XV Convegno del gruppo di studio di Dialisi Peritoneale Bari, 19-20 Marzo 2010 XV Convegno del gruppo di studio di Dialisi Peritoneale Update sulla terapia dell'anemia in PD Francesco Locatelli Dipartimento di Nefrologia, Dialisi e Trapianto di Rene Ospedale “Alessandro Manzoni” di Lecco

Erythropoietin has two erythropoietin receptor binding sites EPO receptor EPO receptor Main Points • This slide shows an X-ray crystallography image of rHuEPO and the EPO receptor.1 • rHuEPO activates RBC progenitor cells by binding and activating the erythropoietin receptor on the cell surface, which is made up of a dimer of erythropoietin receptor molecules Background Information • The Erythropoietin receptor is a member of the superfamily of cytokine receptors. The unique crystal structure that is formed when erythropoietin is complexed to the extracellular ligand-binding domains of the erythropoietin receptor is responsible for optimal signaling through intracellular kinase pathways.1,2 • The complex formed by erythropoietin and the extracellular ligand-binding domain of the erythropoietin receptor is held together by two regions located on opposite faces of erythropoietin. These erythropoietin-receptor interfaces on erythropoietin have been identified as high-affinity and low-affinity binding sites.1 References Syed RS, Reid SW, Culwel Li, et al. Efficiency of signaling through cytokine receptors depends critically on receptor orientation. Nature. 1998;395:511-516. Cheung JY, Miller BA. Molecular mechanisms of erythropoietin signaling. Nephron. 2001;87:215-222. Elliott S, Lorenzini T, Barzilay J, Chang D, Delorme E. Mapping of the active site of recombinant human erythropoietin. Blood. 1997;89:493-502. Elliott S, et al. Blood 89: 493-502, 1997 Syed RS, et al. Nature 395: 511-516, 1998 2

Erythropoietin has two erythropoietin receptor binding sites EPO receptor EPO receptor rHuEPO Main Points • This slide shows an X-ray crystallography image of rHuEPO and the EPO receptor.1 • rHuEPO activates RBC progenitor cells by binding and activating the erythropoietin receptor on the cell surface, which is made up of a dimer of erythropoietin receptor molecules Background Information • The Erythropoietin receptor is a member of the superfamily of cytokine receptors. The unique crystal structure that is formed when erythropoietin is complexed to the extracellular ligand-binding domains of the erythropoietin receptor is responsible for optimal signaling through intracellular kinase pathways.1,2 • The complex formed by erythropoietin and the extracellular ligand-binding domain of the erythropoietin receptor is held together by two regions located on opposite faces of erythropoietin. These erythropoietin-receptor interfaces on erythropoietin have been identified as high-affinity and low-affinity binding sites.1 References Syed RS, Reid SW, Culwel Li, et al. Efficiency of signaling through cytokine receptors depends critically on receptor orientation. Nature. 1998;395:511-516. Cheung JY, Miller BA. Molecular mechanisms of erythropoietin signaling. Nephron. 2001;87:215-222. Elliott S, Lorenzini T, Barzilay J, Chang D, Delorme E. Mapping of the active site of recombinant human erythropoietin. Blood. 1997;89:493-502. Elliott S, et al. Blood 89: 493-502, 1997 Syed RS, et al. Nature 395: 511-516, 1998 3

The ideal ESA Effective Safe Flexible administration route Less frequent administration schedule Cheap Key points Despite years of using erythropoiesis-stimulating agents (ESAs) there is still room to improve the pattern of practice in the treatment of anaemia It is possible to optimize the use of ESAs by careful selection of the type of ESA used, the best route of administration and the frequency of administration It is also posssible to consider potential cost-savings associated with the use of the different ESAs available in the market

Currently available ESAs Recombinant human erythropoietin (rHuEPO) Epoetin alfa Epoetin beta Long-acting ESAs Darbepoetin alfa Different molecular structure Increased biological activity Key points Currently available erythropoiesis-stimulating agents (ESAs): epoetin alfa, epoetin beta, epoetin delta and darbepoetin alfa Darbepoetin alfa has been engineered to contain five N-linked carbohydrate chains (two more than recombinant human erythropoietin [rHuEPO]) Compared with rHuEPO, darbepoetin alfa has a longer serum half-life (approximately three-fold) and greater in vivo potency, even though it has a lower affinity for the erythropoietin receptor Darbepoetin alfa can, therefore, be administered less frequently to obtain the same biological response Background There is a direct relationship between the ESA molecule's sialic acid-containing carbohydrate content and its serum half-life and in vivo biological activity, but an inverse relationship with its receptor-binding affinity Increasing the carbohydrate content should, therefore, lead to a molecule with enhanced biological activity Hyperglycosylated rHuEPO analogues such as darbepoetin alfa, were, therefore, developed Reference Egrie JC et al. Br J Cancer 2001;84(1):3–10 CERA Different mechanism of action Different molecular structure Increased biological activity

Biochemical and biological properties of rHuEPO and glycosylation analogs Receptor binding rHuEPO 3 N-linked carbohydrate chains Up to 14 sialic acids 30,400 daltons ~40% carbohydrate 4 N-linked carbohydrate chains Up to 18 sialic acids 33,750 daltons ~46% carbohydrate Biological activity Serum half-life NESP 5 N-linked carbohydrate chains Up to 22 sialic acids 37,100 daltons ~51% carbohydrate The 4- and 5-N linked chain glycosylation analogs of rHuEPO are biochemically distinct from rHuEPO. They have increased molecular weight, sialic acid content and negative charge. The two extra carbohydrate chains on Aranesp® increase the molecular weight by about 22% and the maximum number of sialic acid residues from 14 to 22. In tests of in vivo efficacy, the magnitude of the hematocrit rise correlated with the number of carbohydrate chains and the sialic acid content of the molecules. Egrie JC, Browne JK. Nephrol Dial Transplant. 2001;16(suppl 3):3-13

Hb concentrations at 4-week intervals 7 8 9 10 11 12 13 14 15 rHuEPO NESP Hb (g/dL) Patient numbers: 37 35 35 33 34 33 31 rHuEPO NESP 129 128 127 127 123 121 106 77 51 25 1 5 9 13 17 21 37 49 Study week Locatelli F et al. Kidney Int 2001; 60: 741-747

Weeks since withheld dose Time for Hb to return to £12 g/dL after dose withheld due to Hb >14 g/dL 16 rHuEPO (n = 13) NESP (n = 31) 15 14 13 Hb (g/dL) 12 11 10 9 –6 –5 –4 –3 –2 –1 1 2 3 4 5 6 7 8 9 10 11 Weeks since withheld dose Locatelli F et al. Kidney Int 2001; 60: 741-747

Percentage of Hb values >14 g/dL Mean % of Hb values > 14 g/dL DA administration Q2W was not associated with an increased frequency of Hb >14 g/dL Percentage of Hb values >14 g/dL 5 4 3 2 1 2.6 Mean % of Hb values > 14 g/dL 1.3 Key points Patients who switched to the darbepoetin alfa once-every-2-week (Q2W) regimen were less likely to experience haemoglobin (Hb) levels above 14 g/dL, compared with the once-weekly (QW) patients: Proportion of patients (95% CI) with Hb levels >14 g/dL was 1.3% (0.28, 2.22) for Q2W patients and 2.6% (1.33, 3.86) for QW patients Reference Locatelli F et al. Nephrol Dial Transplant 2006;21(Suppl 4):SP411 abstract and poster Q2W (n=153) QW (n=153) Locatelli F et al. Nephrol Dial Transplant 2005;20 S.5:MP181

16 (10) 2007

Serum half life of ESAs IV SC Agent Population Mean (± SE) half-life (h) IV SC Epoetin alfa Healthy volunteers1 6.8 ± 0.6 19.4 ± 2.5 Epoetin beta 8.8 ± 0.5 24.2 ± 2.6 Darbepoetin alfa Peritoneal dialysis patients2 25.3 ± 2.2 48.8 ± 5.2 C.E.R.A. Healthy volunteers3 133 ± 9.8 137 ± 21.9 Peritoneal dialysis patients4 134 ± 19 139 ± 20 Pharmacokinetic properties of ESAs in healthy volunteers and peritoneal dialysis patients In healthy volunteers and patients with chronic kidney disease (CKD) receiving peritoneal dialysis, C.E.R.A. has a prolonged and comparable half-life of approximately 130 h following IV and SC administration. This is considerably longer than the half-lives reported for epoetin (alfa and beta) and darbepoetin alfa. C.E.R.A’.s prolonged serum half-life suggests that it can be administered at extended intervals. 1. Halstenson et al. Clin Pharmacol Ther. 1991:50:702-712 2. Macdougall et al. J Am Soc Nephrol. 1999;10:2392-2395 3. Dougherty et al. ASCO 2004 4. Macdougall et al. ASN 2005 Halstenson CE et al. Comparative pharmacokinetics and pharmacodynamics of epoetin alfa and epoetin beta. Clin Pharmacol Ther. 1991;50:702-712. Macdougall IC et al. Pharmacokinetics of novel erythropoiesis stimulating protein compared with epoetin alfa in dialysis patients. J Am Soc Nephrol. 1999;10:2392-2395. Dougherty FC et al. C.E.R.A. (Continuous Erythropoiesis Receptor Activator): dose-response, pharmacokinetics and tolerability in phase I multiple ascending dose studies. J Clin Oncol 2004, 22:(Suppl 15)14S (abstract 6692). Macdougall IC et al. Pharmacologic profile of C.E.R.A. (Continuous Erythropoietin Receptor Activator) in chronic kidney disease (CKD) patients (pts) following intravenous (IV) and subcutaneous (SC) administration. Poster presented at 38th Annual Meeting of the American Society of Nephrology, November 8-13, 2005 (J Am Soc Nephrol. 2005;16:[abstract SA-PO926]).

Understanding how C.E.R.A. is different Receptor binding properties Pharmacokinetic properties CERA is different to other erythropoietic stimulating agents (ESAs), having different receptor binding properties. Different pharmacologic profile

Administration schedule C.E.R.A: Dose independent of schedule Core study, PP population, n=124 (BA16286) Mean (SE) change in Hb (g/dL) at 6 wk n.s. QW Q3W Q4W -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 Administration schedule Locatelli et al. Curr Med Res Opin 2007;23:969–979

Primary efficacy analysis C. E. R. A Primary efficacy analysis C.E.R.A. up to once-monthly as effective as epoetin TIW-QW Non-inferiority limit: C.E.R.A. groups vs epoetin 0.004 -0.215 0.223 C.E.R.A. Q2W ITT -0.213 0.031 0.276 0.025 0.270 -0.220 C.E.R.A. Q2W C.E.R.A. Q4W PP -0.173 0.275 0.051 C.E.R.A. Q4W eheanc11_1001 Table 20 page 84 CSR eheanc11_4001 Table 21 page 85 CSR For the non-inferiority test, the mean change in Hb between the baseline and evaluation periods was assessed for C.E.R.A. 1x/2 weeks and 1x/4 weeks and adjusted relative to the mean change in Hb between the baseline and evaluation periods for epoetin. The lower limits of 97.5% confidence interval for the mean difference in Hb between baseline and evaluation periods between the treatment groups for the C.E.R.A. 1x/2 weeks and 1x/4 weeks were both greater than ‑0.75 g/dL, indicating that both C.E.R.A. 1x/2 weeks and 1x/4 were clinically non-inferior to the epoetin reference group. -1.00 -0.75 -0.50 -0.25 0.00 0.25 0.50 0.75 1.00 Difference in adjusted group mean Hb (g/dL) change between baseline and evaluation (97.5% CI) P<0.0001 for all comparisons Levin NW et al. Lancet 2007, 370 (9596): 1415-1421 16

IV C.E.R.A. once- and twice-monthly maintains stable Hb over one year 4 8 12 16 20 24 28 32 36 40 44 48 52 C.E.R.A. Q2W IV C.E.R.A. Q4W IV Epoetin TIW-QW IV Mean (SD) Hb (g/dL) Months Weeks ehe11p_4170 Stable Hb levels continued to be maintained with C.E.R.A. throughout the year-long study. Results are shown for the ITT population. Reference Levin NW, Fishbane S, Zeig S, Nassar G, Moran J, Villa G, Dougherty FC. Intravenous (IV) C.E.R.A. (Continuous Erythropoietin Receptor Activator) administered once every 2 weeks or once monthly maintains haemoglobin (Hb) levels in patients with chronic kidney disease (CKD) on dialysis. Nephrol Dial Transplant. 2006;21(Suppl 4):iv11 (abstract SO023). Levin NW et al. Lancet 2007, 370 (9596): 1415-1421 17

SC C.E.R.A. once-monthly and twice-monthly as effective as epoetin 3x/wk PROTOS: primary efficacy analysis (PP population) Non-inferiority lower 97.5% CI limit 0.141 - 0.098 0.380 C.E.R.A. 1x/2wk - 0.262 0.217 - 0.022 C.E.R.A. 1x/4wk eheanc11_1001 Table 20 BA16740 section 3-3.2 efficacy CE 04-04-06 eheanc11_4001 Table 21 BA16740 section 3-3.2 efficacy CE 04-04-06 - 1.00 - 0.75 - 0.50 - 0.25 0.00 0.25 0.50 0.75 1.00 Difference in mean adjusted Hb versus epoetin (g/dL) P < 0.0001 for all comparisons Sulowicz, Locatelli Clin J Am Soc Nephrol. 2007 Jul;2(4):637-46

Stable Hb maintenance with once-monthly SC C. E. R. A Stable Hb maintenance with once-monthly SC C.E.R.A. PROTOS: ITT population Mean (SD) Hb (g/dL) C.E.R.A. 1x/2wk C.E.R.A. 1x/4wk Epoetin 1-3x/wk Conversion to IV C.E.R.A. once monthly maintains steady Hb levels ehe11p_4170 Months Weeks 4 8 12 16 20 24 28 32 36 40 44 48 52 Sulowicz, Locatelli Clin J Am Soc Nephrol. 2007 Jul;2(4):637-46

IV C.E.R.A. twice-monthly as effective as darbepoetin 1x/wk STRIATA: primary efficacy analysis (PP population) Non-inferiority lower 95.0% CI limit 0.180 -0.049 0.408 0.180 P < 0.0001 - 0.049 0.408 eheanc11_1001 eheanc11_4001 <<Slide to be animated>> - 1.00 - 0.75 - 0.50 - 0.25 0.00 0.25 0.50 0.75 1.00 Difference in mean adjusted Hb (g/dL) Canaud….Locatelli et al. ERA - EDTA 2006 Canaud….Locatelli et al. Advance Access Published Nephrol Dial Transplant. 2008 Jul 31

Stable Hb maintenance with twice-monthly IV C. E. R. A Stable Hb maintenance with twice-monthly IV C.E.R.A. STRIATA: ITT Population Mean (SD) Hb (g/dL) C.E.R.A. 1x/2wk Darbepoetin 1x/wk Conversion to IV C.E.R.A. once monthly maintains steady Hb levels ehe11p_4170 Months Weeks 4 8 12 16 20 24 28 32 36 40 44 48 52 Canaud….Locatelli et al. ERA - EDTA 2006 Canaud….Locatelli et al. Advance Access Published Nephrol Dial Transplant. 2008 Jul 31

SC C.E.R.A.: Smooth and steady Hb increase with a high response rate ARCTOS: ITT population Mean (SD) Hb (g/dL) Response rate (%) 95% CI C.E.R.A. 1x/2wk 97.5 93.8-99.3 Darbepoetin alfa 1x/wk 96.3 92.1-98.6 16 C.E.R.A. 1x/2wk Darbepoetin alfa 1x/wk 15 14 13 12 11 10 9 8 7 Months BL 1 2 3 4 5 6 Final visit Weeks 4 8 12 16 20 24 Macdougall, … Locatelli et al. Clin J Am Soc Nephrol. 2008 Mar;3(2):337-47

Hb concentrations during 24-week intervals Locatelli F et al. Kidney Int 2001; 60: 741-747 Macdougall, … Locatelli et al. Clin J Am Soc Nephrol. 2008 Mar;3(2):337-47 Mean (SD) Hb (g/dL) 16 15 C.E.R.A. 1x/2wk 14 Darbepoetin alfa 1x/wk 13 rHuEPO 12 11 10 9 8 7 Months BL 1 2 3 4 5 6 Final visit Weeks 4 8 12 16 20 24

Fewer patients exceed Hb 13 g/dL with C. E. R. A Fewer patients exceed Hb 13 g/dL with C.E.R.A. than with darbepoetin alfa ARCTOS: ITT population 40 P < 0.0001 30 Patients (%)* 20 10 C.E.R.A. 1x/2wk Darbepoetin alfa 1x/wk *Patients with ≥1 Hb value >13 g/dL during first 8 weeks Macdougall, … Locatelli et al. Clin J Am Soc Nephrol. 2008 Mar;3(2):337-47

Weeks since treatment interruption Decline in Hb after withholding treatment Long C.E.R.A. half-life does not affect Hb decline following dose interruption – pooled analysis of maintenance studies Mean (SD) Hb (g/dL) C.E.R.A. Q4W (n=59) 16 Epoetin (QW to TIW) or darbepoetin alfa (QW or Q2W) (n=126) 15 14 13 12 11 10 -3 -2 -1 1 2 3 4 5 6 Weeks since treatment interruption (time 0) Safety populations Locatelli F. et al Kidney Intern. S. 2008 : Heifets & Dougherty. WCN 2007

Half life comparison 140 120 SC IV 100 Hours 80 60 40 20 Methoxy polyethylene glycol-epoetin beta’s half-life is much longer than that of darbepoetin alfa 137 h vs 38 h when given SC 133 h vs 25 h when given IV 140 120 SC 100 IV 80 Hours 60 40 CERA’s half-life is much greater than that of darbepoetin alpha (Aranesp®): 137 hours compared to 38 hours when injected SC 133 hours compared to 25 hours when dosed IV 20 Epoetin Epoetin Darbepoetin Methoxy polyethlene glycol-epoetin beta alfa beta alfa Halstenson. Clin Pharmacol Ther. 1991;50:702 Macdougall. J Am Soc Nephrol. 1999;10:23925 Reigner. Nephrol Dial Transplant. 2003;18(suppl 4):167 Abstract M527

Half life of epoetins t (hours) Intravenous Subcutaneous 6.8* 19.4* *Healthy volunteers †Peritoneal dialysis patients t ½ (hours) Intravenous Subcutaneous Epoetin alfa 6.8* 19.4* Epoetin beta 8.8* 24.2* Darbepoetin alfa † 25.3 48.8 3:1 2:1 Methoxy polyethylene glycol-epoetin beta 130 133 1:1 Hematide 75 ~80 Halstenson. Clin Pharmacol Ther. 1991;50:702 Macdougall. J Am Soc Nephrol. 1999;10:2392 Reigner. Nephrol Dial Transplant. 2003;18(suppl 4):167. Abstract M527 Woodburn. Blood. 2004;104:2904

Ht response to EPO or placebo in PD patients Nissenson AR et al. J Am Soc Nephrol 5:1517-1529, 1995

Cosa può influenzare la scarsa risposta agli ESAs in DP? * ERI: EPO resistance index Wei M, et al. Int Urol Nephrol. 2007;39(3):935-40.

ESAs in dialisi peritoneale Sono stati condotti pochi trials sull'uso degli ESA's nei pazienti in dialisi peritoneale La via di somministrazione di ESAs in dialisi peritoneale deve, per ragioni pratiche, essere SC Le dosi di ESAs necessarie per mantenere livelli di Hb nel range suggerito dalle linee guida in dialisi peritoneale sono ridotte rispetto ai pazienti in emodialisi

Open-label study description* Treatment + evaluation period Once monthly (QM) dosing of darbepoetin alfa was effective in CKD patients not on dialysis Reference Open-label study description* Treatment + evaluation period Hb study target range Primary endpoint Ling1 n=98 Q2W DA → QM DA 29 weeks 10-12 g/dl % of patients within Hb study target range Agarwal2 n=152 33 weeks 11-13 g/dl % of patients with Hb ≥11 g/dl Disney3 n=66 10-13 g/dl Maintaining mean Hb ≥10 g/dl Sousa4 n=71 18 months Effectiveness and safety of DA QM Hoggard5 n=442 QW/Q2W rHuEPO → QM DA 28 weeks % of patients converting from EA QW who preferred DA QM at week 21 Ling B, Walczyk M, Agarwal A, Carroll W, Liu W, Brenner R. Darbepoetin alfa administered once monthly maintains hemoglobin concentrations in patients with chronic kidney disease. Clin Nephrol 2005;63:327-34. Agarwal AK, Silver MR, Reed JE, et al. An open-label study of darbepoetin alfa administered once monthly for the maintenance of haemoglobin concentrations in patients with chronic kidney disease not receiving dialysis. J Intern Med 2006;260:577-85. Disney A, Jersey PD, Kirkland G, et al. Darbepoetin alfa administered monthly maintains haemoglobin concentrations in patients with chronic kidney disease not receiving dialysis: a multicentre, open-label, Australian study. Nephrology (Carlton) 2007;12:95-101. Sousa American Society of Nephrology 2006; Abstract SA-PO218. Hoggard J, Crouch T, McMurray S, et al. Preference for monthly darbepoetin alfa dosing in patients with chronic kidney disease not receiving dialysis. Curr Med Res Opin 2006;22:2023-30. 1Ling B et al. Clin Nephrol 2005;63:327-34; 2Agarwal AK et al. J Intern Med 2006;260:577-85; 3Disney A et al. Nephrology (Carlton) 2007;12:95-101; 4Sousa American Society of Nephrology 2006; Abstract SA-PO218; 5Hoggard J et al. Curr Med Res Opin 2006;22:2023-30.

Limited evidence with once-monthly dosing Drug Administration interval (route of administration) Target Hb (g/dL) Population (no. patients enrolled) Trial design Reference Epoetin alfa QW, Q2W Q3W, Q4W (SC) ≥11 CKD not on dialysis (n=519) Randomised Provenzano et al 2005 Darbepoetin alfa Q4W (SC) 10-12 CKD not on dialysis (n=97) Single arm, non-randomised Ling et al 2005 ≥10 CKD not on dialysis (n=66) Disney et al 2007 CKD not on dialysis (n=152) Agarwal et al 2006 Q3W, Q4Wa (IV and SC) 10-13 Dialysis patients (n=54) Jadoul et al 2004 ESAs, erythropoiesis-stimulating agents; Hb, haemoglobin; IV, intravenous; SC, subcutaneous aDosing once a month is not indicated for darbepoetin alfa in dialysis, except in Switzerland 32

Limited evidence of efficacy in HD patients for once-monthly darbepoetin alfaa Hb (g/dL) Dose (g/wk) Patients on darbepoetin alfa Q2W converted to Q3W dosing and, if Hb stable (10-13 g/dL), to Q4W dosinga 522 patients originally recruited Limited conversion to Q4W dosinga: Of 54 patients entering the study, 36 patients were converted to Q4W dosing Limited maintenance on Q4W dosinga: Of 36 patients converting to Q4W dosing, 30 patients maintained Hb >10 g/dL over 20 weeks Hb 13.0 100 Dose 12.5 Q3W dosing (n=44) Q4W dosinga (n=30) 80 12.0 11.5 60 11.0 40 10.5 10.0 20 9.5 9.0 –2 4 8 12 16 20 26 30 34 38 42 Study week aDosing once a month is not indicated for darbepoetin alfa in dialysis, except in Switzerland Jadoul et al. Nephrol Dial Transplant 2004;19:898-903

Labelled dosing information on maintenance therapy dosing interval MIRCERA Once-monthly maintenance dosing in CKD patients on dialysis and not on dialysis Darbepoetin alfa In HD patients Once weekly or once every 2 weeks maintenance dosing In Switzerland in selected HD patients, also once monthly In CKD patients not on dialysis Stepwise expansion from QW over Q2W to QM for those patients stable on previous dose interval and by doubling the dose CKD, chronic kidney disease; HD, haemodialysis; QW, weekly; Q2W, every 2 weeks; QM, once monthly ARANESP, Summary of Product Characteristics, 2006; MIRCERA, Summary of Product Characteristics, 2007

The PATRONUS study: a randomised comparison of the efficacy and safety of MIRCERA® with darbepoetin alfa using once-monthly dosing in haemodialysis patients 35

PATRONUS study objectives Primary To compare the efficacy of once-monthly IV MIRCERA® with that of darbepoetin alfa* in the maintenance of Hb levels in HD patients with chronic renal anaemia previously receiving IV darbepoetin alfa maintenance therapy Secondary To assess the safety and tolerability of IV MIRCERA® in HD patients with chronic renal anaemia previously receiving IV darbepoetin alfa maintenance treatment * Darbepoetin alfa is not licensed for once monthly treatment in dialysis in the UK. 36

PATRONUS compared once-monthly MIRCERA® and darbepoetin alfa maintenance treatment Evaluation (wk 50-53) Screening period MIRCERA® 1x/month (n=245) MIRCERA® 1x/month Primary end point Darbepoetin alfa 1x/week R Darbepoetin alfa 1x/2 weeks (n=245) Darbepoetin alfa* 1x/month 4 weeks 26 weeks 26 weeks R, randomisation * Darbepoetin alfa is not licensed for once monthly treatment in dialysis in the UK.

PATRONUS primary end point Primary end point: superiority assessment of the difference in the proportion of responders between groups Responders are patients with an average Hb decrease from baseline of <1.0 g/dL and an average Hb >10.5 g/dL during evaluation Study was powered to detect an absolute difference of 15% in the primary end point between the 2 treatment groups Assumed 60% of patients would respond to MIRCERA® vs 45% to darbepoetin alfa Target Hb range: 11–13 g/dL * Darbepoetin alfa is not licensed for once monthly treatment in dialysis in the UK.

Analysis population Primary efficacy analysis used the intent-to-treat population, defined as all randomised patients In case of withdrawals, the last Hb value in the second treatment period was used for the Hb assessment (last value carried forward) To correct for any increase in Hb caused by RBC transfusion, the Hb values measured within 3 weeks after a transfusion were replaced by the Hb value measured immediately before the transfusion Safety population included all patients who received >1 dose of MIRCERA® or darbepoetin alfa RBC, red blood cell * Darbepoetin alfa is not licensed for once monthly treatment in dialysis in the UK.

Darbepoetin alfa to MIRCERA® dose conversion schedule Weekly darbepoetin alfa dose (g/week) MIRCERA® starting dose (g/month) <40 120 40–80 200 >80 360 * Darbepoetin alfa is not licensed for once monthly treatment in dialysis in the UK. 40

Darbepoetin alfa starting doses Two changes needed: Double Week – 1 dose Double Week 25 dose Week 1 Week 27 4 week baseline period 26 weeks 26 weeks * Darbepoetin alfa is not licensed for once monthly treatment in dialysis in the UK.

PATRONUS: A multicentre, multinational trial Country Patients enrolled France 95 Italy 86 Spain 80 Canada 65 Germany 33 Belgium 31 UK 22 Portugal 21 Australia 15 Austria Switzerland 11 Finland 9 Denmark 7 Total 490 * Darbepoetin alfa is not licensed for once monthly treatment in dialysis in the UK.

Baseline patient characteristics MIRCERA® n=245 Darbepoetin alfa Male, n (%) 148 (60) 156 (64) Race, n (%) Caucasian 233 (95) 225 (92) Black 5 (2) 12 (5) Other 7 (3) 8 (3) Mean age, years (SD) 66.2 (13.6) 65.5 (13.9) Mean weight, kg (SD) 72.3 (15.1) 73.8 (16.9) Mean baseline Hb, g/dL (SD) 12.09 (0.56) 12.07 (0.55) Mean time since first dialysis, years (SD) 4.20 (5.92) 4.15 (5.55) Dosing Median darbepoetin alfa dose at week before randomisation, g (IQR) 30.0 (20-40) 20.0 (15-40) Median study drug dose in month 1, g/month (IQR) 120.0 (120-200) 100 (60-160) SD = standard deviation; IQR = Interquartile Range

Once-monthly MIRCERA® exhibited a superior response rate compared with once-monthly darbepoetin alfa* Primary end point ® * *P<0.0001 64.1% 64.1% Response rate (%) 40.4% 40.4% CI, confidence interval * Darbepoetin alfa is not licensed for once monthly treatment in dialysis in the UK. 44

Superiority limit: MIRCERA® vs darbepoetin alfa Once-monthly MIRCERA® shows a 59% higher likelihood of response compared with once-monthly darbepoetin alfa* Primary end point Superiority limit: MIRCERA® vs darbepoetin alfa 1.59* 1.33 1.90 −0.75 1.00 1.25 1.50 1.75 2.0 Relative risk of response at evaluation for MIRCERA® vs darbepoetin alfa *P<0.0001 * Darbepoetin alfa is not licensed for once monthly treatment in dialysis in the UK. 45

Weeks (of trial treatment) Only once-monthly MIRCERA® maintained Hb levels during the second 26-week treatment period Evaluation (wk 50-53) 16.0 MIRCERA® (median, IQR) Darbepoetin alfa (median, IQR) 15.0 14.0 13.0 12.0 Median Hb value (g/dL) 11.0 10.0 9.0 8.0 7.0 Baseline 4 8 12 16 20 24 28 32 36 40 44 48 52 Weeks (of trial treatment)

Darbepoetin alfa dose increased by >30% during the second 26-week treatment period Secondary end point Median (IQR) treatment dose, g/month MIRCERA® n=211 Darbepoetin alfa n=219 Week 27 200 (120-313) 150 (80-280) Months 11 and 12 196 (120-351) 225 (106-400) 0% +35% The median MIRCERA® dose was virtually unchanged during the second 26-week treatment period whereas darbepoetin alfa substantially increased by 35% * Darbepoetin alfa is not licensed for once monthly treatment in dialysis in the UK. 47

MIRCERA® dose was unchanged during the second 26-week treatment period 150 MIRCERA (median, IQR) Darbepoetin alfa (median, IQR) Secondary end point 125 100 75 Trial treatment dose change (%) 50 25 −25 −50 7 8 9 10 11 12 Months (of trial treatment) * Darbepoetin alfa is not licensed for once monthly treatment in dialysis in the UK.

Most safety parameters were similar between the two PATRONUS study groups MIRCERA®, n (%) n=245 Darbepoetin alfa, n (%) n=244 AEs 222 (90.6) 217 (88.9) SAEs 99 (40.4) 94 (38.5) AEs leading to withdrawals 3 (1.2) 7 (2.9) Withdrawals 58 (23.7) 96 (39.3) Withdrawals due to insufficient response 10 (4.1) 48 (19.7) Deaths* 14 (5.7) *includes 3 patients who died after withdrawal due to other events AEs, adverse event; SAEs, serious adverse events Similar incidence of AEs in both study arms higher rate of constipation with MIRCERA® Most common AEs in both arms were hypertension (14.7 vs 10.7%), procedural hypotension (8.6 vs 11.1%) and nasopharyngitis (10.2 vs 8.2%) Fewer withdrawals with MIRCERA® than with darbepoetin alfa * Darbepoetin alfa is not licensed for once monthly treatment in dialysis in the UK. 49

SAEs (>5% in either group) were comparable between study groups in PATRONUS MIRCERA® n=245 n (%) Darbepoetin alfa n=244 n (%) All body systems 99 (40.4) 94 (38.5) Infections and infestations 29 (11.8) 27 (11.1) Injury, poisoning and procedural complications 26 (10.6) 18 (7.4) Cardiac 16 (6.5) 16 (6.6) Vascular 14 (5.7) Gastrointestinal 11 (4.5) Nervous system 7 (2.9) 13 (5.3) Neoplasms 10 (4.1) 5 (2.0) Metabolism and nutrition 4 (1.6) Respiratory, thoracic and mediastinal * Darbepoetin alfa is not licensed for once monthly treatment in dialysis in the UK. 50

Clinical Nephrology, Vol 73 – n 2/2010 (94-103)

Conclusions MIRCERA® was shown to be superior to darbepoetin alfa as once-monthly treatment in the dialysis setting Significantly more patients responded with MIRCERA® compared with darbepoetin alfa MIRCERA® maintained Hb levels within a tight target range during the second 26-week treatment period Mean Hb levels for patients receiving darbepoetin alfa fell to below the lower target (11 g/dL) over the same 26-week period, despite substantial dose increases This is the first large, randomised, prospective head-to-head study that has shown one ESA to offer superior efficacy compared with another Carrera F et al. WCN 2009 Milano, poster M558 * Darbepoetin alfa is not licensed for once monthly treatment in dialysis in the UK.

Hb (or at least high levels) vs ESA (or at least high levels)