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Erythropoiesis-stimulating agents (ESA) are now central to the treatment of renal anemia and are associated with improved clinical outcomes. It is well known that erythropoietin (EPO) is a key regulator of erythropoiesis through its promotion of red blood cell production. In order to investigate the role of ESA on iron metabolism, we analyzed the regulation of the iron regulatory hormone hepcidin by ESA treatment in a bone marrow transplant model in mouse. After treating C57BL/6 mice with continuous erythropoietin receptor activator (C.E.R.A.), recombinant human epoetin-β (rhEPO), or recombinant human carbamylated epoetin-β (rhCEPO), we investigated serum hepcidin concentrations and parameters of erythropoiesis. Serum hepcidin concentrations after rhEPO treatment were analyzed in mice subjected to total body irradiation followed by bone marrow transplantation. C.E.R.A. administration caused long-term downregulation of serum hepcidin levels. Serum hepcidin levels in rhEPO-treated mice decreased significantly, whereas there was no change in rhCEPO-treated mice. The reduction in circulating hepcidin levels after rhEPO administration was not observed in irradiated mice. Finally, bone marrow transplantation recovered the response to rhEPO administration that downregulates hepcidin concentration in irradiated mice. These results indicate that ESA treatment downregulates serum hepcidin concentrations, mainly by indirect mechanisms affecting hematopoietic activity in bone marrow cells.

Introduction Acquired hemophilia A (AHA) is caused by autoantibodies against factor (F)VIII, and is characterized by severe, spontaneous bleeding, which can be life‐threatening. Emicizumab, an anti‐FIXa/FX bispecific antibody, significantly reduces bleeding events in congenital hemophilia A (HA) with and without inhibitors. The known pathophysiological mechanisms and current preclinical data in HA suggest that emicizumab could provide effective treatment for AHA, but the coagulation activities of emicizumab in these patients remain unknown. Aim To evaluate the coagulant effects of emicizumab in plasma from AHA patients. Methods and Results Tissue factor‐triggered thrombin generation assays using normal plasma preincubated with anti‐FVIII monoclonal antibodies recognizing different epitopes demonstrated that 20 µg/mL emicizumab recovered the depressed peak levels of thrombin generation to 46% to 72%. Further studies were devised, therefore, to simulate the clinical course in AHA patients, including during the acute phase for severe bleeding requiring FVIII‐bypassing therapy, and during the subacute/chronic phase with less bleeding. Various concentrations of emicizumab were used to represent the potential changes in plasma levels based on the half‐life of the antibody (~30 days). The ex vivo addition of emicizumab to plasma samples from AHA patients (n = 16) increased peak thrombin in all cases, irrespective of the inhibitor epitope specificity. Thrombin generation at 20 and 100 µg/mL emicizumab was restored to (median) 43.9% and 92.2%, respectively. Differences were evident in some cases, however, and recovery rates appeared likely to be greater in patients with type 2 inhibitor than those with type 1. Conclusion Emicizumab improved ex vivo coagulation potential in plasma from AHA patients.

BackgroundHepcidin, a key regulator of iron metabolism, is produced mainly by interleukin-6 (IL-6) during inflammation. A mechanism linking cancer-related anemia and IL-6 through hepcidin production is suggested. To clarify the hypothesis that overproduction of IL-6 elevates hepcidin levels and contributes to the development of cancer-related anemia, we evaluated anti-IL-6 receptor antibody treatment of cancer-related anemia in an IL-6–producing human lung cancer xenograft model.MethodsNude mice were subcutaneously inoculated with cells of the IL-6–producing human lung cancer cell line LC-06-JCK and assessed as a model of cancer-related anemia. Mice bearing LC-06-JCK were administered rat anti-mouse IL-6 receptor antibody MR16-1 and their serum hepcidin levels and hematological parameters were determined.ResultsLC-06-JCK–bearing mice developed anemia according to the production of human IL-6 from xenografts, with decreased values of hemoglobin, hematocrit, and mean corpuscular volume (MCV) compared to non–tumor-bearing (NTB) mice. LC-06-JCK–bearing mice showed decreased body weight and serum albumin with increased serum amyloid A. MR16-1 treatment showed significant inhibition of decreased body weight and serum albumin levels, and suppressed serum amyloid A level. There was no difference in tumor volume between MR16-1-treated mice and immunoglobulin G (IgG)-treated control mice. Decreased hemoglobin, hematocrit, and MCV in LC-06-JCK–bearing mice was significantly relieved by MR16-1 treatment. LC-06-JCK–bearing mice showed high red blood cell counts and erythropoietin levels as compared to NTB mice, whereas MR16-1 treatment did not affect their levels. Serum hepcidin and ferritin levels were statistically elevated in mice bearing LC-06-JCK. LC-06-JCK–bearing mice showed lower values of MCV, mean corpuscular hemoglobin (MCH), and serum iron as compared to NTB mice. Administration of MR16-1 to mice bearing LC-06-JCK significantly suppressed levels of both serum hepcidin and ferritin, with increased values of MCV and MCH.ConclusionsOur results suggest that overproduction of hepcidin by IL-6 signaling might be a major factor that leads to functionally iron-deficient cancer-related anemia in the LC-06-JCK model. We demonstrated that inhibition of the IL-6 signaling pathway by MR16-1 treatment resulted in significant recovery of iron-deficiency anemia and alleviation of cancer-related symptoms. These results indicate that IL-6 signaling might be one possible target pathway to treat cancer-related anemia disorders.

Background Emicizumab prophylaxis is a promising treatment that reduces bleeding events in severely affected patients with hemophilia A (PwHA). It is anticipated that emicizumab could be similarly effective in mild/moderate PwHA (PwMHA) although this effect has not been investigated. Aim We evaluated ex vivo coagulant effects of emicizumabin PwMHA. Methods Clot waveform analysis (CWA) triggered by prothrombin time/activated partial prothrombin time-mixed reagents was utilized to examine coagulant effects of emicizumabin factor (F)VIII-deficient plasma mixed with recombinant (r)FVIIIand in native plasmas from 16 PwMHA. The CWA parameter, adjusted-|min1| (Ad|min1|), was used. Increases in Ad|min1| (ΔAd|min1|) mediated by emicizumab were calculated from the slopes of regression lines in the presence of rFVIII. Results Ad|min1| in FVIII-deficient plasma with various concentrations of rFVIII negatively correlated with ΔAd|min1|by adding emicizumab, and these data were defined as standard reference values. Ad|min1| (4.57 ± 0.50) in 16 PwMHA increased to 5.05 ± 0.54 and 5.37 ± 0.60 by adding emicizumab at 50 and 100 μg/mL, respectively, but remained lower than the normal range (7.22 ± 0.21). ΔAd|min1| levels were 1.5 to 2-fold higher in five cases and 0.4 to 0.6-fold lower in four cases, compared with reference values determined by rFVIII. In some cases, genetic analyses suggested that specific point mutations could have contributed to these findings. Further studies using rFVIII mutants indicated, however, that the differences in ΔAd|min1| were not related to individual FVIII gene defects. Conclusion Emicizumab enhances coagulation potential in PwMHA. Assessment of ex vivo coagulant activity of emicizumab could be helpful for predicting coagulant potentials prior to treatment in these patients.