Mechanisms of Interferon-associated Bone Marrow Suppression with or without Ribavirin. K. Zhdanov

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1 EASL endorsed conference White Nights of Hepatology 2012 Adverse events during antiviral therapy: how to predict, manage and monitor June 7-8 Saint-Petersburg Mechanisms of Interferon-associated Bone Marrow Suppression with or without Ribavirin K. Zhdanov

2 Multiple factors can cause or contribute to the development of cytopenia in patients with chronic viral hepatitis Portal Hypertension / Hypersplenism Cirrhosis Antibodies to blood cells Cytopenia HCC / Chemotherapy Viral Suppression / Antiviral therapy Decreased TPO, EPO, G-СSF Levels/Activity Afdhal N. et al. J. Hepatol., 2008

3 Hematologic events during Peg-IFN + Ribavirin therapy Hematologic events, Discontinuation of treatment, and Dose reduction Peg-IFN α-2a/rbv (%) Peg-IFN α-2b/rbv (%) Neutropenia 31,5 25,8 Neutrophil counts < 750 mm Neutrophil counts < 500 mm 3 5,9 2,8 Thrombocytopenia 31* 8** Platelet counts < mm 3 4* 4** Platelet counts < mm 3 < 1* < 1** Anemia 33,6 33,9 Hemoglobin < 100 g/l 29,6 30,7 Hemoglobin < 85 g/l 3,8 2,5 Serious hematologic disorders 0,8 0,4 McHutchison J. et al. N Engl J Med 2009; 361: * Sulkowski M. Clin Liver Dis 2005; 9: **Poynard T. et al. Gastroenterology 2009; 136:

4 Suppression of haematopoiesis during therapy of chronic hepatitis C Haemoglobin (mg/dl) EOT Leucocytes (10 3 мcl) EOT 4 24 IFN-a2b 5 MU TIW IFN-a2b 5 MU TIW + RBV Peg-IFN-a2a 180 мсg + RBV Peg-IFN-a2b 1,5 мсg/kg + RBV Schmid M., Kreil A., Jessner W., Homoncik M., Datz C., Gangl A., Ferenci P., Peck-Radosavljevic M. Gut, Platelets (10 3 мcl) EOT 4 24

5 Mechanisms of cytopenia during Peg-IFN/RBV therapy Bone marrow suppression Maturation and proliferation suppression of bone marrow progenitor cells leads to a 30-50% decline in the neutrophil and platelet counts and a lesser decline in hemoglobin Antiviral therapy Inhibition of growth factors Appearance of antibodies Immediate effect on mature circulating cells This leads to a 14-38% decline in the platelet and neutrophil counts and a 4% decline in the hemoglobin Shiffman M. et al. In book Advanced Therapy for Hepatitis C, 2012, P

6 Mechanisms of interferon-associated bone marrow suppression JAK Tovey M., Lallemand C. Pharmaceuticals, 2010 Masumi A. et al. Advances in Hematopoietic Stem Cell Research, 2012

7 Mechanisms of IFN action Bekisz J. et al. Pharmaceuticals,2010

8 Mechanisms of interferon-associated bone marrow suppression JAK CYTOPENIA Аctivation of genes transcription Apo 1 Proteins translation Аро 2 FAS TNF TRAIL PK TNF ОАS PK IRF-2 EN-L Receptor Sca1 expression of bone marrow stem cells АPOPTOSIS in bone marrow stem cells Decreased proteins synthesis in haematopoiesis cells Increased production of bone marrow stem cells, their depletion Reduced production, differentiation, maturation of bone marrow cells Tovey M., Lallemand C. Pharmaceuticals, 2010 Masumi A. et al. Advances in Hematopoietic Stem Cell Research, 2012 Inhibition of factors expression: GATA1, p45, MaFg Decreased differentiation and maturation

9 Mechanisms of interferon-associated bone marrow suppression JAK CYTOPENIA Аctivation of genes transcription Apo 1 Proteins translation Аро 2 FAS TNF TRAIL PK TNF ОАS PK IRF-2 EN-L Decreased proteins synthesis in haematopoiesis cells Reduced production, differentiation, maturation of bone marrow cells Tovey M., Lallemand C. Pharmaceuticals, 2010 Masumi A. et al. Advances in Hematopoietic Stem Cell Research, 2012

10 Mechanisms of interferon-associated bone marrow suppression JAK CYTOPENIA Аctivation of genes transcription Apo 1 Proteins translation Аро 2 FAS TNF TRAIL PK TNF ОАS PK IRF-2 EN-L Receptor Sca1 expression of bone marrow stem cells Increased production of bone marrow stem cells, their depletion Reduced production, differentiation, maturation of bone marrow cells Tovey M., Lallemand C. Pharmaceuticals, 2010 Masumi A. et al. Advances in Hematopoietic Stem Cell Research, 2012

11 Interferon regulatory factor-2 (IRF-2) regulates cell growth and differentiation through the target gene promoters Colony-forming assay showed reduced numbers of megakaryocyte progenitors in cells derived from bone marrow of IRF-2/mice vs wild-tipe/mice Masumi A. et al., Advances in Hematopoietic Stem Cell Research, 2012

12 Mechanisms of interferon-associated bone marrow suppression JAK CYTOPENIA Аctivation of genes transcription Apo 1 Proteins translation Аро 2 FAS TNF TRAIL PK TNF ОАS PK IRF-2 EN-L АPOPTOSIS in bone marrow stem cells Tovey M., Lallemand C. Pharmaceuticals, 2010 Masumi A. et al. Advances in Hematopoietic Stem Cell Research, 2012

13 Mechanisms of interferon-associated bone marrow suppression JAK CYTOPENIA Reduced production, differentiation, maturation of bone marrow cells Tovey M., Lallemand C. Pharmaceuticals, 2010 Masumi A. et al. Advances in Hematopoietic Stem Cell Research, 2012 Inhibition of factors expression: GATA1, p45, MaFg Decreased differentiation and maturation

14 IFN-α2b suppressed expression of transcription factors regulating late-stage megakaryopoiesis Human bone marrow derived CD34 + cells were incubated with 10 ng/ml TPO for 7 days. Large megakaryocytes were enriched by velocity sedimentation, followed by 6 days of incubation with 10 ng/ml TPO and 0, 1, or 10 ng/ml IFN-α2b. mrna expression of GATA1, p45nf-f2, MafG, vwf was analyzed using quantitative PCR. Yamane A et al. Blood 2008;112:

15 Ultrastructure of megakaryocytes cultured with or without IFN-α2b. (A) Ultrastructures of representative human megakaryocytes сultured with 10 ng/ml TPO, 10 ng/ml TPO + 10 ng/ml IFN-α2b, and 10 ng/ml TPO + 10 ng/ml IFN-α2b + 1 μg/ml NIP-004. Type 1 - immature megakaryocytes; Type 2 - intermediate megakaryocytes; Type 3 - fully matured megakaryocytes. The definition of each type is in Electron microscopy. Original magnification, N indicates nucleus; Dm, demarcation membranes. (B) The percentage of each type of megakaryocytes. IFN-α2b decreased type 3 megakaryocytes and increased type 2 compared with control (TPO) with statistically significant differences, indicating that IFN-α2b inhibits the development of the demarcation membrane system of megakaryocytes (*P <.05 vs TPO). NIP-004 prevented the inhibitory effects of IFN-α2b on the development of the demarcation membrane system of megakaryocytes (#P <.05 vs TPO + IFN). Yamane A et al. Blood 2008;112:

16 Mechanisms of interferon-associated bone marrow suppression JAK CYTOPENIA Аctivation of genes transcription Apo 1 Proteins translation Аро 2 FAS TNF TRAIL PK TNF ОАS PK IRF-2 EN-L Receptor Sca1 expression of bone marrow stem cells АPOPTOSIS in bone marrow stem cells Decreased proteins synthesis in haematopoiesis cells Increased production of bone marrow stem cells, their depletion Reduced production, differentiation, maturation of bone marrow cells Tovey M., Lallemand C. Pharmaceuticals, 2010 Masumi A. et al. Advances in Hematopoietic Stem Cell Research, 2012 Inhibition of factors expression: GATA1, p45, MaFg Decreased differentiation and maturation

17 Interferon-associated bone marrow suppression (with or without ribavirin) Stem cell Multipotent progenitor cell Committed progenitor cell Immature cell Mature cell 1 Cytokines and growth factors (TPO, G-CSF, EPO) Ribavirin 1. Suppression of late erythroid precursors 2. Leads to an increase in EPO release, which enhances red cell and platelet production 3. Causes alterations in the erythrocyte membrane known to be associated with erythrophagocytic extravasclar destruction and haemolysis IFN 1. Sequestration of mature cells 2. Reduced maturation and production of progenitor cells 3. Reduced secretion of growth factors 4. Enhanced secretion of cytokines, which inhabit the proliferation and maturation of progenitor cells 1 Peripheral capillary beds Shiffman M. et al. In book Advanced Therapy for Hepatitis C, 2012, P

18 EPO and TPO during therapy of chronic hepatitis C EPO (miu/ml) EOT TPO (pg/ml) EOT 4 24 IFN-a2b 5 MU TIW IFN-a2b 5 MU TIW + RBV Peg-IFN-a2a 180 мсg + RBV Peg-IFN-a2b 1,5 мсg/kg + RBV Schmid M., Kreil A., Jessner W., Homoncik M., Datz C., Gangl A., Ferenci P., Peck-Radosavljevic M. Gut, 2005

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20 In the majority of patients treated with IFN/RBV, the decrease in Hb results from RBV-related hemolysis and IFN-related bone marrow suppression of erythroprogenitor cells, representing a mixed anemia 250 IFN may limit the ability of the bone marrow to respond to ongoing hemolysis (reticulocytes, x 103 μl) 58% 42% RBV-related hemolysis Bone marrow suppression 50 0 RBV P=0,002 IFN/RBV 4,3% P<0,001 2,4% De Franceschi L. et al. Hepatology, 2000 Rendo P. et al. Antiviral Ther, 2000 reticulocytes

21 Lowest absolute neutrophil count in patients receiving IFN or Peg-IFN combined with RBV 46% 38% 36% 16% 24% 21% 5% 8% 1% 5% Normal > 2000/mcL Grade /mcL Grade /mcL Grade /mcL Grade 4 < 500/mcL IFN/RBV Peg-IFN/RBV Fried MW et al. N Engl J Med, 2002

22 Lowest platelet count during treatment with IFN/RBV or Peg-IFN with or without RBV 88% 67% 48% 29% 8% 17% 4% 16% 10% 4% 6% 0% 0% 0% 0% Normal > /mcL Grade /mcL Grade /mcL Grade /mcL Grade 4 < 25000/mcL IFN/RBV Peg-IFN/RBV Peg-IFN Sulkowski M. Clin Liver Dis, 2005

23 CONCLUSION Antiviral therapy with interferon alfa and ribavirin induces anaemia, leucopenia and thrombocytopenia, necessitating dose reduction in some cases and discontinuation sometimes. The main mechanisms leading to cytopenia during antiviral therapy seems to be bone marrow suppression by IFN-α and red cells hemolysis by ribavirin. Suppressive action can be observed for pluripotent progenitor cells of all lineages after activation of the latent cytoplasmic signal transducers and activators of transcription, formation of a transcription complex together with IFN regulatory factors, and activation of a specific set of genes that encode the effector molecules responsible for mediating the biological activities of IFN-α. Except main bone marrow suppression, IFN-α has an immediate effect on mature circulating cells, reduces the secretion of hematopoietic growth factors and enhances secretion of cytokines, which inhibit the proliferation and maturation of bone marrow progenitor cells. Ribavirin (except main red cells haemolysis) can lead to suppression of late erythroid precursors in the bone marrow.