The metabolic effects of excess iron and its assessment

1. The metabolic effects of excess iron and its assessment 3rd Pan-European Conference on Haemoglobinopathies & Rare Anaemias Limassol, 24 – 26 October 2012 Ioav Cabantchik Institute of Life Sciences The Hebrew University of Jerusalem

2. guidelines “One enlarges science in two ways: by adding new facts and by simplifying what already exists”(Le Cahier Rouge) Claude Bernard (1813-1878) “In Science, try to make things as simple as possible, but not simpler” Albert Einstein (1879–1955)

3. Fe • Iron is life essential • respiration • energy production • substrate conversion • Hb synthesis • O2transport • DNA synthesis • neurotransmittersynthesis • Transcriptionfactors • Oxidation/reduction Iron is deleterious • Respiration • labile • Fe(II)  Fe(III) O2● + H2O2 O2 + OH− + OH● • oxidative cell damage Iron deficiency nutritional, acquired, inherited • Iron overload/accumulation • systemic (primary/secondary) • or regional, associatedwithmaldistributionof the metal • systemic (anemia) and/or • regional (tissues/ cells) Organisms control iron uptake and storage to meet metabolic needs without incurring into regional or systemic inbalance

4. IRON HOMEOSTASIS lessons from cellular, animals and human studies • ORGANISMS CONTROL IRON LEVELS BY BALANCING duodenum Spleen hepcidin IRP hamp LCI Labile cell iron post / translational \ co cells systemic via expression of TfR1 and of DMT1 iron uptake via repression of ferroportin • BUT ALSO TO COPE WITH COLATERAL DAMAGE EXERTED BY IRON AND O2 iron storage via expression of ferritin via expression of ferritin

5. OH. Coping with the inevitable O2 H+ Controlling labile cell iron LCI Controlling formation of Reactive O Species (ROS) e- H2O 1% of the 3 kg of O2 consumed daily (33 g/d) turns into Reactive O Intermediates (ROI) respiration labile cell iron LCI Limiting the uptake O2÷ Fe(III) Fe(II) SOD superoxide dismutase Neutralizing/ storing the excess H2O2 Catalase H2O+O2 Antioxidants (bilirubin, uric acid, ascorbic acid, vitamin E, GSH) Chelators are tools designed to safely extract labile iron. GPX glutathione peroxidase and thereby lead to depletion of cellular iron pools GSH GSSG NADPH NADP+ GR glutathione reductase Radical scavengers Cell Damage (proteins, DNA, membranes)

6. PATHOLOGICAL IRON ACCUMULATION IN VARIOUS DISORDERS Iron accumulates naturally in various organs and excessively in various disorders • Systemic iron accumulation • Regional iron accumulation Elevated plasma iron levels (>70 % transferrin saturation) & and body (liver) stores (> 500 ng/ml plasma ferritin) hypertrophic cardiomyopathy & interstitial myocardial fibrosis hypointensity- dentate nuclei tiger-eye - globuspalidus FRDA NBIA Pearl stained biopsy of liver Pearl stained biopsy of heart T2* MRI (4 msec) of highly IO heart Iatrogenic Liver and spleen iron accumulation in CKD patients supplemented with polymeric iron-saccharate Protein, DNA and lipid oxidation products

7. HOW DO WE KNOW WHEN/IF IRON ACCUMULATION LEADS TO DAMAGE • Systemic iron accumulation • Regional iron accumulation Inert iron pools Protein, DNA and lipid oxidation products LIP (labile iron pool) IRON TOXICITY Fe accumulating in labile forms , can prompt (ROI) reactive O intermediates to form excessive toxic (ROS) reactive O species Iron accumulates naturally in various organs and excessively in various disorders Fe(II) Fe(III) ROIs derive from respiration enzymatic O2●− + H2O2 ROS derive from metal catalyzed ROI oxido-reductions O2 + OH− + OH● non-enzymatic Oxidative damage ensues when ROS formation overrides cell antioxidant measures coping with ROI & ROS oxidative damage chelators antioxidants

8. What is labile iron (LI) • LI is a form of ionic Fe (+2 or +3) that is • chemically active : • exchangeable between (bio)ligands and/or (bio)metals • redox-active Fe(II↔III): in biosystems, undergoes conversion by bio-redox agents and catalyzes bioreactions • engages in the formation of reactive O species (ROS) by reacting with O2 or with reactive O intermediates (ROI) (O2, H2O2) that are products of metabolism/respiration • chelatable what is labile iron (LI)’s role in the biomedical scence

9. LCI as bona fide indicator of cell and systemic iron status response to signals 1. ferritin levels 2. [Tf] (& TfR) levels Fe(II) 5. Ingress of permeant Fe 6. Ingress of chelators Fe(II) Fe(II) LCI NADH 3. Redoxstatus of cells 4. Genes of Fe and heme metabolism phamacological Fe(II) ? toxicological Infiltration of NTBI hereditary and transfusionalsiderosis

10. Measuring labile iron in plasma and in cells • LABILE PLASMA IRON (LPI) • the redox-active, chelatableand • membrane permeant • component of non • transferrinbound iron (NTBI) • LABILE CELL IRON (LCI) • the metabolically and redox- • active and chelatable • component of cellular iron TBI ~50 µM LPI ~1 µM LCI ~1 µM TCI ~60 µM LPI and LCI are the direct targets of chelators

11. Measuring labile cell iron LCI in living cells 5- no chelator as redox-active and chelatable iron 4- DFP 3- non-fluorescent oxidizable precursors become fluorescent by ROS generated from labile cell iron prompted with H2O2 chelator  DFR DHR oxidation (r.u.) 2- turn-on probe H2O2  con 1- 0- │ 0 │ 10 │ 20 │ 30 │ 40 min LCI=∆F LCI=∆F Fe bc→ac Fluorescence DFP pretreated chelator turn-off/on probe H2O2 R DHR fluorescent quenched Fe as directly chelatable iron (DCI) Breuer, Epsztejn, Glickstein & Cabantchik, 95-98

12. Labile iron is dynamically monitored y Calcein green (CALG) in fluids and cells after addition of chelator L1→ Fe FL1-H 120- 80- 40- CALG -Fe (fluorescent) +Fe (non-fluorescent) 0.5  0 Fe (µM) microscope imaging counts in cells in solution CALG-histonenuclear targeting CALG-beads flow cytometry CALG-AM cytosol targeting CALG Fe(II)/Fe(III) LH 4- AM 101 102103 104 Fluorescence (485-515) FL1-H 2- CALG↘ DFO 2’ 0 F recovery ∆F is ~ [Fe} F dequenching ≡ Fe binding ↖RPA Breuer, Epsztejn, Glickstein & Cabantchik, 95-98

13. b0= 0.35 ± 0.01; b1= 87± 7; r2=0.997 bd4-ova--CALG FC analysis of CALG-loaded cells before and after addition of permeantchelator. Breuer and Cabantchik, 2006 chelator Fe ΔFL (a.u.) K½/ ΔFm = 0.005 ± 0.0004 1/ ΔFm = 0.011 ± 0.0003 r2=0.999 bead bead Fe ΔF quenched fluorescent ΔF ≡ [LCI] Shifts in fluorescence ΔF are higher in blood cells of hyoertranfused patients. Prusand Fibach2008 (thalass. patients) Doulias..& Galaris 2008 (ox. stress and age) 400- -CALG -chelator +chelator 200- 0- l 100 l 101 l 102 l 104 l 103 How can shifts in CALG fluorescence intensity ΔF obtained in cells be converted into [LCI]? Breuer and Cabantchik, 2010 F intensity CALG beads

14. LABILE CELL IRON (LCI): target of sytemic iron overload • the metabolically and redox-active and directly chelatablecomponent of cellular iron pools (= LIP). NTBI • NON TRANSFERRIN BOUND IRON (NTBI): mediator of iron overload LCI Iron that outpours into circulation causing plasma iron to rise and surpass transferrin’s binding capacity generates iron forms not bound to transferrin Hershko et al 78, 79: “… NTBI might be relevant to the pathogenesis of tissue damage and the protective effect of chelation…” • Aplastic anemia (AA) • Bone-marrow transplantation • Chemotherapy • Hereditary hemochromatosis (HH) • Thalassemiamajor (TM) • Thalassemiaintermedia (TI) • Myelodyplastic syndrome (MDS) • Sickle cell disease (SCD) a variety of chronic metabolic disorders such as diabetes

15. PLASMA NTBI possible clinical value • for defining the degree of systemic iron overload • (diagnostically/ prognostically) ? • for predicting tissue iron overload and end organ toxicity? • for assessing the efficacy of chelation in maintaining plasma free of iron sources implicated in tissue iron overload?

16. Measuring plasma NTBI total NTBI, mostly protein adsorbed Hershko el 1978 ; Hider, Porter et al 01) extraction & filtration plasma transferrin 40 mM Fe3+ NTA 2 µM Fe3+ 1. extraction via “non-mild” chelation (80 mM NTA) 3. Detection with sensor ( HPLC) Fe3+ Fe3+ 2. filtration Accurate, sensitive, reproducible. Laborious; might mobilize Fe from Tf and Fe-chelates

17. NTBI is detected in plasma when transferrin saturation exceeds 70% represents ~1–10 % of the (~ 40 µM) TIBC • is composed of complexes of : • iron-citrate and phosphates • iron and/or of iron-ligands bound to proteins • the iron in the complexes can be : • exchanged with other metals or ligands • chelated by agents with high binding affinity for the metal • reduced by natural reductants and • translocated across membranes via resident transporters/channels these properties define the labile components of NTBI in plasma NTBI is heterogeneous: its composition differs according to sources, levels attained and also following chelation. (Fe-chelate complexes can be measured as NTBI by some assays!).

18. Measuring plasma NTBI labile components LPI and DCI TBI Fe3+ High throughput fluorescence assays 40 µM LPI DCI Fe chelator blocks NTBI ascorbate prompts NTBI to redox-cycle  ROS and oxidize a non-fluorescent probe Fe3+ 2 µM a fluorescent-chelator binds LPI bead plasma factors that affect LPI/DCI (albumin, citrate, uric acid) are eliminated with 0.1-0.5 mM NTA) DHR ROS oxidizes probe Turn-off Probe plate reader Turn-on Probe R Fe3+ LPI: labile plasma iron, Espósitoet al 2003 feROS, DCI: Directly chelatable iron, Breuer et al 98, 02 feRISK, flow cytometer bead

19. LPI in non chelated beta-thal/HbEpatients correlations Pootrakul P. et al. 2004 Blood 104 p. 1504 LPI appears when transferrin saturation exceeds 70-80% LPI correlates with   serum ferritin  iron stores RBC membrane Fe  cell accumulated Fe

20. LPI as early indicator of chelation efficacy

21. Deferasirox on β-thalassemiapatients period of treatment required to attain basal LPI levels Measure of chelation activity attained 2h after absorption of the drug Measure of chelation activity retained in plasma 24 h after drug intake Post administration (2h) Preadministration (trough value) * p< 0.01 (n=13) * * trough levels attained normal range within 16 weeks * * week * 0 4 16 28 40 52 Daar S., et al. 2009 E.J. Haem. 82 p. 454 Cabantchik May 2012

23. Which chelation regimen confers daily protection from LPI appearance in thalassemia major patients under different chelation regimens? LPI (group mean of n=20 ± stdev) 45% 50% Deferiprone (3x 25 mg/k/d po) : diurnal fluctuations in LPI and high stdev Deferrioxamine (O/N sc ): significant rise in diurnal LPI and high stdev Combination (continued sequential DFP and DFO): maintained low LPI over 24 h Deferasirox (switched from DFO or DFP after 24 h drug washout) show low LPI over 24h 95% 88% Zanninelli G. et al. 2009 Br. J. Haem. 147 p. 744

24. 6 6 3 3 0 0 −3 40 60 80 100 120 40 60 80 100 120 Do NTBI/LPI levels correlate with established clinical parameters? LPI µM (< 10 h washout)1 NTBI µM (> 24 h washout)2 Transferrin saturation (%) All patients with iron toxicity-related cardiomyopathies have NTBI/LPI; however, the reverse is not the case 1. Zanninelli G, et al. 2009 Brit J. Hematol. 147, 744. 2. Piga A, et al. 2009, Am J Hematol. 84:29-33.

25. Summary • A single LPI measurement 2 h after drug intake provided a measure for the ability of the drug to attain levels sufficient for instantaneous elimination of LPI • A single LPI measurement taken at trough chelator levels in plasma (~24 h after administration of DFR, 10-12 h of DFP and 12-14 h of DFO) provided an indication for the ability of a chelation regimen to maintain LPI at basal levels (< 0.2 ± 0.1 µM) at a given day in the course of treatment of thalassemia major, thalassemiaintermedia, SCD or MDS patients. • Repeated (monthly) LPI measurements in the course of treatment indicated that LPI reached basal levels while serum ferritincontinue to decline and was correlated with long term reduction in liver iron concentration

26. When and how should iron overload be treated? The routine treatment is administration of iron chelators, but clinicians must make some important decisions … … when should chelator treatment be initiated? … which chelation regimen to use? … how should chelation efficacy/efficiency be evaluted/modified? These decisions are too often based on indicators of iron overload that have delayed-response, like serum ferritin levels. Iron overload in organs can be imaged by MRI methods, but these are expensive and not routinely available. • Development of early alert markers of emerging iron overload that: • respond to changes in iron status with minimal delay • are based on readily available technology .