1. Erythropoietin Mimetic Peptides, Other approaches and New IV Iron Preparations: What Data Do We Have? What Data Do We Need?
Anatole Besarab MD, FACP
Director of Clinical Research
Division of Nephrology and Hypertension
Henry Ford Hospital
Detroit, MI

2. Disclosure
Dr Besarab has received grant support, been a consultant or has received honoraria
ESA Related Iron Related
Affymac American Regency Labs
Akebia
Amgen Colorado Labs
Aventis Pharmaceuticals
Bayer Rockwell Int
Fibrogen
Hoechst-Marion-Rousell Schein
Medgenics
Roche Watson

3. The development of ESAs and IV iron preparations over the last 20 years
Macdougall IC and Ashenden M. Adv Chronic Kidney Dis 2009;16:117−130

4. Erythropoietin and Iron: Both Important
About 3-4
Days
Proliferation, maturation & survival (apoptosis)
Iron dependence
Erythropoietin
Erythroblast
Iron
About 10-14
days
Erythropoietin (EPO) is needed in the first stage (from progenitor cells in the burst-forming and colony –forming unit-erythroid to proerythroiblast) but not the second precursor cell stage
Main Points
Erythropoietin is the essential growth factor required for RBC development.
Erythropoietin stimulates the survival, differentiation, and proliferation, of early progenitor cells at the BFU-E and CFU-E stages, allowing them to mature into RBCs. CFU-E cells have significantly more erythropoietin (EPO) receptors than BFU-E and depend on erythropoietin for survival.
Background Information
 All hematopoietic cells are derived from an original pool of pluripotent stem cells. The most primitive progenitor cells committed exclusively to the erythroid cell line are the burst-forming units-erythroid (BFU-E) cells.
 In contrast, colony-forming units-erythroid (CFU-E) are more differentiated. Stimulated CFU-E cells can form reticulocytes within about 8 days.
The most important functional differences between BFU-E and CFU-E cells is that the latter have significantly more EPO receptors, and depend on erythropoietin for survival. In vitro experiments have shown that CFU-E cells survive only a few hours in the absence of erythropoietin.
EPO is critical for multiplication and differentiation processes of BFU-E and CFU-E where increasing dependence and sensitivity to EPO is manifest.
EPO receptors expressed by CFU-E and proerythroblast stages diminish during the final stages of erythroid differentiation such that reticulocytes and erythrocytes are devoid of EPO receptors
 Iron is not an erythropoietic stimulant and does not replace the need for exogenous erythropoietin in patients on dialysis. Erythropoietin and iron work synergistically—erythropoietin is the essential stimulus for RBC production, and iron is an important substrate that supports the proper formation of the heme portion of Hb.
 Note that it takes about days to get a cell to maximally incorporate iron into Hb, and only 3-4 days in which to do it in. since the process is a continuous one. one should never have a state wherein iron delivery for hemoglobin synthesis is ever limited
References
Hillman RS. Finch CA. Red Cell Manual, 7th ed. Philadelphia, PA: F.A. Davis Company; 1996:Chapter 1.
Ogawa M. Differentiation and proliferation of hematopoietic stem cells. Blood 1993;81:2844–2853.
Papayannopoulou T, D’Andrea AD, Abkowitz JL, Migliaccio AR. Biology of erythropoiesis, erythroid differentiation, and maturation. In: Hoffman R, Benz, EJ, Shattil SJ, et al. Hematology Basic Principles and Practice, 4th ed. Philadelphia, PA; Elsevier Churchill Livingstone :
About 15-21
Days
About 1-2 Days
EPO dependence
Pluripotent Stem Cell
Burst-Forming Unit-Erythroid Cells (BFU-E)
Colony-Forming Unit-Erythroid Cells (CFU-E)
Proerythro- blasts
Erythro- blasts
Reticulocytes
RBCs
Hillman RS, et al. Red Cell Manual, 7th ed. Philadelphia, PA: F.A. Davis Company; 1996:chap 1. Papayannopoulou T, et al. In: Hoffman R, et al. eds. Hematology: Basic Principles and Practice, 4th ed. Philadelphia, PA: Elsevier Churchill Livingstone; 2005:chap 20. Brock. Iron Metabolism in Health and Disease. W.B. Saunders Co; 1994

5. Erythropoiesis is affected by local levels of cytokines and interleukins
IL-6
TGF-β
TNF-α
TRAIL
IL-6, interleukin-6; TGF-b; transforming growth factor-b; TNF-a; tumour necrosis factor-a; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand 7 7

6. Why is there a need for new ESAs or new Approaches ?
1) Failure to achieve optimal benefits
2) Risks: are these the same for all ESA irrespective of duration of action?
3) PRCA: probably a non issue with commercial well studied agents but may be with some “me too” generics
4) High Cost
5) Need to reduce Hb variation
Maybe be influenced by the pharmacokinetic duration of action of the ESA
Coordinating the sequential steps of erythrocyte production
6. Fear of iron: decreased but have we gone too far?

7. Endogenous Erythropoietin (EPO) and Current Erythropoiesis-Stimulating Agents (ESAs): Same Mechanism of Action
BCL-XL
Caspase-3
Gene transcription
Nucleus
Cytochrome c
release
Mitochondria
Erythropoiesis
“survival”
“proliferation”
“differentiation”
Adapted from: Eur Arch Psychiatry Clin Neuro .2001;251:

8. Hematide (Affymax / Takeda)
Synthetic peptide-based ESA (dimeric, PEGylated)
No structural homology with EPO
Activates EPO receptors
Currently completed Phase 3 studies
Hematide immunologically distinct from EPO
Anti-EPO Abs do not cross-react with/neutralise Hematide
PEGylation
Spacer Linker
Peptide
EpoR
EpoR
Cell surface 2

9. Hematide (peginesatide)
14 patients in the UK, France, and Germany
Ab-mediated PRCA
Treated with Hematide 0.05 mg/kg q4wks 60
14.0 50
13.0 40
12.0
% ofPatients Receiving RBC Transfusions
During Each Study Month 30
Mean (SD) Hb Concentration (g/dL)
11.0 20
10.0 10
9.0
8.0 BL 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
Study Months
Macdougall IC et al, N Engl J Med Nov 2009.

10. Hematide Pharmacokinetics: half-life
Agent
Population
Mean (± SE) Half-life (h) IV SC
Hematide
Healthy volunteers1 Predialysis CKD patients1
35-68
39-104
Epoetin alfa
Healthy volunteers2
HD, CAPD patients3,6
6.8 ± 0.6
6.5 ± 12.7
19.4 ± 2.5
Epoetin beta
8.8 ± 0.5
24.2 ± 2.6
Darbepoetin alfa
Peritoneal dialysis patients3
25.3 ± 2.2
48.8 ± 5.2
CERA
Healthy volunteers4
Peritoneal dialysis patients5
133 ± 10
134 ± 19
137 ± 22
139 ± 20
Hematide’s serum half-life is similar to darbepoetin alfa, longer than epoetin alfa or beta, and shorter than CERA
1. Hematide Investigators Brochure (Affymax data on file)
2. Halstenson et al. Clin Pharmacol Ther. 1991;50:
3. Macdougall et al. J Am Soc Nephrol. 1999;10:
4. Dougherty et al. ASCO 2004.
5. Macdougall et al. Clin J Am Soc Nephrol. 2006;1:
6. Boelart et al. Peritoneal Dialysis Int.1989; 9:95-98; Kindler et al. Nephrol. Dial Transplant. 1989; 4: 13

11. AFX01-04: Correction of Anemia (Hgb ≥11 g/dL & Hgb Increase ≥ 1 g/dL From BaseLine)36 77 92 80 95
100 69 64 93 10 20 30 40 50 60 70 90
4 Weeks
8 Weeks
12 Weeks
Duration of Treatment
Correction of Anemia (% of Patients)
0.025 mg/kg SC (n=15)
0.05 mg/kg SC (n=45)
0.075 mg/kg SC (n=15)
0.05 mg/kg IV (n=15)
Same as Dr. Fishbane’s presentation. 14 14 14

12. A Phase 2 Study of the Safety and Efficacy of Hematide™/Peginesatide for the Maintenance Treatment of Anemia in Patients With Chronic Kidney Disease Who Are on Hemodialysis or Do Not Require Dialysis and Were Previously Treated With Darbepoetin Alfa

13. Study Design: Conversion Study
Open-label, single arm, switching from darbepoetin alfa
Primary Objective:
To determine whether peginesatide treatment can maintain Hb levels following conversion from darbepoetin alfa treatment
Secondary Objectives:
Proportion of patients maintaining Hb target of g/dL after conversion To Evaluate the safety and tolerability of peginesatide
Titration
1 to 18
Evaluation
19-24
Period: Screening
Week: to -1
n~ 182
n ~ 80
n = 102
Peginesatide Q4W IV or SC
Visits Every Other Week
Visits Every Week
Enrollment

14. Primary and Secondary Endpoints
Value (95% CI)
Primary Endpoint
Mean (95% CI) change in Hb from Baseline to the Evaluation Period, g/dL 92
0.04 (–0.14, 0.23)
Secondary Endpoints
Patients with Hb within 10.0–12.0 g/dL during Evaluation, % (95% CI)
71% (61%, 81%)
Patients with Hb change within ±1 g/dL from Baseline during Evaluation, % (95% CI)
74% (64%, 83%)
Red blood cell transfusions, % (95% CI)
101
4% (0.0, 8.3)
Time Period (range of weeks) n=
Few patients discontinued the study
No particular safety concerns were raised However, this study was not designed to definitively determine the safety of peginesatide.

15. Hematide (peginesatide) - the Phase 3 clinical trial programme

16. Hematide™ Phase 3 Clinical Trial Summary
Study
Description
Sample Size (Hematide vs Control)
Geographic Origin
PEARL 1 AFX01-11
Comparison with darbepoetin alfa for the correction of anemia in patients not on dialysis (SC)
~300:150 US
PEARL 2 AFX01-13
US/ Europe
EMERALD 1 AFX01-12
Comparison with epoetin alfa for the maintenance treatment of anemia in HD patients previously treated with EPO (IV)
~500:250
EMERALD 2 AFX01-14
Comparison with epoetin alfa for the maintenance treatment of anemia in HD patients previously treated with EPO (IV/SC)

17. PEARL Study Design 0 to 24 Hematide™ 0.025 mg/kg Q4W SC
Period: Screening
Week: to 0
Correction
0 to 24
Evaluation
25 to 36
Long-term
37 to 52+
n~900
n=450
n=150
Darbepoetin 0.75 mcg/kg Q2W SC
Hematide™ mg/kg Q4W SC
Hematide 0.04 mg/kg Q4W SC
Randomization
(1:1:1 ratio)
Enrollment period
52 weeks
~104 weeks
First patient
Last patient
End of study

18. (following a 1-week ESA-free interval)
EMERALD Study Design
Period: Screening
Week: to 0
Titration
0 to 28
Evaluation
29 to 36
Long-term
37 to 52+
n~1260
n=630
n=420
n=210
Hematide Q4W IV
(following a 1-week ESA-free interval)
EPO 1-3x per week IV
Randomization
(2:1 ratio)
Enrollment period
52 weeks
~104 weeks
First patient
Last patient
End of study

19. Composite Safety Endpoint (CSE) Results
Total Events Adjudicated: Events in 734 Patients
Dialysis+Non-Dialysis
Peginesatide (N=1722)
Control (N=869)
Patients with CSE Events
385 (22%)
188 (22%)
Hazard Ratio
1.06
90% CI / 95% CI
(0.91, 1.23) / (0.89, 1.26)
solid line = peginesatide dotted line = control 25 25

20. CSE Overall and by Dialysis, Non-Dialysis: Event-Free Rate (Kaplan-Meier Survival)
peginesatide (N=656)
darbepoetin (N=327)
Patients with CSE Events
142 (22%)
56 (17%)
Hazard Ratio
1.34
90% CI
(1.03, 1.73)
95% CI
(0.98, 1.82)
solid line = peginesatide dotted line = darbepoetin
Dialysis
peginesatide (N=1066)
epoetin (N=542)
Patients with CSE Events
243 (23%)
132 (24%)
Hazard Ratio
0.95
90% CI
(0.79, 1.13)
95% CI
(0.77, 1.17)
solid line = peginesatide dotted line = epoetin 26

21. CSE (=“On Study”) vs. “On Drug” Sensitivity Analysis27

22. Conclusions
Peginesatide, administered once monthly, increases and maintains Hb levels in CKD patients not on dialysis and to maintain Hb levels in CKD patients on dialysis
For the CSE analysis pooled across dialysis and non-dialysis populations, the noninferiority criterion are met for peginesatide vs. comparator ESA
However, CSE findings in dialysis and non-dialysis are distinct
CSE findings in the dialysis population indicate a similar CV safety profile of peginesatide and epoetin
Differences in the CSE in non-dialysis patients for peginesatide compared to darbepoetin are being further evaluated 28

23. Current Status
Company has announced plans for new drug application only for hemodialysis patients at this time to be submitted some time in Q2 of 2011

24. Differences Between Hematide and Other ESAs
Long Acting
Intermediate
Acting
2.
1.
3.
4.
Short Acting
Hematide
Mircera
Aranesp
Eprex/Espo/Epogin/
Neo-Recormon
Chemically synthetic peptide, not a protein drug √
Dialysis:
Q4W Maintenance Dose
Pre-Dialysis:
Q4W Correction Dose
Simple Conversion
Potential for Room Temperature Stability
Potential for Time and Cost Saving
No PRCA
Treat PRCA
This is the current version. Because several portions are revised, I have used our template, not Affymax-Takeda template. 30

25. Activation of HIF under hypoxic conditions
HIF stabilisers
Hypoxia-Inducible
Factor (HIF)
Activation of HIF under hypoxic conditions
Gene eg EPO
P300
HIF-PH
HIF-α O2
HIF-β
HRE
Inhibition of HIF under normal conditions
HIF-PH
HIF-α
Proteasome OH O2
VHL
Proteasomal degradation

26. New ESAs – what’s in the pipeline?
Hematide (peginesatide); EPO-mimetic peptide
Amino acid sequences unrelated to native EPO; immunologically distinct; used to treat PRCA
Results: hematologic effects comparable to other ESAs
HIF (Hypoxia inducible factor) stabilisers
AKA: prolyl hydroxylase inhibitors
Orally-active inhibitors of HIF prolyl hydroxylase
have been synthesized (e.g. FG-2216; FG-4592 – FibroGen)
Increase EPO levels, even in CKD patients
Stimulate erythropoiesis AT MUCH lower peak EPO levels
Suppress hepcidin levels
Concerns: specificity (several hundred genes are HIF dependent)
In clinical trials (Akebia, Glaxo, Bayer)
GATA 2 inhibitors [acts through same pathway as HIFs but just distally]
GATA-2 inhibitors (K-11706, K-7174) Inhibit GATA acetylation OR Stimulate GATA de-acetylation
Still in animal models
EPO gene therapy
EPO-secreting modified fibroblasts
Hypoxia-dependent EPO-associated adenovirus
Phycoerythrin specific EPO encoding B-lymphocyte
mRNA
HIF ß α
GATA-2
EPO gene
5’ACGTGC
GATA
prolyl hydroxylase
inhibitor
GATA-2 inhibitors (K-11706, K-7174)
Inhibit GATA acetylati OR
Stimulate GATA de-acetylation2
HIFα pro
HIFα
EPO
prolyl
hydroxylase
All increase serum EPO and haematocrit levels in animal models

27. Baseline Serum Iron (Mean ± SEM)
FG-4592, A NOVEL ORAL HIF PROLYL HYDROXYLASE INHIBITOR, ELEVATES HEMOGLOBIN IN ANEMIC STAGE 3-4 CKD PATIENTS Besarab et al
Treatment
Baseline Serum Iron (Mean ± SEM)
% Taking PO Iron
Placebo
71.1 ± 4.1
39%
1.5 mg/kg FG-4592
70.4 ± 4.0
57%
2.0 mg/kg FG-4592
73.0 ± 3.9
55%
rHuEPO (Schwartz)
72.2 ± 8.7
100% 33 33

28. Mol Ther 2005; 12:

29. Biopump therapy Therapy outline
Ex vivo genetic manipulation of dermal tissue structures for autologous production and delivery of therapeutic proteins
Therapy outline
Harvesting
Ex-vivo genetic manipulation using viral vector
Pre- implantation analysis
Secretion levels, Dosing, Viability, Sterility
Subcutaneous implantation after days
Ablation or removal – to reduce dose, if required
Now in Phase II clinical trials (Besarab et al.)

30. EPODURE was safe and doseable; no antigenic response
Erythropoiesis Sustained 12 months by the EPODURE Biopump in Patients with Chronic Kidney Disease: Further Results of Phase I/II Proof of Concept TrialErythropoiesis Sustained 12 months by the EPODURE Biopump in Patients with Chronic Kidney Disease: Further Results of Phase I/II Proof of Concept Trial Besarab et al
EPODURE was safe and doseable; no antigenic response
Clinical feasibility demonstrated in 12 pts [4 previously ESA dependent
Single EPODURE administration can raise and maintain Hemoglobin levels for months without any injections of ESAs
10-12 pts responded. Two that did not were resistant to ESA

31. What about Iron Iron Deficiency Common in ESRD and CKD Blood loss
Poor iron absorption
Interference with iron absorption
Acid blockers
Phosphate binders
Other

32. Iron ESRD History Prior to ESAs, Iron overload common
Due to blood transfusions
Was always difficult to prove “toxicity” from the stored” iron unless ferritins > 3000 ng/ml.
In ESA era, absolute and functional iron deficiency developed
1993
Iron deficiency common
50% of pts with TSAT < 20%
Oral iron could not keep up with erythropoietic needs (parenteral iron gap)
1996 First KDOQI stresses need for IV iron
2010
60-80% of HD pts treated with IV iron
Mean Serum ferritin approx 585 ng/ml (min recommended is 200 ng/ml)

33. Iron or EPO Debate EPO IRON
Combine summary with previous
EPO
Data to support IV Iron in Patients with Serum Ferritin (SFe) > 500 ng/mL
Current practice (mean SFe in U.S. HD pts was ng/mL in 2007)
Besarab (RCT); DRIVE Study (RCT)
Kalantar-Zadeh (Observational): Lowest all-cause and CV death risks with SFe:
IV iron < 400 mg/mo associated with improved survival; doses >400 mg/mo correlated with ’d death risk
Hemochromatosis unlikely [need acc of 20 g]
No association with infection when controlled for time-dependent effects
Do not give during active infection
Less costly to achieve same Hb
NO black box warnings (Fe sucrose & gluconate) vs. ESAs & Fe dextran
IRON
EPO & iron have multiple functions
→ essential at physiological levels
→ harmful at high levels
Correlation of M &M with higher ESA dose is statistical
→ may be clinically unrelated
→ both caused by underlying systemic disorders
Cons for iron:
Adverse effects of Fe: chronic in nature
IV iron safety data from short-term trials
IV iron can increase oxidative stress
→ IV iron should be used with caution to minimize adverse long-term consequences, particularly in inflamed patients

34. Iron Efficacy and Safety
Variety of studies indicate 20%-60% reduction in ESA dose requirements
Improvement in achieved Hb
1000 mg ~ 1 g/dl Hb
Generally well tolerated
Acute hypotensive reactions
?immune response
?free iron
Other
No interventional studies on effect on higher level outcomes: morbidity and mortality
Long term safety not established
No obvious signal of harm from epidemiologic studies on large data bases
Unclear benefit beyond Hb and ESA dose

35. Current IV Iron Agents (USA)
Iron dextran
Low molecular weight (brand and generic)
High molecular weight (brand only)
Sodium ferric gluconate
Brand and generic
Iron sucrose
Ferumoxytol
Brand only

36. Current Knowledge of Iron preparations
Iron dextran
Low molecular weight may be safer
Brand and generic versions may not be equivalent
Iron sucrose study in Europe1
Unclear if differences in other IV iron agents influence
Efficacy
Safety
1. Rottenbourg et al NDT 2011: doi /ndt/gfr024 (in print)

37. Targeting Higher Transferrin Saturation and Ferritin Levels Using Ferumoxytol
510 mg Ferumoxytol x 2 if TSAT < 30% and ferritin < 500 ng/ml to 690 patients of a SDO (Mid Atlantic)
ESA protocol left unchanged
TSAT 24% to 31%, ferritin 349 to 542 at 3 mo, both decreased toward baseline by 6 months
Experience at Satellite Health: 61/12,105 doses ; AE rate* per dose administered (0.5 %); Allergic 10/61; anaphylactoid 4/61; hypotension 8/61
For those with baseline Hb > 10 g/dl, EPO dose from 7200 to 5500 U 3x/wk

38. What is on the horizon? Parenteral Non-conventional Oral ?
Ferric carboxymaltose
Iron isomaltoside 1000
Non-conventional
Dialysate: Sodium Ferric Pyrophosphate
Oral ?
Ferric citrate

39. Ferric carboxymaltose
Can administer large doses at one time
Up to 750 mg IV
Currently approved in Europe
Phase 3 studies in US
FCM vs. iron sucrose

40. Need for additional knowledge on IV iron
Are there true benefits related to patient outcomes?
Is there any risk to long term administration?
Oxidative stress
Inflammation

41. A new way of administering iron to HD patients?1

42. Sodium Ferric Pyrophosphate
Delivered by dialysate
Advantages
Convenience
Delivery matches needs
As iron lost with dialysis, replaced
Slower delivery than IV
Avoids free iron surges
May be well tolerated
Phase 2 studies indicate that IV iron may still be required at times
Phase 3: CRUISE is underway

43. Hepcidin binding to ferroportin
JAK2; Janus kinase 2
Krushner JP. Blood 2010;115:3425–3426
Kushner JP. Blood 2010;115:17:3425–6.
JAK2, Janus Kinase 2

44. Hepcidin regulates iron flow into plasma
Spleen
Hepcidin
Liver
Hepcidin
Red blood cells
Bone marrow
Duodenum
Hepcidin
Plasma Fe-Tf
Ganz T. J Am Soc Nephrol 2007;18:394–400; Ganz T & Nemeth E. Am J Physiol Gastrointest Liver Physiol 2006;290:G199–203

45. Regulation of hepcidin by inflammation
Plasma Fe-Tf
Inflammation
Liver
Spleen
Hepcidin
Hepcidin
Red blood cells
Bone marrow
Duodenum
Hepcidin
Ganz T. J Am Soc Nephrol 2007;18:394–400

46. MAb against hepcidin effective in combination therapy for anaemia of inflammation14 14 12 12
Hb (g/dL)
Hb (g/dL) 10 10
p<0.01
p<0.01
p<0.01
p<0.01 8 8 6 13 6 13
Day
Day
3X Ab ESA
3X Ab 2.7
4X control Ab
4X Ab ESA
3X control Ab + ESA
4X Ab ESA
4X control Ab + ESA
4X Ab 2.7.
5X Ab ESA
Ab 2.7 restored response to ESA treatment in hHepc knock in AI mice
Ab Ab Ab Ab Ab
Ab Ab Ab Ab
Ab Ab Ab
Administration time of BA, anti-hepcidin (or control) antibody, and ESA or saline control
Sasu B et al. Blood 2010;115:3616–3624

47. Conclusions
We have used ESA therapy and IV iron compounds to treat renal anemia for 20 years
We still do not know the optimum way to use these products in CKD patients as they progress from moderate to end stage renal disease and thereafter
Several novel therapeutic strategies for managing renal anemia are on the horizon
BA16738 – 132 patients in CERA arm SC, 52 week treatment period (18 week correction, 10 week evaluation, 24 week extension) 264
BA16736 – 126 patients in the CERA arm IV, 52 week treatment period (24 week correction, 28 week evaluation) 168
BA16739 – 310 patients in 2 CERA arms IV, 52 week treatment period 465
BA16740 – 310 patients in 2 CERA arms SC, 52 week treatment period 465
BA17284 – 132 patients in the CERA arm pre-filled syringes, 36 week treatment period 264
BA17283 – 132 patients in the CERA arm IV, 52 week treatment period 264
Total 897 IV patients
Total 729 SC patients

48. We know we must individualize…
We know we must individualize
One size does not fit all!!!!

49. Thank you