Dawley rats. The study was carried out over a period of 21days, divided into pre-treatment (day 0–7), treatment (day 8–12) and post-treatment (day 13–21) periods. Parasitaemia, HCT, RBC count, Hgb, plasma TNF-α, IL-6 and IL-10 concentrations were monitored in non-infected and infected rats following a once-off application of an OA-pectin patch (34mg/kg). Animals treated with drug-free pectin and CHQ (30mg/kg, p.o) twice daily for 5 consecutive days acted as negative and positive controls respectively. Infected control animals exhibited increased percentage parasitaemia, TNF-α, IL-6, IL-10 and a reduction in HCT. Interestingly, OA-pectin patch application cleared the malaria parasites and increased HCT values back to normalcy. Furthermore, TNF-α, IL-6 and IL-10 were reduced by day 12 of the study. These findings suggest that the OA-pectin patch delivers therapeutic doses of OA which are able to attenuate cytokine release and ameliorate anaemia during malaria infection. Therefore, transdermally delivered OA may be a potent therapeutic agent for malaria and amelioration of anaemia.

Biosimilar epoetin alfa increases haemoglobin levels and brings cognitive and socio-relational benefits to elderly transfusion-dependent multiple myeloma patients: results from a pilot study

Anaemia is a complication reported in up to 70% of the multiple myeloma patients (MM), with remarkable clinical, cognitive and socio-relational consequences. Anaemia relates to the course of MM, normalizing in patients during remission and reappearing in relapsing/non-responding patients. In a pilot study with 31 patients with MM and transfusion-dependent anaemia, we evaluated the effects of Binocrit (biosimilar epoetin alfa) on transfusions, haemoglobin levels, mental status (mini-mental state evaluation) and the patients' social-relational functioning and quality of life (QoL). Within a 12-week interval, patients received 40.000U Binocrit once a week. Binocrit significantly decreased the incidence of transfusion, regardless of the patients' transfusion history, and significantly increased haemoglobin levels (before-and-after-treatment median haemoglobin values = 8.20 vs. 9.40g/dl, respectively; Wilcoxon Z test, p<.001). A comparatively greater increment in haemoglobin levels among patients who responded to first vs. additional lines of chemotherapy was also observed. Importantly, we additionally found moderate-to-strong positive associations between increments in haemoglobin levels and corresponding increments both in psychological well-being and QoL (FACT-An scores) and the patients' cognitive status (mini-mental state evaluation scores). After statistically controlling for possible concurrent benefits of anti-myeloma therapy, increments in haemoglobin levels clearly predicted both increments in socio-relational FACT-An scores (Spearman's rho=0.60, p<.001) and in cognitive functioning scores (Spearman's rho=0.49, p<.006). Binocrit thus appears as an effective, well-tolerated agent for the management of myeloma anaemia, whose documented benefits include amelioration of anaemia, reduction in transfusion, and improvements in the patients' social-relational functioning and cognitive well-being.

The application of quantitative transfusion of hemoglobin in neonates

Objective To establish the quantitative transfusion of hemoglobin method and explore the application of this method in neonatal transfusion. Methods This study selected 93 cases of anemic neonates who had no other underlying diseases from the Neonatal Department of Maternal and Child Health (MCH) in Dalian, and they were the principle of weight and age divided into two groups. The first group (control group: conventional method) was transfused and injected the red blood cells that were leukocyte-reduced, irradiated and washed twice by 0.15 U/kg under the normal reservation. The study measured the hemoglobin value before transfusion and within 24 h after being transfused, and then detected the hemoglobin, volume, and hematocrit of a small amount of blood during the preparation, and finally calculated the utilization rate of hemoglobin in neonates who were transfused. For the second group (observation group: quantitative erythrocyte injection of hemoglobin), it calculated in reverse how much hemoglobin the children needed to be supplemented, based on the doctor′s expected hemoglobin values achieved after children being transfused and the utilization rate of hemoglobin obtained from the first group. Then, according to the calculation, it prepared a small amount of blood of the red blood cells that were leukocyte-reduced, irradiated and washed, made the quantitative injection of hemoglobin twice, and measured the hemoglobin value before transfusion and within 24 h after being transfused. At last, it analyzed statistically the results. Results Both two methods effectively improved the children′s anemia (P 0.05) for two methods in improving the anemia of children, but the quantitative transfusion of hemoglobin (s=6.6, cv=4.6%) could basically reach the doctor′s expected hemoglobin value, and it avoided the situations in the first method (s=14.45, vc=8.6%), like that after transfusion a minority of hemoglobin was too high or it did not reach complete amelioration of anemia of the children. Conclusions Both traditional transfusion method and quantitative transfusion of hemoglobin method can improve the anemia of the children, but the latter one is more suitable for the clinical needs and achieves the true quantitative transfusion. Key words: Erythrocyte transfusion; Blood transfusion; Hemoglobins; Infant, newborn