Mechanisms of Interferon-Associated Bone Marrow Suppression with Or Without Ribavirin

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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 Multiple factors can cause or contribute to the development of cytopenia in patients with chronic viral hepatitis

Portal Hypertension / Cirrhosis Hypersplenism

Antibodies Cytopenia HCC / to Chemotherapy blood cells

Viral Decreased TPO, Suppression / EPO, G-СSF Antiviral therapy Levels/Activity

Afdhal N. et al. J. Hepatol., 2008 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 mm3 27 22 Neutrophil counts < 500 mm3 5,9 2,8

Thrombocytopenia 31* 8** Platelet counts < 50000 mm3 4* 4** Platelet counts < 25000 mm3 < 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: 580-593 * Sulkowski M. Clin Liver Dis 2005; 9: 439-451 **Poynard T. et al. Gastroenterology 2009; 136: 1618-1628 Suppression of haematopoiesis during therapy of chronic hepatitis C

18 8 16 7 14 6 12 5 10 4 8 3 6 2 4 1 3 2 Haemoglobin (mg/dl) Leucocytes (10 мcl) 0 0 0 4 8 12 EOT 4 24 0 4 8 12 EOT 4 24

IFN-a2b 5 MU TIW 250 200 IFN-a2b 5 MU TIW + RBV 150 Peg-IFN-a2a 180 мсg + RBV 100 Peg-IFN-a2b 1,5 мсg/kg + RBV 50 Platelets (103 мcl) 0 Schmid M., Kreil A., Jessner W., Homoncik M., Datz C., Gangl A., 0 4 8 12 EOT 4 24 Ferenci P., Peck-Radosavljevic M. Gut, 2005 Mechanisms of cytopenia during Peg-IFN/RBV therapy

Maturation and proliferation suppression of bone marrow progenitor cells leads to Bone marrow suppression 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.160-168 Masumi Masumi al. A. et Advances Hematopoieticin Cell Stem Research, 2012 Tovey

M., Lallemand Mechanisms of interferon

STAT 1 C. Pharmaceuticals, C. Pharmaceuticals, 2010 JAK

STAT 1 bone marrow suppression

- associated

Mechanisms of IFN action

Bekisz J. et al. Pharmaceuticals,2010 Mechanisms of interferon-associated bone marrow suppression

Аctivation of genes transcription •Apo 1 Proteins translation •Аро 2 •FAS АPOPTOSIS •TNF •TRAIL in bone marrow •PK •TNF stem cells •ОАS •PK Decreased proteins •IRF-2 synthesis in Receptor •EN-L haematopoiesis cells Sca1 expression

of bone marrow JAK

Increased production

stem cells of bone marrow stem

cells, their depletion

STAT 1 STAT

Reduced production, differentiation, maturation of bone marrow cells

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

Аctivation of genes transcription •Apo 1 Proteins translation •Аро 2 •FAS •TNF •TRAIL •PK •TNF •ОАS •PK Decreased proteins •IRF-2 synthesis in

•EN-L haematopoiesis cells

JAK

STAT 1 STAT

Reduced production, differentiation, maturation

of bone marrow cells CYTOPENIA

Tovey M., Lallemand C. Pharmaceuticals, 2010 Masumi A. et al. Advances in Hematopoietic Stem Cell Research, 2012 Masumi Masumi al. A. et Advances Hematopoieticin Cell Stem Research, 2012 Tovey

M., Lallemand Mechanisms of interferon

STAT 1 C. Pharmaceuticals, C. Pharmaceuticals, 2010 JAK

STAT 1 bone marrow suppression

• • • • • • А IRF ОА PK TNF Аро Apo 1 ctivation

S -

Sca1 expression of bone marrow marrow bone of

stem cellsstem Receptor of genes transcription

of bone marrow cells differentiation Reduced production

• • • • • Proteins translation EN PK TNF TRAIL FAS

- L

associated

Increased Increased production of bone marrow marrow bone stem of cells, cells, depletion their , maturation ,

CYTOPENIA 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 Mechanisms of interferon-associated bone marrow suppression

Аctivation of genes transcription •Apo 1 Proteins translation •Аро 2 •FAS АPOPTOSIS •TNF •TRAIL in bone marrow •PK •TNF stem cells •ОАS •PK

•IRF-2 •EN-L

JAK

STAT 1 STAT

CYTOPENIA

Tovey M., Lallemand C. Pharmaceuticals, 2010 Masumi A. et al. Advances in Hematopoietic Stem Cell Research, 2012 Masumi Masumi al. A. et Advances Hematopoieticin Cell Stem Research, 2012 Tovey

M., Lallemand Mechanisms of interferon

STAT 1 C. Pharmaceuticals, C. Pharmaceuticals, 2010 JAK

STAT 1 bone marrow suppression

GATA1, p45, Inhibition of factorsexpression:

MaFg of bone marrow cells differentiation Reduced production

- associated , maturation ,

differentiation and differentiation

maturation Decreased

CYTOPENIA 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;112:542-550 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, ×3000. 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;112:542-550 Mechanisms of interferon-associated bone marrow suppression

of bone marrow JAK

Increased production

stem cells of bone marrow stem

cells, their depletion

STAT 1 STAT

Reduced production, differentiation, maturation of bone marrow cells

Inhibition of factors expression: CYTOPENIA GATA1, p45, MaFg Decreased differentiation and Tovey M., Lallemand C. Pharmaceuticals, 2010 maturation Masumi A. et al. Advances in Hematopoietic Stem Cell Research, 2012 Interferon-associated bone marrow suppression (with or without ribavirin)

Multipotent Committed Stem cell progenitor cell progenitor cell Immature cell Mature cell 1

Cytokines and growth factors (TPO, G-CSF, EPO)

2 2 4 3 Ribavirin IFN 1. Suppression of late erythroid 1. Sequestration of mature cells precursors 2. Reduced maturation and Peripheral 2. Leads to an increase in EPO production of progenitor cells release, which enhances red cell 3. Reduced secretion of growth 1 capillary and platelet production factors beds 3. Causes alterations in the 4. Enhanced secretion of erythrocyte membrane known cytokines, which inhabit the to be associated with proliferation and maturation of erythrophagocytic extravasclar progenitor cells destruction and haemolysis

Shiffman M. et al. In book Advanced Therapy for Hepatitis C, 2012, P.160-168 EPO and TPO during therapy of chronic hepatitis C

35 200 180 30 160 25 140

20 120 100 15 80 10 60 40 5 EPO (mIU/ml) 20 TPO (pg/ml) 0 0 0 4 8 12 EOT 4 24 0 4 8 12 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

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

IFN may limit the ability of the bone marrow to respond to ongoing hemolysis (reticulocytes, x 103 μL) 58% 42% 250

200

150 RBV-related hemolysis Bone marrow suppression 100 P=0,002 50 4,3% 0 P<0,001 RBV IFN/RBV 2,4%

De Franceschi L. et al. Hepatology, 2000 reticulocytes Rendo P. et al. Antiviral Ther, 2000 Lowest absolute neutrophil count in patients receiving IFN or Peg-IFN combined with RBV

46%

38% 36%

24% 21% 16%

8% 5% 5% 1%

Normal Grade 1 Grade 2 Grade 3 Grade 4 > 2000/mcL 1500-2000/mcL 1000-1500/mcL 500-1000/mcL < 500/mcL

IFN/RBV Peg-IFN/RBV

Fried MW et al. N Engl J Med, 2002 Lowest platelet count during treatment with IFN/RBV or Peg-IFN with or without RBV

88%

67%

48%

29%

17% 16% 8% 10% 4% 4% 6% 0% 0% 0% 0%

Normal Grade 1 Grade 2 Grade 3 Grade 4 > 100000/mcL 75000-100000/mcL 50000-75000/mcL 25000-50000/mcL < 25000/mcL

IFN/RBV Peg-IFN/RBV Peg-IFN

Sulkowski M. Clin Liver Dis, 2005 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.