Protocol. This trial protocol has been provided by the authors to give readers additional information about their work.

Transcription

1 Protocol This trial protocol has been provided by the authors to give readers additional information about their work. Protocol for: Solomon SD, Uno H, Lewis EF, et al. Erythropoietic response and outcomes in kidney disease and type 2 diabetes. N Engl J Med 2010;363:

2 Date: 17 April 2008 Page 1 of 96 Title: Trial to Reduce Cardiovascular Events with Aranesp Therapy TREAT Amgen Protocol Number (Darbepoetin alfa) Trial Sponsor: Amgen Inc. One Amgen Center Drive Thousand Oaks, CA , USA Date: Superceding Version: Superceding Version 2: Amendment 1: Superceding Version: Amendment 2: Amendment 3: 2 February February March May June May April 2008

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4 Date: 17 April 2008 Page 3 of 96 Protocol Synopsis Title Trial to Reduce Cardiovascular Events with Aranesp Therapy (TREAT) Study Phase 3 Indication Treatment of anemia in subjects with chronic kidney disease (CKD). Primary Objective Secondary Objective(s) Study Design To assess the effect of anemia therapy with darbepoetin alfa on the composite event comprising all-cause mortality and cardiovascular (CV) events (including myocardial ischemia, congestive heart failure [CHF], myocardial infarction [MI], and cerebrovascular accident [CVA]) in subjects with both CKD and type 2 diabetes mellitus (DM). To assess the effect of darbepoetin alfa on the time to end-stage renal disease (ESRD). To assess the effect of anemia therapy with darbepoetin alfa on CV mortality and the individual components of the composite event: all-cause mortality and CV events. To assess the effect of darbepoetin alfa on the change in the rate of decline in glomerular filtration rate (egfr) and patient-reported fatigue. This is a multicenter (United States, Australia, Canada Latin America, and Europe), randomized, double-blind, controlled study. Study Population: Subjects with hemoglobin (Hb) 11.0 g/dl, CKD, egfr 20 ml/min and 60 ml/min, and type 2 DM. Duration: The study will conclude when approximately 1203 patients have experienced a primary event. The Data and Safety Monitoring Committee will monitor the overall event rate, and Amgen and the Executive Committee will set the study termination date based on the anticipated date of occurrence of 1203 primary endpoints. Given an assumed placebo event rate of 12.5% and an anticipated treatment effect of 20%, approximately 4000 subjects will be enrolled. It is anticipated that the enrollment period will be approximately 2 years, and subjects will be followed for approximately 4.5 years from the date that the first subject is randomized. Eligible subjects will be randomized in a 1:1 ratio to either Group A or Group B. Group A (treatment): subjects will receive darbepoetin alfa to achieve and maintain a target Hb of 13.0 g/dl. Group B (control): subjects will receive placebo if the

5 Date: 17 April 2008 Page 4 of 96 Hb is 9.0 g/dl. If the Hb is < 9.0 g/dl, subjects in this treatment group will receive rescue therapy with darbepoetin alfa until their Hb is 9.0 g/dl. Primary Endpoint and Secondary Endpoints Sample Size Primary Endpoint: Time to the composite event comprising all-cause mortality and CV events including: Myocardial ischemia CHF MI CVA Key Secondary Endpoints Time to ESRD or all-cause mortality Other Secondary Endpoints Time to all-cause mortality Time to CV mortality Time to myocardial ischemia Time to MI Time to CVA Time to CHF Time to ESRD Rate of decline in egfr relative to baseline Change in patient reported fatigue relative to baseline at week 25 Other Endpoints of Interest Time to cardiac revascularization Change in urine protein/urine creatinine ratio relative to baseline Health resource utilization (HRU) per patient follow-up time Change in patient reported outcomes (PRO), other than fatigue, relative to baseline at week 25 Change in physical and mental composite scores of the SF-36 from baseline Change from baseline in distance walked in six minutes Safety Endpoints Adverse events Changes in laboratory parameters and blood pressure Anti-erythropoietic protein seroreactivity Approximately 4000 subjects

6 Date: 17 April 2008 Page 5 of 96 Summary of Subject Eligibility Criteria Investigational Product Dosage and Administration Control Group Route of Administration Procedures Demographics 18 years old Signed, written informed consent to participate in this study Disease Condition Subjects must have: Hb 11.0 g/dl CKD egfr 20 ml/min and 60 ml/min Type 2 DM Mean transferrin saturation 15% Medical History Subjects must not have: A prior kidney transplant or be anticipating or scheduled for a living related-donor kidney transplant Uncontrolled hypertension Erythropoietic protein (eg, rhuepo; Procrit, Eprex, Neorecormon, Epogen, Aranesp ) use within the 12 weeks prior to randomization History of CV events within 12 weeks prior to randomization including: o Myocardial ischemia o Hospitalization for CHF o MI o CVA Darbepoetin alfa will be presented as a clear, colorless protein solution in pre-filled syringes (PFS) with the following concentrations: 10, 15, 20, 30, 40, 50, 60, 80, 100, 150, 200, and 300 µg. Placebo will be presented as a clear, colorless solution in PFS identical in appearance to those containing darbepoetin alfa. Group B Subcutaneous (SC) Injection Screening All screening evaluations used to establish subject eligibility will be performed following signing of the informed consent and up to 2 weeks prior to randomization. Screening evaluations will include the following: Medical history Physical examination Blood pressure

7 Date: 17 April 2008 Page 6 of 96 Blood samples for hematology, serum chemistry, and iron stores egfr measured using the Modification of Diet in Renal Disease (MDRD) equation Spot urine protein/urine creatinine ratio Randomization Subjects successfully completing screening evaluations, will be randomized in a 1:1 ratio to either Group A or Group B. Randomization will be stratified by center, level of baseline proteinuria (spot urine protein/urine creatinine ratio) and history of CV disease. Study Assessments The following assessments will be completed during the course of the trial. All assessments will be completed prior to dosing at the specified timepoints. Blood draw for anti-erythropoietic protein seroreactivity prior to the first dose of investigational product 12-lead electrocardiogram on week 1, and annually thereafter Blood pressure and heart rate on week 1, and every 2 weeks thereafter during correction, and monthly during maintenance Hb on week 1, and every 2 weeks thereafter during correction, and monthly during maintenance Blood draw for serum ferritin and transferrin saturation [Tsat] on week 1, and every 12 weeks thereafter. Blood draw for HbA 1C, serum chemistry (BUN, creatinine, albumin, potassium, C-reactive protein [CRP]), and a lipid panel (cholesterol, triglycerides, HDL, and LDL) on week 1, and every 24 weeks thereafter egfr determination using the MDRD equation on week 1, and every 24 weeks thereafter NYHA Classification determination at weeks 13, 25, 49 and annually thereafter Physical examination, including measurements of waist and hip circumference on week 49 and annually thereafter Spot urine protein/urine creatinine ratio on week 1, and every 24 weeks thereafter Completion of the FACT-Fatigue, EQ-5D and HRU questionnaire on weeks 1, 13, 25, and every 24 weeks thereafter Completion of the Short Form (SF)-36

8 Date: 17 April 2008 Page 7 of 96 questionnaire on weeks 1, 25, 49, and 97 Six-minute walk test on weeks 1, 25, 49, and 97 in a subset of 1000 subjects. Details of subject hospitalization, including diagnosis and length of stay, will be collected at every study visit Details of concomitant medications including: anticoagulants, analgesics/antipyretics (eg, aspirin, Plavix, non-steroidal anti-inflammatory agents), antidepressants, iron therapy, cardiac drugs (e.g. dyslipidemic agents, antiarrythmics), diuretics, antidiabetic agents, oral corticosteroid therapy, hormone replacement therapy (HRT), vitamin B 12, and folate will be collected at every study visit Adverse events will be documented by the investigator along with his/her assessment of severity and relationship to investigational product. Adverse events will be collected from the day the subject receives the first dose of investigational product through one week (7 days) following the last dose of investigational product. Adverse events resulting in death will be collected during the entire study or until 30 days after the last dose of investigational product, whichever is later. Safety data will be monitored throughout the study and periodically reviewed by an independent Data and Safety Monitoring Committee (DSMC). The following End of Study assessments will be performed at study completion, or at the time of withdrawal (subject terminates early from the study): Physical examination, including weight and measurements of waist and hip circumference Blood pressure, temperature and heart rate Blood samples for hematology, serum chemistry, Tsat, ferritin, lipid panel, HbA 1c, antierythropoietic protein seroreactivity Spot urine protein/urine creatinine ratio egfr determination Completion of the PRO and HRU questionnaires Subjects who no longer receive investigational product for any reason, including those who initiate renal replacement therapy (RRT) (eg, dialysis, renal transplantation) during the course of the study will continue to be followed monthly for the duration of the study. If administration of investigational product is discontinued, a blood sample for anti-erythropoietic protein seroreactivity should be obtained (for subjects initiating RRT, it should be obtained before RRT is

9 Date: 17 April 2008 Page 8 of 96 initiated). Adverse events (other than death) will not be collected after one week (7 days) following the last administration of investigational product. All other procedures, including the collection of endpoints, are expected to be performed as detailed in Sections 7.2 and 7.3 of the protocol. Subjects who have had the administration of investigational product discontinued for any reason, including those who initiate RRT, and do not consent to have all study procedures performed, will consent to having endpoint data collected until completion of the trial. These subjects will remain on study and have this information collected either through a review of their medical records or via telephone contact. Dose and Schedule: The starting dose of investigational product will be 0.75 µg/kg administered once every 2 weeks (Q2W). The dose obtained from this calculation will be rounded to the nearest PFS. Dosing will be provided by contact with an Interactive Voice Response System (IVRS). All subjects randomized to receive darbepoetin alfa will receive investigational product SC Q2W, titrated to achieve the target Hb of 13.0 g/dl. Dose increases in darbepoetin alfa will not occur more frequently than once monthly (QM). After a subject achieves the target Hb, defined as two consecutive Hb levels between g/dl with no change in dose, the frequency of administration will be extended to QM. The initial QM dose of investigational product will be equal to two times the previous Q2W dose. The QM dose of investigational product will be titrated as necessary to maintain the target Hb. Subjects on QM dosing who are receiving the maximum allowable dose (600 µg QM) and have three consecutive Hb values below 11.0 g/dl will have their frequency of administration increased to Q2W. The Q2W dose of investigational product will be equal to 50% of their initial QM dose. To prevent unblinding, Group B subjects will have dose and schedule changes mirroring those that occur for Group A, via a dynamic dose change schedule that imitates Group A dose changes. An IVRS algorithm ensuring this balance will be implemented prior to study start. If the Hb declines below 9.0 g/dl at any time for Group B subjects, therapy with darbepoetin alfa will be instituted with a single dose of 0.45 µg/kg rounded to the nearest PFS. The Hb level will be reassessed at the next monthly visit. If the Hb remains below 9.0 g/dl an additional dose of darbepoetin alfa will be administered, titrated according to Hb rate of rise. Dose increases in

10 Date: 17 April 2008 Page 9 of 96 rescue therapy with darbepoetin alfa will not occur more frequently than QM. This process will continue indefinitely until the Hb is 9.0 g/dl at which time subjects will resume placebo injections. Screening Hb values, which will be used to confirm subject eligibility, will be measured at the study site using venous blood with a hemoglobin point of care device provided by Amgen. The screening Hb values will also be measured by the central laboratory. Hb values from Week 1 through the completion of the study will be measured at the study site using venous blood with a hemoglobin point of care device provided by Amgen. Hb values to determine investigational product dosing must be obtained within 1 week before or on the same day as the investigational product administration visit. These Hb values should be obtained at the site by a third party not directly involved in the study subject s care to preserve blinding of Hb values to study personnel directly involved in subject care. Hb values will be entered into the IVRS, which will calculate and manage the initial doses and all subsequent dose adjustments of investigational product. Statistical Considerations Primary Endpoint Analysis The principal analysis for the primary endpoint employs the intent-to-treat principle and uses a life-table analysis. For each treatment group, Kaplan-Meier curves will be estimated, graphically displayed, and compared using a two-sided log-rank test (stratified by level of baseline proteinuria [spot urine protein/urine creatinine ratio], and history of cardiovascular disease). The familywise type 1 error is 0.05, which will be divided between the analyses of the primary endpoint and the key secondary endpoint. There will be four interim and one final analysis for the primary endpoint, with an overall alpha controlled at Subjects who discontinue investigational product early (for reasons other than withdrawn consent) will be followed for the potential development of the primary endpoint, and will be censored if this does not occur. Subjects completing the study and not experiencing the composite event will be censored. Subjects progressing to RRT will be included in the analysis. Secondary Endpoint Analysis There will be one final analysis for the key secondary endpoint, with the alpha at This will be performed at the time of and be similar to the final analysis of the primary endpoint.

11 Date: 17 April 2008 Page 10 of 96 The other secondary endpoints will be tested using a Hochberg procedure for controlling the type I error rate at a significance level to be based on the outcome of the analyses for the primary and key secondary endpoint. Analysis methods for the other time-to-event secondary endpoints will be similar to that used for the primary endpoint. Analyses for the continuous secondary endpoints will be based on linear models. Safety Analyses The subject incidence of adverse events will be tabulated by system organ class and preferred term for each group. Adverse events considered related to investigational product, including serious adverse events, will be summarized. In addition, the exposureadjusted event rate for each adverse event will be calculated as the number of events per subject-year at risk. Changes in laboratory parameters and blood pressure will be summarized. The proportion of subjects with confirmed antibodies will be tabulated. Interim Analysis An external, unblinded, independent statistical analysis center will perform safety analyses in support of the Data Safety Monitoring Committee (DSMC) and the interim efficacy analyses. The interim results will not be shared with the sponsor until the sponsor is unblinded. The four planned efficacy interim analyses will occur when approximately 20%, 40%, 60%, and 80% of the cumulative composite events have been observed. An O Brien-Fleming alpha-spending function (with truncated symmetric monitoring boundaries) will be used to allocate the interim and final significance levels for the primary hypothesis. Executive Committee An executive committee will provide oversight and advice to ensure efficient conduct and execution of the trial. DSMC An independent committee consisting of members with relevant expertise will be assembled prior to study commencement. Safety data and enrollment trends will be regularly reviewed. The DSMC will participate in the conduct of the planned efficacy interim analyses. The DSMC will advise the executive committee and study sponsor on findings that may impact the conduct of the study, and communicate recommendations based on the results of the efficacy interim analysis.

12 Date: 17 April 2008 Page 11 of 96 Sponsor/Licensee Endpoint Adjudication Committee An external endpoint adjudication committee will be formed prior to study commencement to adjudicate the components of the primary endpoint and ESRD in a blinded fashion. Amgen Inc.

13 Date: 17 April 2008 Page 12 of 96 Study Design and Treatment Schedule Group A N 2000 (Hemoglobin Target 13 g/dl) 1:1 Randomization ~4000 subjects Group B N 2000 (Control) Screening Year 1 Year 2 Year 3 Year 4 Study Start Enrollment ~2 years Treatment ~2.5 years Study End (1203 primary events; approximately year 4.5)

14 Date: 17 April 2008 Page 13 of 96 Study Glossary Abbreviation/Acronym/Term ACE AUC BP BUN CABG CHF CI CFR CKD CRF CRO CRP CT CV CVA dl DM DSMC ECG egfr End of Study EPO; rhuepo ESRD EU FACT FDA g GCP Hb HbA 1C HCG HDL Hematology HRT HRU Definition Angiotensin converting enzyme Area under the curve Blood pressure Blood urea nitrogen Coronary artery bypass graft Congestive heart failure Confidence interval Code of Federal Regulations Chronic kidney disease Case report form; chronic renal failure Contract research organization C-reactive protein Computed tomography Cardiovascular Cerebrovascular accident Deciliter Diabetes mellitus Data and safety monitoring committee Electrocardiogram Estimated glomerular filtration rate Occurrence of 1203 primary events Erythropoietin; recombinant human erythropoietin End stage renal disease Europe Functional assessment of chronic illness therapy Food and Drug Administration Gram Good clinical practice Hemoglobin Hemoglobin A 1C Human chorionic gonadotropin High-density lipoprotein PLT, WBC, and reticulocytes Hormone replacement therapy Health resource utilization

15 Date: 17 April 2008 Page 14 of 96 ICH GCP IEC/IRB ITT IVRS IND NKF K/DOQI NSAID Kg LDL MDRD MI Monthly MRI μg PCI PIN PRO PFS PLT PTCA Q2W QM Registration Randomization RBC RRT Screen Failures Screening SC SCr SF-36 Study Day 1 Subject ID Number International Conference on Harmonisation Tripartite Guideline on Good Clinical Practice Independent Ethics Committee/Institutional Review Board Intent-to-treat Interactive voice response system Investigational New Drug Application National Kidney Foundation Kidney Disease Outcomes Quality Initiative Non-steroidal anti-inflammatory Kilogram Low-density lipoprotein Modification of Diet in Renal Disease Myocardial infarction Every 4 weeks Magnetic resonance imaging Microgram Percutaneous coronary intervention Personal identification number Patient reported outcomes (EQ-5D, FACT-fatigue, and SF-36) Pre-filled syringes Platelets Percutaneous coronary angioplasty Every 2 weeks Once monthly Assignment of an identification number by IVRS Assignment of treatment by IVRS Red blood cell Renal replacement therapy Subjects who are ineligible for the study based on any of the screening assessments Study period between signing informed consent and randomization (2 weeks = Weeks 2 and 1) Subcutaneous injection Serum creatinine Short form 36 health survey questionnaire Defined as the first day investigational product is administered to the subject A unique 9-digit identification number. The first 2 digits will be the protocol identifier; the next 4 digits will represent the site; the last 3 digits will identify the subject

16 Date: 17 April 2008 Page 15 of 96 TREAT Tsat UK ULN USRDS WBC Withdrawal Trial to Reduce Cardiovascular Events with Aranesp Therapy Transferrin saturation United Kingdom Upper limit of normal United States Renal Data System White blood cell Subjects may withdraw consent for either treatment with investigational product only or they may withdraw consent for further participation in all activities associated with the study. Subjects who withdraw from treatment with investigational product will continue to be followed monthly per the schedule of procedures for the collection of study data, including the occurrence of composite events.

17 Date: 17 April 2008 Page 16 of 96 Table of Contents Page PROTOCOL SYNOPSIS... 3 STUDY DESIGN AND TREATMENT SCHEDULE STUDY GLOSSARY OBJECTIVES Primary Secondary BACKGROUND AND RATIONALE Disease Darbepoetin alfa Rationale Clinical Hypothesis EXPERIMENTAL PLAN Study Design Number of Centers Number of Subjects Estimated Study Duration SUBJECT ELIGIBILITY Prescreening Screening Inclusion Criteria Exclusion Criteria SUBJECT ENROLLMENT Subject Registration Screen Failures Randomization and Treatment Assignment TREATMENT PROCEDURES Investigational Product Dosage, Administration, and Schedule Dosage Adjustments Monthly Dosing Subjects Who Do Not Maintain Target Hb on QM Dosing Rescue Therapy Concomitant Therapy Proscribed Therapy During Study Period STUDY PROCEDURES Screening (Weeks 2 and 1)... 39

18 Date: 17 April 2008 Page 17 of Treatment Period (Week 1 to Approximately Week 235) End of Study (When 1203 Primary Events are Observed or Approximately Week 235 [+ 2 weeks]) Subjects Who Are No Longer Receiving Treatment With Investigational Product, Including Subjects Requiring Renal Replacement Therapy (RRT) Six-Minute Walk Testing Third Party Responsibilities Access to Hb data Endpoint Determination Definition of Endpoints Data Collection REMOVAL AND REPLACEMENT OF SUBJECTS Removal of Subjects ADVERSE EVENT REPORTING Definitions Adverse Events Serious Adverse Events Adverse Event Profile of Darbepoetin alfa Reporting Procedures for All Adverse Events Serious Adverse Event Reporting Procedures Data and Safety Monitoring Committee (DSMC) STATISTICAL CONSIDERATIONS Study Design Health Economic Considerations Subjects Sets, Study Endpoints, and Analyses Primary Endpoint Analysis Secondary Endpoint Analyses Analyses of Other Endpoints of Interest Analyses of Functional Capacity Sub-study Endpoints Sample Size Considerations Sample Size Consideration for the Primary Endpoint Sample Size Consideration for Functional Capacity Sub-study Access to Individual Subject Treatment Assignments Interim Analysis and Early Stopping Guidelines Planned Methods of Analysis General Approach/Considerations Analysis of Efficacy Endpoints Additional Analyses Safety Analyses Study Oversight... 58

19 Date: 17 April 2008 Page 18 of Data and Safety Monitoring Committee (DSMC) Executive Committee Endpoint Adjudication Committee INVESTIGATIONAL PRODUCT Darbepoetin alfa Access to Blinded Investigational Product Assignment REGULATORY OBLIGATIONS Informed Consent Independent Ethics Committee/Institutional Review Board Prestudy Documentation Requirements Subject Confidentiality Investigator Signatory Obligations ADMINISTRATIVE AND LEGAL OBLIGATIONS Protocol Amendments and Study Termination Study Documentation and Storage Study Monitoring and Data Collection Language Publication Policy Compensation REFERENCES APPENDICES Appendix A. Schedule of Assessments Appendix B. Adverse Event Assessments Appendix C. Sample Serious Adverse Event Form Appendix D. Pharmacy Guide Appendix E. Pregnancy Notification Worksheet Appendix F. Sample Addendum to Informed Consent Appendix G. Proposed Endpoint Definitions.81 Appendix H. Iron Treatment Algorithm 84 Appendix J. Health Economic Questionnaire Appendix K. Prescreening Hemoglobin Consent Template 95

20 Date: 17 April 2008 Page 19 of OBJECTIVES 1.1 Primary To assess the effect of anemia therapy with darbepoetin alfa (Aranesp ) on the composite event comprising all-cause mortality and cardiovascular (CV) events (including myocardial ischemia, congestive heart failure [CHF], myocardial infarction [MI], and cerebrovascular accident [CVA]) in subjects with both chronic kidney disease (CKD) and Type 2 diabetes mellitus (DM). 1.2 Secondary To assess the effect of anemia therapy with darbepoetin alfa on CV mortality and the individual components of the composite event: all-cause mortality, and CV events. To assess the effect of darbepoetin alfa on the time to end-stage renal disease (ESRD), the change in the rate of decline in glomerular filtration rate (egfr), and patient reported fatigue. 2. BACKGROUND AND RATIONALE 2.1 Disease Analyses from The Third National Health and Nutrition Examination Survey (NHANES III) indicate that CKD is a common and important public health concern. 1 Approximately 4.7% of the United States population (8.3 million individuals) have moderate to severely decreased kidney function, defined as an estimated glomerular filtration rate (egfr) <60 ml/min/1.73 m 2 and approximately 20 million have kidney disease, as evidenced by proteinuria or egfr < 90 ml/min/1.73 m 2. Historically, much of the importance and research in CKD has focused on disease progression and the risk of developing ESRD. Landmark trials, demonstrating that inhibition of angiotensin 2 in patients with CKD and diabetes retards kidney disease progression, have guided management of patients with CKD. 2-5 Recent data, however, highlight the mortality risk associated with CKD. In fact, the overwhelming majority of patients with CKD do not survive to ESRD. This is evident in the discrepancy in size of the CKD population compared to the prevalent ESRD population, which comprised 406,000 prevalent cases in Recent analyses of a

21 Date: 17 April 2008 Page 20 of 96 cohort of 13,796 patients with CKD from the Kaiser Permanente health maintenance organization, demonstrates that death is a competing event with ESRD (Figure 1). 7 While the frequency of death and ESRD progressively increased among patients with more severe renal impairment, the proportion that died consistently exceeded the proportion that developed ESRD at every level of kidney function. The implications of these new observations are profound for both future clinical investigation and medical practice. Figure 1. Frequency of Death and ESRD During 5-Year Follow-up, Kaiser Permanente Cohort 70 Percentage of Patients </= ESRD Death GFR (ml/min/1.73 m 2 ) Adapted from Keith D, et al. ASN Meeting; Nov 3, Diabetes is a leading cause of CKD. Data from the 5% Medicare Sample cohort revealed diabetes was present in 37.1% of patients with CKD. 8 Diabetes is also recognized as the most common cause of ESRD, accounting for kidney failure in 44.5% of this patient population. 6 Finally, the prevalence of diabetes has increased by 30% to 50% over the past decade, with substantial implications for growth of the CKD and ESRD populations. 8 A large body of data associates CKD and diabetes with increased risk for mortality and cardiovascular disease compared with the general population. A prospective study of 10,940 patients with hypertension demonstrated that higher baseline serum creatinine

22 Date: 17 April 2008 Page 21 of 96 had significant prognostic value for mortality over 8 years of follow-up. 9 Other studies have shown that elevated risk of cardiovascular disease is common early in the course of CKD and this risk increases in concert with progressive kidney impairment Studies of patients with CKD have demonstrated an inverse relationship between kidney function and left ventricular hypertrophy, an established mortality risk factor The importance of diabetes as a cardiovascular risk factor is supported by a study that evaluated the seven-year incidence of myocardial infarction in 1,059 patients with diabetes and 1,373 subjects without diabetes (Figure 2). 19 First, this study confirms previous observations that a prior history of myocardial infarction greatly increases the risk of subsequent myocardial infarction. This finding highlights the importance of cardiovascular risk modification in patients with a known history of cardiovascular disease and myocardial infarction. Such patients are aggressively managed with established interventions including dietary and lifestyle modifications, aspirin, beta-blockade, lipid lowering and interruption of the renin-angiotensin aldosterone axis. What is particularly noteworthy, however, is the observation that patients with diabetes and no previous history of myocardial infarction were found to have a similar risk for subsequent myocardial infarction as non-diabetic patients with a prior history of myocardial infarction. This finding underscores the need to ensure that all patients with diabetes, many of whom also have kidney disease, receive appropriate cardiovascular risk factor modification in addition to emphasis on optimizing diabetes control and traditional end organ damage.

23 Date: 17 April 2008 Page 22 of 96 Figure 2. Incidence of Myocardial Infarction in Diabetic and Non-diabetic Subjects With and Without Prior History of Myocardial Infarction Over Seven Years of Follow-Up 50 Without Diabetes n = 1,373 With Diabetes n = 1,059 p < p < % Percentage of Patients n = % n = 1, % n = % n = No Prior MI* Prior MI No Prior MI Prior MI Adapted from Haffner et al. N Engl J Med. 1998;339:229. Risk for cardiovascular disease in patients with CKD can be attributed, in part, to the high burden of traditional risk factors including diabetes, hypertension and dyslipidemia in this population. 20, 21 Recent evidence suggests, however, that anemia is an important and independent risk factor for cardiovascular disease in this population, of similar magnitude as traditional risk factors. 16, 22, 23 A study of Canadian CKD patients demonstrated independent associations between hemoglobin, systolic blood pressure and relative risk of left ventricular growth and hypertrophy. 24 For every 0.5 g/dl decrease in hemoglobin and every 15 mmhg increase in systolic blood pressure the relative risk of left ventricular growth increases by 33%. Of particular importance is the finding that a relatively modest difference in hemoglobin was associated with the same relative risk as a large difference in systolic blood pressure. Anemia is associated with cerebrovascular disease. The Atherosclerosis Risk in Communities Study (ARIC) sought to prospectively identify cardiovascular risk factors among 13,716 middle-aged subjects. 25 Patients with CKD were found to have an

24 Date: 17 April 2008 Page 23 of 96 increased hazard ratio (95% CI) for cerebrovascular accident of 1.81 (1.26, 2.02), compared to patients without CKD. In a cohort that had anemia in addition to CKD, the hazard ratio for stroke increased to 5.43 (2.04, 14.41) suggesting that increased risk of cerebrovascular accident in patients with CKD is exacerbated by the presence of anemia. Anemia has been shown to be a risk factor for mortality, independent of CKD. A retrospective analysis of the Studies of Left Ventricular Dysfunction (SOLVD) database revealed that a 2% lower hematocrit and 10 ml/min/1.73 m 2 lower estimated GFR were each independently associated with a 6% higher risk for mortality. 26 These findings were confirmed in a population of 665 Medicare beneficiaries with congestive heart failure, where the impact of CKD and anemia were similar to that in the SOLVD database. 27 Additional studies of the 5% Medicare Sample cohort illustrated that over a 2-year follow-up period, CKD, anemia, diabetes and congestive heart failure were all risk factors for mortality (Figure 3). 8 Importantly, patients with CKD and anemia were found to have an equivalent mortality risk as patients with diabetes and congestive heart failure.

25 Date: 17 April 2008 Page 24 of 96 Figure 3. Relative Risk of Death During 2-Year Follow-up, 5% Medicare Random Sample Cohort Relative Risk None DM only Anemia only CKD only DM/CKD only DM/Anemia only CHF only DM/CKD/Anemia only DM/CHF only CKD/Anemia only CHF/Anemia only DM/CHF/Anemia only only CHF/CKD/Anemia only DM/CHF/CKD only DM/CHF/CKD/Anemia Collins, AJ. Adv Stud in Med. 2003;3(3C):S14-S17. The increased risk of mortality and cardiovascular disease in this population call attention to the importance of identifying and implementing cardiovascular risk modification strategies in patients with CKD. Treatment of anemia to achieve hemoglobin levels of approximately 13 g/dl in patients with congestive heart failure and impaired kidney function has been shown to have beneficial impact on congestive heart failure severity and hospitalization in several uncontrolled trials. 28, 29 An analysis of the Greater Boston Chart Audit demonstrated that patients with CKD and a baseline hemoglobin of < 10.0 g/dl who subsequently received erythropoietic therapy had a 60% reduction in their adjusted hazard of cardiovascular hospitalization compared with similar patients who did not receive erythropoietic therapy (Figure 4).

26 Date: 17 April 2008 Page 25 of 96 Figure 4. Hazard Ratio for Hospitalization for Cardiovascular Disease in Patients with Anemia and CKD With and Without Erythropoietin Treatment, Greater Boston Chart Audit Hazard Ratio* for Cardiovascular Hospitalization < Hct (%) at Entry to Clinic No EPO EPO treated * N = 160; Adjusted for age, sex, race, DM, GFR and ACE-I Source: Greater Boston Chart Audit Similar findings were obtained from an analysis from the University of Pittsburgh Medical Center health system. Patients with CKD, diabetes and hemoglobin 10 g/dl who received erythropoietic therapy had a 53% reduction in their risk of mortality and nonfatal cardiovascular events when compared to similar patients who did not receive erythropoietic therapy (Figure 5).

27 Date: 17 April 2008 Page 26 of 96 Figure 5. Survival Over Four Years in Patients with Anemia, CKD and Diabetes With and Without Erythropoietin Treatment, University of Pittsburgh Medical Center Health System Survival In Patients with Index Hb <10 13 g/dl Survival to Composite Cum Survival Endpoint (Death or CV Event).8 12 g/dl.7.6 untreated Event Hb Group Hazard Ratio 0.47 ( ) censored censored.1 LE LE 10-censored Months Months to Composite Event Source: UPMC Data Analysis In summary, CKD is a highly prevalent threat to public health. Patients with CKD are more likely to die than reach ESRD, and patients with CKD and diabetes are at much higher risk for mortality and cardiovascular disease than the general population. In patients with CKD, anemia is an important risk factor for cardiovascular disease, like hypertension, diabetes and dyslipidemia.

28 Date: 17 April 2008 Page 27 of Darbepoetin alfa Darbepoetin alfa is an erythropoietic stimulating protein that works via the same mechanism of action as endogenous erythropoietin (EPO). Research indicates that the sialic acid-containing carbohydrate of EPO determines its serum half-life. Endogenous EPO and recombinant human erythropoietin (rhuepo) have three N-linked glycosylation sites, while darbepoetin alfa was designed to have five N-linked glycosylation sites. As a result, darbepoetin alfa has an approximately three-fold greater biological activity and serum half-life than rhuepo. These unique properties allow darbepoetin alfa to be administered at extended intervals compared to rhuepo. Additional details of the chemistry, preclinical pharmacology, pharmacokinetics, toxicology, and clinical experience with darbepoetin alfa are contained in the Investigator s Brochure Rationale The evidence suggesting that anemia is an independent risk factor for CV disease in CKD, though substantial, is based largely on associative data and uncontrolled studies. 5, 10, 11, 13, 16, 20, 22, 23 No direct evidence is available that conclusively demonstrates that treating anemia will reduce CV morbidity and mortality in patients with CKD. The availability of such data would be invaluable in raising awareness of the importance of anemia therapy in CKD. Therefore, the need exists for a robust, prospective clinical trial to provide definitive evidence that anemia is a modifiable risk factor for mortality and cardiovascular morbidity in the high-risk population of patients with CKD and diabetes. Data also suggest that anemia therapy may have a beneficial impact on progression of CKD. 31 While compelling, these data are not definitive and need to be validated in a randomized controlled trial. TREAT is a large, randomized, prospective and double-blind clinical trial of patients with CKD and type 2 DM in which the effect of the treatment of anemia with darbepoetin alfa on all-cause mortality and non-fatal CV events will be studied. TREAT will also evaluate the effect of the treatment of anemia with darbepoetin alfa in reducing the risk of progression to ESRD in patients with CKD and type 2 DM.

29 Date: 17 April 2008 Page 28 of Clinical Hypothesis Treatment of anemia with darbepoetin alfa in subjects with CKD and Type 2 DM will decrease mortality and CV morbidity. Treatment of anemia with darbepoetin alfa in subjects with CKD and Type 2 DM will delay the progression to ESRD. 3. EXPERIMENTAL PLAN 3.1 Study Design This is a randomized, double-blind, multicenter (United States, Australia, Canada, Latin America, and Europe) study. Eligible subjects will be randomized in a 1:1 ratio to either Group A or Group B. Group A subjects will receive darbepoetin alfa to achieve and maintain a hemoglobin (Hb) target of 13.0 g/dl. Subjects randomized to Group B will receive placebo if the Hb is 9.0 g/dl. If the Hb is < 9.0 g/dl, subjects in this treatment group will receive rescue therapy with darbepoetin alfa as described in Section Group A N = 2000 (Hemoglobin Target of 13 g/dl) 1:1 Randomization ~4000 subjects Group B N = 2000 (Control) Screening Year 1 Year 2 Year 3 Year 4 Study Start Enrollment 2 years Treatment 2.5 years Study End (1203 primary events; approximately year 4.5) All study subjects should receive standard of care for their comorbid conditions. Ideally, comorbid conditions should be managed in an attempt to achieve the following targets, as recommended by the American Diabetic Association and the National Kidney Foundation Kidney Disease Outcomes Quality Initiative (K/DOQI) guidelines: HbA 1c 7.0% Systolic blood pressure < 130 mmhg Diastolic blood pressure < 80 mmhg

30 Date: 17 April 2008 Page 29 of 96 Proteinuria (spot urine protein/urine creatinine ratio) < 500 mg/day LDL < 100 mg/dl The study endpoints are defined in Section Number of Centers Approximately 700 centers in the United States, Canada, Australia, Latin America, and Europe will participate in this study. Sites that do not randomize a subject within 2 months of site initiation may be closed. 3.3 Number of Subjects Participants in this study shall be referred to as subjects. Approximately 4000 subjects may be randomized into this study. Refer to Section 10.4 for additional information regarding the calculation of the sample size. 3.4 Estimated Study Duration The study is event driven and will conclude when approximately 1203 patients have experienced a primary event. The DSMC will monitor the overall event rate, and Amgen and the Executive Committee will set the termination date for the study based on the anticipated date of occurrence of 1203 primary endpoints. All subjects will be followed from randomization through the date of study termination (eg, when approximately 1203 subjects have experienced a primary event). It is anticipated that the enrollment period will be approximately 2 years, and the follow-up period will be approximately 4.5 years from the date that the first subject is randomized. Based on the accumulating event rate in the pooled treatment groups, the Executive Committee may choose to alter the sample size in order to complete the trial in approximately four and a half years from the start of the recruitment phase. 4. SUBJECT ELIGIBILITY 4.1 Prescreening Sites may utilize the hemoglobin point of care device (Hemocue ) provided for this trial to obtain prescreening hemoglobin values for potential subjects. The site must obtain written informed consent from the potential subject prior to performing the hemoglobin test using an abbreviated consent form (Appendix K). If the hemoglobin result is 11.0 g/dl, the site may proceed with obtaining written informed consent from the

31 Date: 17 April 2008 Page 30 of 96 subject or legally acceptable representative for participation in the TREAT study using the full TREAT consent form. 4.2 Screening Before any study specific procedure is performed, the subject or legally acceptable representative must give written informed consent for participation in this study. Investigators will be expected to maintain a screening log of all potential study candidates that includes limited information about the potential candidate (age, sex, race), date, and outcome of the screening process (eg, enrolled into study, reason for ineligibility, or refused to participate). 4.3 Inclusion Criteria Before any study-specific procedure, the appropriate informed consent must be obtained (see Section 12.1) years old Clinical history of type 2 DM defined as: 32 a. hyperglycemia not requiring insulin therapy or, b. hyperglycemia requiring insulin when either i. The period between diagnosis and the initiation of insulin therapy is > 1 year and/or, ii. C-peptide levels are elevated above normal Clinical history of CKD (at any point prior to registration) defined as: a. creatinine clearance or egfr 20 ml/min and 60 ml/min or, b. serum creatinine 1.2 mg/dl for all females, 1.5 mg/dl for black males, and 1.3 mg/dl for non-black males An egfr during the screening period 20 ml/min and 60 ml/min measured by the following MDRD equation: egfr = 186 x [Serum creatinine] x [Age] x [0.742 if subject is female] x [1.210 if subject is black] Mean Hb 11.0 g/dl (mean of 2 values taken at least 6 days apart during the screening period, using the hemoglobin point of care device) Mean Tsat 15% (mean of 2 values taken at least 6 days apart during the screening period).

32 Date: 17 April 2008 Page 31 of Exclusion Criteria Anticipating or scheduled for a living related-donor kidney transplant, or a prior recipient of a kidney transplant Uncontrolled hypertension defined as diastolic BP >110 mm Hg or systolic BP > 180 mmhg on 2 separate occasions during screening Use of any erythropoietic protein (eg, rhuepo; Procrit, Eprex, Neorecormon, Epogen, Aranesp ) within 12 weeks of randomization Prior history (within 12 weeks of randomization) of CV events including: a. Myocardial ischemia b. Hospitalization for CHF c. MI d. CVA Grand mal seizure within the 12 weeks prior to randomization Major surgery within 12 weeks prior to subject randomization (excluding vascular access surgery) Currently receiving intravenous antibiotic therapy for systemic infection Known HIV positive Clinical evidence of current malignancy with the exception of basal cell or squamous cell carcinoma of the skin and cervical intraepithelial neoplasia Currently receiving systemic chemotherapy and/or radiotherapy Active bleeding Hematologic disease (eg, sickle cell disease, myelodysplastic syndromes, hematologic malignancy); myeloma; hemolytic anemia, thalassemia Subject is currently enrolled in or has not yet completed at least 30 days since ending other investigational product trial(s), or subject is receiving other investigational agent(s) Subject is pregnant (eg, positive HCG test) or is breast-feeding. Females must have a negative serum pregnancy test (or definitive evidence to demonstrate lack of pregnancy) within 21 days prior to randomization if they are of childbearing potential, unless there is a documented history of amenorrhea Subject is not using adequate contraceptive precautions, as determined by the investigator Subject has known sensitivity to any of the products to be administered during dosing Subject has previously entered this trial (this does not include subjects who rescreen).

33 Date: 17 April 2008 Page 32 of Subject will not be available for follow-up assessments Subject has any disorder (excluding illiteracy or visual impairment) that compromises the ability of the subject to give written informed consent and/or to comply with study procedures. Subjects are ineligible for participation in the functional capacity sub-study if (in addition to the above) they: 1. Have intrinsic walking limitations (eg, amputation, stroke) 2. Do not consent to participate in the sub-study 5. SUBJECT ENROLLMENT Before subjects may be entered into the study, Amgen requires a copy of the site s written Independent Ethics Committee/Institutional Review Board (IEC/IRB) approval of the protocol, informed consent form, and all other subject information and/or recruitment material, if applicable (see Section 12.2). For the purpose of this trial the following definitions will apply: Registration-when the subject has signed the informed consent and been assigned a subject identification number by the interactive voice response system (IVRS) Randomization-when the subject has been assigned to treatment by IVRS All subjects or legally acceptable representatives must personally sign and date the consent form before registration. A sample of the informed consent is provided in Appendix F. 5.1 Subject Registration After obtaining informed consent, the Investigator (or assigned designee) will contact an interactive voice response system (IVRS) to register the subject. The following information will be required for registration: subject initials date of birth body weight (kg) confirmation that the subject has signed the informed consent

34 Date: 17 April 2008 Page 33 of 96 Upon receipt of this information, the IVRS will assign a unique 9-digit identification number to each subject. The first two digits will be the protocol identifier (ie, 84); the next four digits will represent the site (eg, 0001, 0002, 0003 ); the last three digits will identify the subject (eg, 001, 002, 003 ). The subject will be considered registered once a subject identification number has been confirmed by the IVRS. All subjects who are registered for the study will receive a subject identification number from IVRS at the time of registration. This number will be used to identify the subject throughout the trial and must be used on all study documentation related to that subject. The subject identification number must remain constant throughout the entire trial; it must not be changed at the time of rescreening, enrollment, or randomization. 5.2 Screen Failures Registered subjects who are ineligible for the study based on any of the screening assessments will be considered screen failures. The screen failure will be entered into IVRS, including the reason for the screen failure. These subjects may begin screening procedures again 2 weeks after the date of the last screening assessment. This date will be provided by IVRS when the screen failure data is entered. Rescreened subjects need not be registered again via IVRS as the originally assigned subject identification number will still apply, however the rescreening attempt will need to be entered into IVRS. 5.3 Randomization and Treatment Assignment Registered subjects who successfully complete all screening evaluations and fulfill all eligibility criteria will be randomized in a 1:1 ratio to Group A or Group B. Randomization will be stratified by center, level of baseline proteinuria (spot urine protein/urine creatinine ratio 1 g/day, or < 1 g/day) and history of CV disease. The investigator (or assigned designee) will contact IVRS to randomize a subject within 7 days after the date of completion of the last screening assessment. The following information will be required for randomization: subject identification number confirmation that the subject has met all eligibility criteria screening Hb values screening egfr value screening TSAT values

35 Date: 17 April 2008 Page 34 of 96 baseline proteinuria (screening spot urine protein/urine creatinine ratio) history of CV disease Upon receipt of this information, IVRS will confirm the successful randomization of the subject into the trial. The first box of investigational product to be administered will be assigned during the week 1 medication assignment call that is made by the third party individual at the site. This information will be entered into the case report form. 6. TREATMENT PROCEDURES Darbepoetin alfa is the only investigational product administered in this study. The first dose of investigational product must be administered within 3 days of subject randomization. 6.1 Investigational Product Dosage, Administration, and Schedule Darbepoetin alfa will be provided as a clear, colorless, sterile protein solution in prefilled syringes (PFS) in the following strengths: 10, 15, 20, 30, 40, 50, 60, 80,100, 150, 200, and 300 µg. Placebo will be provided as a clear, colorless, sterile solution in PFS identical to those containing the active drug. The starting dose of investigational product will be 0.75 µg/kg, estimated from the subject s body weight at the time of registration. The dose obtained from this calculation will be rounded to the nearest PFS and administered by subcutaneous (SC) injection once every 2 weeks (Q2W). IVRS will calculate the initial dose for each subject at the time of randomization and will provide the site the box number of the investigational product kit assigned. All subjects randomized to receive darbepoetin alfa will receive investigational product SC Q2W (± 3 days) until the Hb target of 13.0 g/dl has been achieved. During this time the dose of investigational product will be titrated incrementally by PFS until the target is reached. The Hb target is defined as 2 consecutive Hb levels (that must include the most recent assessment) between 12.0 and 13.5 g/dl with no change in dose. Once at target, the frequency of administration will be extended to once monthly (QM ± 5 days). The initial QM dose of investigational product will be twice the previous Q2W dose. During the remainder of the trial, the dose of the investigational product will be adjusted as necessary to maintain the target Hb. The maximum dose of investigational product administered will be 300 µg Q2W or 600 µg QM.

36 Date: 17 April 2008 Page 35 of 96 To prevent unblinding, Group B subjects will have dose and schedule changes mirroring those that occur within Group A, via a dynamic dose change schedule that imitates the Group A dose changes. An IVRS algorithm ensuring this balance will be implemented prior to study start. Additional details regarding the administration of investigational product may be found in the pharmacy guide in Appendix D. 6.2 Dosage Adjustments All dose changes will be managed using IVRS. Hb values to determine investigational product dosing must be obtained within 1 week before the investigational product administration visit or on the same day as the visit. These Hb values should be obtained at the site by a third party not directly involved in the study subject s care using a hemoglobin point of care device provided by Amgen. Hb values will be entered into the IVRS, which will calculate and assign the appropriate dose and box of investigational product. The IVRS will also notify the site if the dosing schedule for the subject is to be changed from Q2W to QM. The dose of investigational product will be adjusted beginning at week 5 to achieve the target Hb. Dose increases in darbepoetin alfa will not occur more frequently than QM. The dose will be adjusted based jointly on Hb levels and Hb rate of rise per 2-week period as follows: Hb (g/dl) Hb rate of rise (g/dl/2 weeks) Dose Adjustment < 0.5 Increase to next higher PFS < < 1.0 Maintain dose 1.0 Decrease to next lower PFS < 0.5 Maintain dose < < 1.0 Maintain dose 1.0 Decrease to next lower PFS

37 Date: 17 April 2008 Page 36 of Any Decrease to next lower PFS > 14.0 Any Administer placebo until Hb value is below 13.0, then resume investigational product at next lower PFS Subjects receiving 10 µg of investigational product Q2W who require a dose decrease per the above table, will have their dosing interval extended to QM dosing at 10 µg QM. Subjects receiving 10 µg of active investigational product Q2W who require placebo administration per the above table, will resume dosing at 10µg QM when their Hb value is below 13.0 g/dl Missed Doses If a subject misses a scheduled dose of investigational product, the missed dose should be administered as soon as possible during that dosing week (defined as ± 3 days from scheduled dose). If this is not possible, the missed dose must be recorded on the case report form. The subject will return to the original dosing schedule and day of the week during subsequent weeks. Subjects should not receive more investigational product than the prescribed total Q2W dose Monthly Dosing Once a subject achieves the target Hb, defined as two consecutive Hb levels between 12.0 and 13.5 g/dl with no change in dose, the frequency of administration will be extended to QM. The initial QM dose of investigational product will be equal to two times the previous Q2W dose. Dose adjustments will be made to maintain the target Hb, based jointly on the Hb level and calculated rate of rise per 2-week period as follows: Hb (g/dl) Hb rate of rise (g/dl/2 weeks) Dose Adjustment < 0.5 Increase to next higher PFS < < 1.0 Maintain dose 1.0 Decrease to next lower PFS < 0.5 Maintain dose

38 Date: 17 April 2008 Page 37 of < < 1.0 Maintain dose 1.0 Decrease to next lower PFS Any Decrease to next lower PFS > 14.0 Any Administer placebo until Hb value is below 13.0, then resume investigational product at next lower PFS Missed Doses During QM Administration If a subject misses a scheduled dose of investigational product, the dose should be administered as soon as possible during that dosing week (defined as within ± 5 days of the scheduled date for QM dosing). If this is not possible, the missed dose must be recorded on the case report form. The subject will return to the original dosing schedule and day of the week during subsequent weeks Subjects Who Do Not Maintain Target Hb on QM Dosing It is anticipated that some subjects will be unable to maintain the Hb target once they have been changed to QM dosing. If 3 consecutive monthly Hb s are below 11.0 g/dl in a subject receiving the maximum QM dose (600 µg) of investigational product, the subject will have the frequency of administration of investigational product increased to Q2W and their dose of investigational product reduced to 50% of their initial QM dose Rescue Therapy If at any time the Hb falls below 9.0 g/dl for Group B subjects, therapy with darbepoetin alfa will be instituted with a single dose of 0.45 µg/kg rounded to the nearest PFS. The Hb level will be reassessed at the next monthly visit; if below 9.0 g/dl an additional dose of darbepoetin alfa will be administered per the following table. Dose increases in rescue therapy with darbepoetin alfa will not occur more frequently than QM. This process will continue indefinitely until the Hb reaches 9.0 g/dl, at which time placebo injections will be resumed.

39 Date: 17 April 2008 Page 38 of 96 Hb (g/dl) Hb rate of rise (g/dl/2 weeks) Dose Adjustment < 9.0 < 0.5 Increase to next higher PFS < , but < 1.0 Maintain dose < Decrease to next lower PFS 9.0 Any Resume placebo administration 6.3 Concomitant Therapy Throughout the study, investigators may prescribe any concomitant medications or treatments deemed necessary to provide adequate supportive care except for those listed in Section 6.4. Iron may be administered according to the algorithm provided in Appendix H to ensure subjects are iron replete, ie, transferrin saturation (Tsat) 20%. Supplemental iron therapy will be recorded on the case report form. Red blood cell (RBC) transfusions are permitted during this study; these data will be recorded on the case report forms. 6.4 Proscribed Therapy During Study Period Subjects are prohibited from participating in other studies using investigational products or devices while participating in TREAT. Subjects should not receive commercial erythropoietic proteins while receiving investigational product during the TREAT study. If subjects withdraw from investigational product for any reason (but continue participation in the TREAT study), it is likely that subjects may receive commercial erythropoietic proteins. Most commonly, this will occur for subjects who commence RRT. If this occurs, administration of commercial erythropoietic proteins will be captured on the case report forms.

40 Date: 17 April 2008 Page 39 of STUDY PROCEDURES All laboratory analyses for this study will be performed at a central laboratory with the exception of the assay for anti-erythropoietic protein seroreactivity, which will be performed by Amgen Inc, and the Hb values used to determine subject eligibility and dosing of investigational product, which will be performed by an independent third party at the investigational site using a hemoglobin point of care device provided by Amgen. The results of all laboratory tests, with the exception of Hb results, will be provided to the investigator by the central laboratory as soon as they are available. A tabular summary of study procedures is provided in Appendix A. The total amount of blood drawn from each subject will be approximately 160 ml/year. The investigator is responsible for ensuring that all assessments are performed according to the protocol. All study-related data will be recorded in the case report form. The only exception to this will be the results of the laboratory tests that are conducted at the central laboratory, which will be electronically captured. Missed visits, test(s) that are not performed, and examinations that are not conducted must be reported as such on the case report forms. 7.1 Screening (Weeks 2 and 1) The subject s historical laboratory data should be prescreened to ensure that they are likely to fulfill the eligibility criteria. The hemoglobin point of care device may be used to obtain a prescreening hemoglobin value once the subject has provided consent for this procedure. Written informed consent for the TREAT study using the full TREAT informed consent form must be obtained prior to performing any screening procedures. The following examinations and assessments will be performed once during the 2-week screening period: Medical history including etiology and duration of CKD and concomitant diseases Physical examination including height, measurement of waist and hip circumference, and body weight (kg) Heart rate, respirations and temperature (oral, axillary or tympanic) NYHA Classification (if applicable) Serum chemistry profile (BUN, creatinine, albumin, potassium, C-reactive protein [CRP]) Hematology (platelets [PLT], white blood count [WBC], reticulocytes) Serum ferritin egfr calculated using the MDRD equation

41 Date: 17 April 2008 Page 40 of 96 Spot urine protein/urine creatinine ratio Serum pregnancy test for women of childbearing potential Concomitant medications The following assessments will be performed twice during the screening period (once during week 2 and once during week 1, at least 6 days apart): BP (sitting, 5 minutes resting) Tsat Hb (utilizing the hemoglobin point of care device provided by Amgen, as well as sending a sample to the central laboratory) If a subject is ineligible for the study based on any of the screening assessments, the subject may be rescreened 2 weeks from the date of the last assessment. 7.2 Treatment Period (Week 1 to Approximately Week 235) Week 1 assessments should be obtained before the first dose of investigational product and are considered pre-treatment assessments for this study. The following examinations and assessments will begin within 3 days of completing randomization and will be performed/obtained at specified time-points during the study: Heart rate, BP, sitting, 5 minutes resting will be measured at week 1, and Q2W (± 3 days) thereafter during titration and monthly (± 5 days) during maintenance. Hb will be measured with the hemoglobin point of care device at week 1, and Q2W (± 3 days) thereafter during correction and monthly (± 5 days) during maintenance. Hb will also be measured by the central laboratory at weeks 1, 13, 25, 37, 49 and annually thereafter (these values will remain blinded to all site personnel). HbA 1C measurements will be performed at week 1, and every 24 (± 2) weeks thereafter. Serum chemistry (BUN, creatinine, albumin, potassium, CRP): will be measured at week 1, and every 24 (± 2) weeks thereafter. Tsat and serum ferritin: will be measured at week 1, and every 12 (± 2) weeks thereafter. Blood sample for lipid panel (cholesterol, triglycerides, LDL, and HDL) at week 1, and every 24 (± 2) weeks thereafter. NYHA classification, if applicable, will be collected at weeks 13, 25, 49 and annually thereafter egfr will be calculated at week 1, and every 24 (± 2) weeks thereafter. Spot urine protein/urine creatinine ratio at week 1, and every 24 (± 2) weeks thereafter. 12-lead electrocardiogram at week 1, and annually thereafter.

42 Date: 17 April 2008 Page 41 of 96 Completion of the EQ-5D and FACT-fatigue (PRO) and HRU questionnaires on weeks 1, 13, 25, and every 24 (± 2) weeks thereafter. Questionnaires will be completed prior to any other study procedures. Completion of the SF-36 questionnaire (PRO) on weeks 1, 25, 49, and 97 (± 2 weeks) Six-minute walk test on weeks 1, 25, 49, and 97 (± 2 weeks) on a subset of 1000 subjects Physical examination, including measurement of waist and hip circumference, and weight, at week 49 and annually thereafter. Details of subject hospitalization, including diagnosis and length of stay, will be collected in the case report form at each study visit. Anti-erythropoietic protein seroreactivity: A blood sample for determining seroreactivity will be drawn on day 1 before the first dose of investigational product. Detailed instructions for blood sample collection and shipment will be provided in the laboratory manual. Details of concomitant medications including: anticoagulants, analgesics/antipyretics (eg, aspirin, Plavix, non-steroidal anti-inflammatory agents), antidepressants, iron therapy, cardiac drugs (eg, dyslipidemic agents, antiarrythmics), diuretics, antidiabetic agents, oral corticosteroid therapy, hormone replacement therapy (HRT), vitamin B 12, and folate will be collected at every study visit RBC transfusions: All RBC transfusions administered will be recorded on the case report form. Commercial erythropoietic agents: Details regarding the use of any erythropoietic agent during the course of the study will be recorded on the case report form. Use of these agents is only permitted for subjects not receiving investigational product (ex. subject on RRT). Adverse events: Information on adverse events that occur from the date of the administration of the first dose of investigational product through one week (7 days) following the last administration of investigational product will be recorded (see Section 9). Adverse events resulting in death will be collected during the entire study or until 30 days after the last dose of investigational product, whichever is later. Serious adverse events will be reported to Amgen or designee within 1 working day of discovery by recording the event(s) on a Serious Adverse Event (SAE) Form (see Appendix C) faxed to the attention of the Safety Specialist at Amgen or designee (see Section 9.3). Note: subjects with an adverse event documenting a current diagnosis of cancer, with the exception of localized basal cell and squamous cell carcinoma of the skin, or intra-epithelial neoplasia of the uterine cervix, must be removed from investigational product treatment. These subjects will continue to be followed in the trial as described in Section 7.4. At week 1, week 49, week 97, week 145, and week 193 duplicate serum and urine samples will be collected and stored at the central laboratory for possible future analysis. No genetic, pharmacogenomic, or DNA testing is planned for these samples.

43 Date: 17 April 2008 Page 42 of End of Study (When 1203 Primary Events are Observed or Approximately Week 235 [+ 2 weeks]) The following End of Study assessments will be performed at the termination of the trial [when 1203 primary events has been observed, approximately week 235 (+ 2 weeks)] or at the time of withdrawal (± 2 weeks) of consent for further participation in the trial: Physical examination 12-lead electrocardiogram Heart rate, BP, respiration and temperature (oral, axillary or tympanic) NYHA Classification (if applicable) Hematology (Hb, reticulocytes, PLT, and WBC) HbA 1c Serum chemistry profile (BUN, creatinine, albumin, potassium, CRP) Serum ferritin and Tsat Lipid panel Blood sample to determine anti-erythropoietic protein seroreactivity Adverse events (new and resolution of previously documented) Spot urine protein/urine creatinine ratio egfr Concomitant medications Completion of the EQ-5D, PRO and HRU questionnaires Also at week 235, duplicate serum and urine samples will be collected and stored at the central laboratory for possible future analysis. No genetic, pharmacogenomic, or DNA testing is planned for these samples. 7.4 Subjects Who Are No Longer Receiving Treatment With Investigational Product, Including Subjects Requiring Renal Replacement Therapy (RRT) Subjects who have had the administration of investigational product discontinued due to any reason, including those who require RRT (eg, dialysis, renal transplantation) during the course of the study, will continue to be followed QM. Administration of investigational product will be discontinued and a blood sample for anti-erythropoietic protein seroreactivity obtained (for subjects initiating RRT, it should be obtained before RRT is initiated). Adverse events (other than death) will not be collected after one week (7 days) following the last administration of investigational product. All other procedures,

44 Date: 17 April 2008 Page 43 of 96 including endpoint data collection, will continue to be performed as detailed in Sections 7.2 and 7.3. Subjects who have had the administration of investigational product discontinued due to any reason, including the initiation of RRT, and do not consent to have all study procedures performed, will consent to having endpoint data collected QM until completion of the trial. These subjects will remain on study and have this information collected either through a review of their medical records or via telephone contact QM. Commercial erythropoietic protein therapy is permitted for subjects who initiate RRT. Use of commercial erythropoietic proteins will be captured on the case report form. Subjects may be removed from investigational product treatment if any of the following occur: subject request administrative decision by the investigator or Amgen pregnancy (report on Pregnancy Notification Worksheet; see Appendix E) Note: females who become pregnant while on study will be removed from investigational product treatment ineligibility significant protocol deviation patient noncompliance adverse event (report on adverse event CRF) Note: subjects who have a current diagnosis of cancer, with the exception of localized basal cell and squamous cell carcinoma of the skin, or intraepithelial neoplasia of the uterine cervix, will be removed from investigational product treatment. 7.5 Six-Minute Walk Testing Six-minute walk testing will be conducted in a subset of 1000 subjects at weeks 1, 25, 49 and 97. The distance ambulated during the six minutes over a specific course as well as the use of pulmonary medications (eg, bronchodilators) will be recorded on the case report form. Details regarding the six-minute walk testing, including the specifications of the course to be used will be provided in a procedure manual supplied to the investigational site.

45 Date: 17 April 2008 Page 44 of Third Party Responsibilities The third party at each site will be responsible for obtaining the Hb value at each visit using venous blood and the hemoglobin point of care device provided by Amgen. This individual should not be directly involved in the care of the study subject. Third party personnel will monitor the Hb values for all study subjects for safety purposes. This individual will inform the Principal Investigator so that he/she may follow-up as clinically indicated when any of the following are seen: Hb value is 7.0 g/dl (the Principal Investigator will be notified of the actual Hb value in this instance) Hb value is 16.0 g/dl (the Principal Investigator will be notified of the actual Hb value in this instance) A decrease in the Hb value of 2.0 g/dl in any 4-week period 7.7 Access to Hb data An individual at Amgen or designee who is not affiliated with the clinical study team will review the Hb data for individual subjects. This data will be reviewed for accuracy of entry into IVRS, for comparison to the central laboratory values to determine if retraining on the use of the hemoglobin point of care device is required at the clinical sites, and to determine if any of the alerts defined in Section 7.6 have occurred. The clinical team will be notified in a blinded fashion if follow-up with the clinical sites is required. 7.8 Endpoint Determination Definition of Endpoints Details regarding the definition of the components of the composite endpoint are provided in Appendix G Data Collection Endpoint data will be collected on specific case report forms. All supporting documentation will be provided to Amgen or designee for each event as it occurs during the course of the trial. These data will be provided to the Endpoint Adjudication Committee for review and confirmation of events as outlined in their charter.

46 Date: 17 April 2008 Page 45 of REMOVAL AND REPLACEMENT OF SUBJECTS 8.1 Removal of Subjects Subjects may withdraw from treatment with investigational product only and continue receiving follow-up until the close of the trial (see Section 7.4). Infrequently, a subject may withdraw from participation in all trial activities, including follow-up for information on the composite event (withdrawal from study). The reasons that subjects may withdraw from study include either withdrawal of consent or lost to follow-up. The reason for withdrawal from study will be recorded on the end-of-study case report form. Subjects have the right to withdraw from the study at any time and for any reason without prejudice to his or her future medical care by the physician or at the institution. Any subject who withdraws consent to participate in all study activities will be removed from further study procedures immediately upon the date of request. The subject may need to undergo additional tests but the data generated should not be included in the subject s study data. Should a subject (or a legally acceptable representative) request or decide to withdraw from the study, all efforts will be made to complete and report the observations as thoroughly as possible up to the date of withdrawal. All information should be reported on the applicable case report forms. Subjects who withdraw from the study will not be replaced. 9. ADVERSE EVENT REPORTING 9.1 Definitions Adverse Events An adverse event is an undesirable medical occurrence (sign, symptom, or diagnosis) or worsening of a pre-existing medical condition (eg, diabetes, congestive heart failure, rheumatoid arthritis) that occurs after initiation of investigational product through one week (7 days) following the last date of investigational product administration whether or not considered to be investigational product related. Adverse events resulting in death will be collected during the entire study or until 30 days after the last dose of investigational product, whichever is later.

47 Date: 17 April 2008 Page 46 of 96 A worsening of an existing medical condition is one that was present at baseline (eg, diabetes, migraine headaches, gout) and became more severe, more frequent, or increased in duration during investigational product treatment. Abnormal laboratory values should not be reported as adverse events; however, any clinical consequences of the abnormality should be reported as adverse events Serious Adverse Events A serious adverse event is defined by regulatory agencies as one that suggests a significant hazard or side effect, regardless of the investigator s or sponsor s opinion on the relationship to investigational product. This includes, but may not be limited to, any event that (at any dose): is fatal is life threatening (places the subject at immediate risk of death) requires in-patient hospitalization or prolongation of existing hospitalization is a persistent or significant disability/incapacity is a congenital anomaly/birth defect Any event that does not exactly meet this definition, yet in the investigator s opinion represents a significant hazard, can be assigned the other significant hazard regulatory reporting serious criteria. Important medical events that may not be immediately life threatening or result in death or hospitalization but that may jeopardize the subject or require intervention to prevent one of the outcomes listed above, or result in urgent investigation, may be considered serious. Examples include allergic bronchospasm, convulsions, and blood dyscrasias. An Emergency Room visit is not considered an inpatient hospitalization and would therefore, not be considered a serious adverse event unless the Emergency Room visit was necessary to treat, in the investigator s opinion, a significant hazard. 9.2 Adverse Event Profile of Darbepoetin alfa As of October 2004, more than 13,000 subjects with anemia in the chronic renal failure (CRF) setting and greater than 10,000 subjects with anemia in the oncology setting have been treated with darbepoetin alfa in clinical trials.

48 Date: 17 April 2008 Page 47 of 96 Darbepoetin alfa is generally well tolerated. Some of the side effects reported in clinical studies were in subjects with kidney disease, dialysis treatment, or cancer with and without chemotherapy and may not have been due to darbepoetin alfa treatment. These events were more common in subjects with CKD than in subjects with cancer. It is not expected that subjects will experience any new side effects that differ from side effects associated with other medications available to treat anemia (eg, Epoetin alfa). Cardiac events (eg, heart attack), circulatory events (stroke, blood clots, including clots associated with blood vessel access for dialysis) and seizure or death have been reported in subjects receiving darbepoetin alfa. The cardiac events may be due to raising the hemoglobin levels and/or rapid increases in hemoglobin. Blood pressure may rise during treatment with darbepoetin alfa and adjustment of blood pressure treatment may be required. It is important that subjects take antihypertensive medications as prescribed. Subjects may experience headache while treated with darbepoetin alfa. Darbepoetin alfa injections may also be associated with stinging or discomfort at the injection site, and rare allergic reactions including rash, itching, hives or more serious reactions including shortness of breath. In one study of Epoetin alfa, a compound related to darbepoetin alfa, in subjects with CKD receiving dialysis, who also suffered from heart disease, there was a higher rate of death, heart attack, and blood clots associated with those subjects who were assigned to maintain a hemoglobin of 14.0 ± 1.0 g/dl. Pure red cell aplasia (PRCA), in association with neutralizing antibodies to native erythropoietin, has been observed in patients treated with erythropoiesis-stimulating proteins, including darbepoetin alfa. Any subject with a loss of response to darbepoetin alfa should be evaluated for the etiology of this loss of effect. Darbepoetin alfa should be discontinued in any subject who shows evidence of PRCA and the subject should be evaluated for the presence of binding and neutralizing antibodies. Amgen should be notified of any subject in whom PRCA is suspected. In patients with PRCA secondary to neutralizing antibodies to other erythropoiesis-stimulating proteins, darbepoetin alfa should not be administered. Other known causes of PRCA include autoimmune disease, infection (eg, parvovirus B19, human immunodeficiency virus), inflammatory disease (eg, rheumatoid arthritis), malignancy, thymoma, and marrow-suppressive drugs. 30 The principal investigator will be notified of any subject at their site who develops neutralizing antibodies to Epoetin alfa or darbepoetin alfa during the course of

49 Date: 17 April 2008 Page 48 of 96 the trial. The investigator will be provided with instructions regarding additional tests and procedures that may need to be completed. Additional information on the safety profile is contained in the Investigator s Brochure and the physicians prescribing information in the physician s package insert. 9.3 Reporting Procedures for All Adverse Events All adverse events occurring after the first dose of investigational product and through one week (7 days) following the last dose of investigational product observed by the investigator or reported by the subject (whether or not attributed to investigational product), will be reported on the case report form. Medically significant adverse events considered related to the investigational product by the investigator or the sponsor will be followed until resolved or considered stable. The following attributes must be assigned by the investigator: description; dates of onset and resolution; severity; assessment of relatedness to investigational product, and action taken. The investigator may be asked to provide follow-up information. All deaths occurring on study must be reported to Amgen or designee. These include deaths within 30 days of the last investigational product dose and deaths up to the last formal follow-up observational period, whichever is longer. All medically confirmed deaths should be reported on a CRF, and the autopsy reports should be sent when available. The investigator should notify the Independent Ethics Committee (IEC)/Institutional Review Board (IRB) of serious adverse events occurring at the site and other adverse event reports received from Amgen or designee, in accordance with local procedures. It will be left to the investigator s clinical judgment whether or not an adverse event is of sufficient severity to require the subject s removal from treatment. However, investigational product must be discontinued in subjects who have a current diagnosis of cancer, with the exception of localized basal cell and squamous cell carcinoma of the skin, or intra-epithelial neoplasia of the uterine cervix. A subject may also voluntarily withdraw from treatment due to what he or she perceives as an intolerable adverse event.

50 Date: 17 April 2008 Page 49 of 96 Subjects removed from treatment will continue to participate in the study and be followed for the collection of follow-up data. The subject must be given appropriate care under medical supervision until symptoms cease or the condition becomes stable. If the subject was permanently withdrawn from investigational product administration due to a serious adverse event, this information must be included in either the initial or follow-up Serious Adverse Event Report Form. The severity of toxicities will be assessed on the following scale with appropriate clinical definitions: 1 = mild, 2 = moderate, 3 = severe, 4 = life threatening, and 5 = fatal. The relationship of adverse events to the investigational product will be assessed by means of the question: Is there a reasonable possibility that the event may have been caused by the investigational product? Answer Yes or No. These grading scales are further described in Appendix B. 9.4 Serious Adverse Event Reporting Procedures All serious adverse events must be reported to Amgen or designee within 1 working day of discovery or notification of the event. Initial serious adverse event information and all amendments or additions must be recorded on a Serious Adverse Event Report form (Appendix C) and faxed to: US and Latin American sites: ICON at Western European sites: ICON Medical United Kingdom at Eastern European sites: ICON Medical Germany at Australian sites: Amgen at Canadian sites: Amgen at or Data and Safety Monitoring Committee (DSMC) An external DSMC will review the safety data on an ongoing basis throughout the course of the trial. Safety data reports will be prepared for the DSMC by an independent statistical group with expertise in safety data reporting for large cardiovascular outcomes trials. Additional details may be found in Section and in the DSMC charter.

51 Date: 17 April 2008 Page 50 of STATISTICAL CONSIDERATIONS 10.1 Study Design TREAT is a randomized, double-blind, controlled, multicenter study evaluating the effect of anemia therapy with darbepoetin alfa on the composite event comprising all-cause mortality and CV morbidity. The study will conclude when approximately 1203 composite events have occurred (approximately 4.5 years after the first subject enrolls). Approximately 4000 subjects will be enrolled. For subjects randomized to Group A, darbepoetin alfa will be administered Q2W until the target Hb is achieved, and then extended to a QM regimen. Subjects randomized to Group B will receive placebo if the Hb is 9.0 g/dl. If the Hb is < 9.0 g/dl, subjects in this treatment group will receive rescue therapy with darbepoetin alfa. To maintain blinding, the dose changes will be balanced between Groups A and B using an IVRS algorithm; for subjects randomized to Group B, a dynamic dose change schedule that imitates the Group A dose changes will be implemented to minimize the possibility of the investigator knowing the treatment group of each subject. Hb values to determine darbepoetin alfa/placebo dosing will be obtained at the site using a hemoglobin point of care device provided by Amgen by a third party not directly involved in the study subject s care to preserve blinding of Hb values to study personnel directly involved in subject care. The IVRS will calculate and manage the initial doses and all subsequent dose adjustments of investigational product. For analyses of the primary endpoint, missing data will not be imputed Health Economic Considerations In support of the importance of darbepoetin alfa therapy in this setting, health care decision-makers will require information on its economic (resource utilization) and humanistic (quality of life) benefits. With the exception of drugs that improve glucose levels, no CV disease product indicated for the prevention of clinical events has demonstrated an ability to improve quality of life preceding the CV event. Feasibility assessments indicate that the improvement in quality of life associated with anemia management could be as important as the decrease in the consumption of health-care resources associated with fewer CV events.

52 Date: 17 April 2008 Page 51 of 96 Quality of life, physical function and resource utilization data will be collected on PRO (EQ-5D, FACT-fatigue, and SF-36) and HRU questionnaires. For PRO and HRU analyses, missing data will be imputed using last observation carried forward (and other methods such as the last rank carried forward to investigate the impact of the imputation procedure) Subjects Sets, Study Endpoints, and Analyses Efficacy Analysis Set: Efficacy analyses will be performed on the intent-to-treat (ITT) subject set, which is defined as all subjects who were randomized. Efficacy analyses will include all follow-up from all randomized subjects. Safety Summary Set: Safety analyses will be performed on the ITT subjects who received at least one dose of investigational product. Safety analyses will be based on follow-up between the first dose of investigational product and one week (7 days) following the permanent discontinuation of blinded investigational product. Adverse events resulting in death will be collected during the entire study or until 30 days after the last dose of investigational product, whichever is later. PRO Analysis Set: PRO analyses will be performed on the ITT subject set. In addition, sensitivity analyses may be carried out using ITT subjects who complete both a screening and week 25 questionnaire. Functional Capacity Sub-study Analysis Set: A sub-study will be conducted in subjects to assess the distance walked in six minutes. The analysis on this endpoint will be conducted on all subjects who elect to participate in this sub-study and complete both baseline and post-baseline assessments Primary Endpoint Analysis The primary endpoint is time to the composite event consisting of all-cause mortality or CV events (myocardial ischemia, CHF, MI, and CVA). The principal analysis for the primary endpoint employs the intent-to-treat principle and uses a life-table analysis. For each treatment group, Kaplan-Meier curves will be estimated and graphically displayed. The two survival functions will be compared using a two-sided log-rank test (stratified by level of baseline proteinuria [spot urine protein/urine creatinine ratio], and history of CV disease), at a significance level, accounting for four interim analyses (which are described in Section 10.6, below).

53 Date: 17 April 2008 Page 52 of 96 The overall type 1 error rate in this study is controlled at an 0.05 level, which will be divided between the primary endpoint and the key secondary endpoint with and 0.002, respectively. This multiplicity adjustment ensures the validity of testing the treatment effect from either the primary endpoint or the key secondary endpoint. After accounting for four interim analyses of the primary endpoint (described in Section 10.6 below), the significance level remaining for the final analyses of the primary endpoint is A supportive analysis of the primary endpoint uses Cox s Proportional Hazard model to evaluate the covariates that may modify the outcome of the primary analysis. The hazard ratio and its corresponding 95% confidence interval will be estimated. The selection of the covariates will be prospectively specified in the statistical analysis plan (prior to study commencement). Subjects who withdraw from treatment with investigational product early, including those who commence RRT, will continue to have all study procedures performed and be followed for the development of the primary endpoint. Such data will be used in the primary analysis. Censoring: Subjects who withdraw from treatment with investigational product early will be followed for the potential development of the primary endpoint and will be censored if the composite event does not occur (or follow-up data are not obtainable). Subjects completing the study and not experiencing the composite event will be censored. Subjects progressing to RRT will be included in the analysis Secondary Endpoint Analyses The key secondary endpoint is: Time to ESRD or all-cause mortality The other secondary endpoints are: Time to all-cause mortality Time to CV mortality Time to myocardial ischemia Time to MI Time to CVA

54 Date: 17 April 2008 Page 53 of 96 Time to CHF Time to ESRD Rate of decline in egfr relative to baseline Change in patient reported fatigue relative to baseline at week 25 There will be one final analysis for the key secondary endpoint, with an alpha at The other secondary endpoints will be tested for statistical significance only if either the primary or secondary endpoint is statistically significant. The type 1 error rate associated with the multiplicity of comparisons for these other secondary endpoints will be controlled according to a Hochberg adjustment at an overall significance level that will depend on the outcome of the primary and key secondary endpoints as described below: If both the primary endpoint and the key secondary endpoint are statistically significant, then the overall significance level for the other secondary endpoints will be If the primary endpoint is statistically significant and the key secondary endpoint is not, then the overall significance level for the other secondary endpoints will be If the primary endpoint is not statistically significant and the key secondary endpoint is, then the overall significance level for the other secondary endpoints will be For the time-to-event secondary endpoints, an analysis similar to the one employed for the primary endpoint will be used. The censoring mechanism will also be similar to the one used for the analysis of the primary endpoint. For the rate of decline in egfr, a linear mixed effects model will be used. Subject fatigue will be measured by the FACT-Fatigue scale as the change from baseline for ITT subjects. Mean changes and standard errors will be computed and compared across the two randomized treatment groups, using a significance level of Ninety-five percent confidence intervals will be calculated for each. A generalized linear model will be applied to evaluate the covariates, particularly baseline FACT-fatigue scores, which may modify the outcome of the secondary analysis. The specific generalized linear model and the selection of the covariates will be prospectively specified prior to study commencement. The coefficient and corresponding 95% confidence interval will be determined.

55 Date: 17 April 2008 Page 54 of Analyses of Other Endpoints of Interest Other endpoints of interest include: Time to cardiac revascularization Change in urine protein/urine creatinine ratio relative to baseline Health resource utilization (HRU) per patient follow-up time Change in patient reported outcomes (PRO), other than fatigue, relative to baseline at week 25 Change in physical and mental composite scores of the SF-36 relative to baseline The analysis of time to revascularization and change in urine protein/urine creatinine ratio relative to baseline will be similar to the analysis of the primary endpoint. The other PRO endpoints include the change from baseline in the EQ-5D at week 25. The changes in functional capacity endpoints include the change from baseline in 2 composite scores of the SF-36 (PCS-36 and MCS-36) at each timepoint. These endpoints will be analyzed using the ITT subjects. Mean change from baseline and standard errors will be computed and compared across the two randomized treatment groups, using a significance level of Ninety-five percent confidence intervals will be calculated for each. A generalized linear model will be applied to evaluate covariates, particularly baseline scores, which may modify the outcome of the secondary analysis. The coefficient and corresponding 95% confidence interval will be determined. The selection of the covariates will be prospectively specified prior to study start. For HRU, a total cost from across the entire study period will be calculated and normalized to monthly follow-up time. Point estimates and 95% confidence intervals will be calculated for each treatment group and for the difference between the treatment groups. A statistical model may be applied to evaluate covariates, which may modify the outcome of the analysis. The coefficient and corresponding 95% confidence interval will be determined. The selection of the covariates will be prospectively specified prior to study start. Although PRO data will be collected throughout the study period and the observation period, some missing data are expected. Imputation rules for missing values due to early withdrawals while on study will be specified prior to study start. Missing PRO data for other reasons will be imputed using last observation carried forward, during the study period. Sensitivity analyses using alternative methods of imputation will be conducted to assess the robustness of study results.

56 Date: 17 April 2008 Page 55 of Analyses of Functional Capacity Sub-study Endpoints The endpoints in the functional capacity sub-study are the changes from baseline in the distance walked during six minutes at each timepoint. These endpoints will be analyzed using analysis of covariance (adjusted for baseline distance walked during six minutes) with the Functional Capacity Sub-study Analysis Set. Mean change from baseline and standard errors will be computed and compared across the two randomized treatment groups, using a significance level of Ninety-five percent confidence intervals will be calculated for each Sample Size Considerations Sample Size Consideration for the Primary Endpoint The sample size calculation for the primary endpoint assumes a Group B composite event rate of approximately 12.5% per year 33, a 2-year enrollment period, and a 15% lost to follow-up rate over the approximately 4.5 year study duration. The true hazard ratio associated with the use of darbepoetin alfa is assumed to be 0.8; however, it is anticipated that attenuation of the treatment effect will occur due to treatment lag at the beginning of the trial and the initiation of therapy with erythropoietic stimulating proteins in all subjects requiring RRT during the course of the study. After accounting for these factors, and based on a two-sided log-rank test using a significance level (which is based on an overall alpha at and accounts for four planned interim analyses with , , , and significance levels respectively), 1203 primary endpoints are require to ensure adequate power (80%) with a total sample size of approximately 4000 subjects Sample Size Consideration for Functional Capacity Sub-study The sample size consideration for the functional capacity sub-study assumes a standard deviation is 537 ft and lost to follow up rate of 15% over the duration of the sub-study. 35 Based on a 2-sided independent t-test with an α of 0.05, approximately 500 subjects per group would be needed to provide 90% power to detect a mean difference between Group A and Group B of 120 ft from baseline Access to Individual Subject Treatment Assignments The individual subject treatment assignments will be maintained by IVRS. This will be provided to the DSMC if requested. Unblinded results including aggregated and subject level data will be presented to the DSMC for safety and interim efficacy data reviews.

57 Date: 17 April 2008 Page 56 of 96 Only the DSMC, and neither the sponsor nor other committees will have access to the treatment assignments before unblinding the final database Interim Analysis and Early Stopping Guidelines An external independent statistical analysis center will perform safety analyses in support of the DSMC and the interim efficacy analyses. The interim results will not be shared with the sponsor. An independent DSMC consisting of members with relevant expertise will be assembled prior to study commencement. Safety data and enrollment trends will be periodically reviewed throughout the study. The DSMC will participate in the conduct of four planned efficacy interim analyses. The DSMC will advise the executive committee and study sponsor on potential findings that may impact the conduct of the study, and communicate recommendations based on the results of the efficacy interim analyses. The four planned efficacy interim analyses of the primary endpoint will occur when approximately 20%, 40%, 60%, and 80% of the cumulative composite events have been observed. An O Brien-Fleming alpha-spending function with truncated symmetric monitoring boundaries 36 is used to allocate significance levels of , , , and at the interims, and a final significance level of for the testing of the primary endpoint. Early stopping rules will be precisely pre-specified prior to study start Planned Methods of Analysis General Approach/Considerations Baseline characteristics, demographics, pre-existing conditions, historical illness, and possible prior treatments will be summarized for each treatment group. Subject disposition and reason for discontinuation will be summarized for each group. Mean dose of investigational product administered at scheduled weeks will be summarized for the treatment group. The number of subjects who received darbepoetin alfa as a rescue therapy and the dosage will be summarized for Group B.

58 Date: 17 April 2008 Page 57 of Analysis of Efficacy Endpoints Analyses will be performed on the time-to-event data for the primary endpoint and its individual components (consisting of all-cause mortality, MI, CVA, CHF, and myocardial ischemia). The analysis of the key secondary endpoint will be similar. Supportive analyses adjusting for covariates use the Cox Proportional Hazards model. 25 The potential modification of the outcome of the primary analysis will be assessed. The selection of covariates will be prospectively specified and the hazards ratio of Group A versus Group B for the primary endpoint will be estimated using Cox model accounting Additional Analyses Subgroup analyses based on sex, race, and age will be performed. Additional exploratory analyses may be performed if deemed necessary. Analyses of recurrent events for the non-fatal CV components of the primary endpoint will be performed. Concomitant medications of interest will be summarized for each group and will be compared between groups to assess the potential impact on the primary endpoint Safety Analyses The subject incidence of adverse events will be tabulated by system organ class and preferred term for each group. Adverse events considered related to investigational product, as well as serious adverse events, will be summarized. Tables and/or narratives of on-study deaths and serious and significant adverse events, including early withdrawals due to adverse events, will also be provided. In addition, the exposureadjusted event rate for each adverse event will be calculated as the number of events per subject-year at risk. Changes in laboratory parameters and BP will be summarized. A summary table of Hb will be provided based on the measurements from the hemoglobin point of care device provided by Amgen. If anti-erythropoietic protein seroreactivity is detected, the number and proportion of subjects with confirmed antibodies will be tabulated.

59 Date: 17 April 2008 Page 58 of Study Oversight Data and Safety Monitoring Committee (DSMC) The DSMC is an independent, multidisciplinary group comprising medical and statistical representatives. Safety data will be reviewed by the DSMC on a periodic basis and will assess enrollment trends. In addition, the DSMC will participate in the review of findings of four planned efficacy interim analyses and provide risk-benefit assessment of the trial conduct Executive Committee An executive committee comprising experts in the renal, diabetic, and cardiovascular fields will provide oversight and advice to ensure the most efficient conduct and execution of the trial. The Executive Committee will consider sample size reexamination in the eventuality that the observed blinded pooled event rate for the primary endpoint falls below its assumed estimate in the design stage Endpoint Adjudication Committee An external endpoint adjudication committee will be formed prior to study commencement to adjudicate the components of the primary endpoint and ESRD in a blinded fashion. The principal analysis of the primary and key secondary endpoints will use the adjudicated results 11. INVESTIGATIONAL PRODUCT 11.1 Darbepoetin alfa Darbepoetin alfa will be presented as a clear, colorless, sterile protein solution in prefilled syringes (PFS) of the following strengths: 10, 15, 20, 30, 40, 50, 60, 80, 100, 150, 200, and 300 µg. Placebo will be presented as a clear, colorless solution in PFS identical to those containing the active drug. The box number of investigational product (active drug or placebo) is to be recorded on each subject s Drug Administration case report form. This must be done in a manner such that a subject s treatment assignment will not be unblinded during the trial.

60 Date: 17 April 2008 Page 59 of 96 Additional information regarding details of the investigational product including labeling, storage, preparation, etc. are provided in the Pharmacy Guide Appendix D Access to Blinded Investigational Product Assignment The identity of investigational product assigned to subject numbers or to individual boxes of investigational product will be maintained by IVRS. Authorized site staff will be provided with a unique Personal Identification Number (PIN) to access the IVRS to obtain unblinding information. This PIN is unique to the individual and must not be shared. A subject s treatment assignment should only be unblinded when knowledge of the treatment is essential for the further management of the subject. Unblinding at the study site for any other reason will be considered a protocol deviation. The principal investigator is strongly encouraged to contact the Amgen study manager before unblinding any subject s treatment assignment, but must do so within 1 working day after the event and must document the unblinding in the subject s case report form. 12. REGULATORY OBLIGATIONS 12.1 Informed Consent Before a subject s participation in the trial, the investigator or designee is responsible for obtaining written informed consent from the subject or legally acceptable representative after adequate explanation of the aims, methods, anticipated benefits, and potential hazards of the study and before any protocol-specific screening procedures or any investigational products are administered. The investigator or designee is also responsible for asking the subject if the subject agrees to have his/her primary care physician informed of the subject s participation in the clinical trial. If the subject agrees to such notification, the investigator should inform the subject s primary care physician of the subject s participation in the clinical trial. The acquisition of informed consent and the subject s agreement or refusal of his/her notification of the primary care physician should be documented in the subject s medical records, and the informed consent form should be signed and personally dated by the subject or a legally acceptable representative and by the person who conducted the informed consent discussion (not necessarily an investigator). The original signed informed consent form should be retained in accordance with institutional policy, and a

61 Date: 17 April 2008 Page 60 of 96 copy of the signed consent form should be provided to the subject or legally acceptable representative. If a potential subject or legally acceptable representative is illiterate or visually impaired the investigator must provide an impartial witness to read the informed consent form to the subject or legally acceptable representative and allow for questions. Thereafter, both the subject or legally acceptable representative and the witness must sign the informed consent form to attest that informed consent was freely given and understood Independent Ethics Committee/Institutional Review Board A copy of the protocol, proposed informed consent form, other written subject information, and any proposed advertising material must be submitted to the IEC/IRB for written approval. A copy of the written approval of the protocol and informed consent form must be received by Amgen or designee before recruitment of subjects into the study and shipment of investigational product. The investigator must submit and, where necessary, obtain approval from the IEC/IRB for all subsequent protocol amendments and changes to the informed consent document. The investigator should notify the IEC/IRB of significant deviations from the protocol or serious adverse events occurring at the site and other adverse event reports received from Amgen or designee, in accordance with local procedures. The investigator will be responsible for obtaining annual IEC/IRB approval/renewal throughout the duration of the study. Copies of the investigator s reports and the IEC/IRB s continuance of approval must be sent to Amgen or designee Prestudy Documentation Requirements The investigator is responsible for forwarding the following documents to Amgen or designee for review before study initiation from Amgen Inc. can occur: Signed and dated protocol signature page (Investigator s Agreement) Copy of approved informed consent form and subject information sheet, if applicable Copy of the IEC/IRB approval of the protocol, consent form, and subject information sheet Up-to-date curricula vitae of principal investigator and all co/subinvestigators The IEC/IRB composition and/or written statement that IEC/IRB is in compliance with regulations

62 Date: 17 April 2008 Page 61 of 96 Laboratory normal ranges and documentation of laboratory certification (or equivalent) Signed study contract Completed FDA form 1572 Completed Financial Disclosure statements for the principal investigator, all subinvestigators, and their spouses (legal partners) and dependent children 12.4 Subject Confidentiality The investigator must ensure that the subject s confidentiality is maintained. On the case report forms or other documents submitted to Amgen or designee subjects should be identified by their initials and a subject study number only. Documents that are not for submission to Amgen or designee (eg, signed informed consent forms) should be kept in strict confidence by the investigator. In compliance with Federal regulations/ich GCP Guidelines, it is required that the investigator and institution permit authorized representatives of the company, of the regulatory agency(s), and the IEC/IRB direct access to review the subject s original medical records for verification of study-related procedures and data. Direct access includes examining, analyzing, verifying, and reproducing any records and reports that are important to the evaluation of the study. The investigator is obligated to inform and obtain the consent of the subject to permit named representatives to have access to his/her study-related records without violating the confidentiality of the subject Investigator Signatory Obligations Each clinical study report should be signed by the investigator or, in the case of multicenter studies, the coordinating investigator. The coordinating investigator, identified by Amgen, will either be: a recognized expert in the therapeutic area an investigator who provided significant contributions to either the design or interpretation of the study an investigator contributing a high number of eligible subjects

63 Date: 17 April 2008 Page 62 of ADMINISTRATIVE AND LEGAL OBLIGATIONS 13.1 Protocol Amendments and Study Termination Protocol amendments must be made only with the prior approval of Amgen. Agreement from the investigator must be obtained for all protocol amendments and amendments to the informed consent document. The IEC/IRB must be informed of all amendments and give approval for all amendments including any amendments likely to affect the safety of the subjects or the conduct of the trial. The investigator must send a copy of the approval letter from the IEC/IRB to Amgen or designee. Both Amgen and the investigator reserve the right to terminate the study, according to the study contract. The investigator should notify the IEC/IRB in writing of the trial s completion or early termination and send a copy of the notification to Amgen or designee Study Documentation and Storage The investigator should maintain a list of appropriately qualified persons to whom he/she has delegated trial duties. All persons authorized to make entries and/or corrections on case report forms will be included on the Amgen Delegation of Authority Form. Source documents are original documents, data, and records from which the subject s case report form data are obtained. These include but are not limited to hospital records, clinical and office charts, laboratory and pharmacy records, diaries, microfiches, radiographs, and correspondence. The investigator and study staff are responsible for maintaining a comprehensive and centralized filing system of all study-related (essential) documentation, suitable for inspection at any time by representatives from Amgen and/or applicable regulatory authorities. Elements should include: Subject files containing completed case report forms, informed consents, and supporting copies of source documentation Study files containing the protocol with all amendments, investigator s brochure, copies of prestudy documentation (see Section 12.3), and all correspondence to and from the IEC/IRB and Amgen

64 Date: 17 April 2008 Page 63 of 96 Study files containing the proof of receipt, Investigational Product Accountability Record, Return of Investigational Product for Destruction, Final Investigational Product Reconciliation Statement, and all drug-related correspondence In addition, all original source documents supporting entries in the case report forms must be maintained and be readily available. No study document should be destroyed without prior written agreement between Amgen and the investigator. Should the investigator wish to assign the study records to another party or move them to another location, he/she must notify Amgen in writing of the new responsible person and/or the new location Study Monitoring and Data Collection The Amgen representative or designee and regulatory authority inspectors are responsible for contacting and visiting the investigator for the purpose of inspecting the facilities and, upon request, inspecting the various records of the trial (eg, case report forms and other pertinent data) provided that subject confidentiality is respected. The Amgen monitor or designee is responsible for inspecting the case report forms at regular intervals throughout the study to verify adherence to the protocol; completeness, accuracy, and consistency of the data; and adherence to local regulations on the conduct of clinical research. The monitor should have access to subject medical records and other study-related records needed to verify the entries on the case report forms. The investigator agrees to cooperate with the monitor to ensure that any problems detected in the course of these monitoring visits, including delays in completing case report forms, are resolved. In accordance with ICH GCP and the sponsor s audit plans, this study may be selected for audit by representatives from Amgen s clinical quality assurance department (or designees). Inspection of site facilities (eg, pharmacy, drug storage areas, laboratories) and review of study-related records will occur to evaluate the trial conduct and compliance with the protocol, ICH GCP, and applicable regulatory requirements. All paper case report forms should be typed or filled out with a black ball-point pen and must be legible.

65 Date: 17 April 2008 Page 64 of 96 Corrections to paper forms will be made by a single line stroke through the error and insertion of the correction above or beside the error. The change must be initialed and dated by the investigator or a member of the study staff authorized by the investigator on the Amgen Delegation of Authority Form. No erasures, correction fluid, or tape may be used. Corrections to electronic forms will be automatically documented through the software s audit trail. To ensure the quality of clinical data across all subjects and sites, a clinical data management review will be performed on subject data received at Amgen. During this review, subject data will be checked for consistency, omissions, and any apparent discrepancies. In addition, the data will be reviewed for adherence to the protocol and GCP. To resolve any questions arising from the clinical data management review process, data queries and/or site notifications will be sent to the site for completion and return to Amgen or designee. The principal investigator will sign and date the indicated places on the case report form. These signatures will indicate that the principal investigator inspected or reviewed the data on the case report form, the data queries, and the site notifications, and agrees with the content. Amgen s clinical data management department or designee will correct the database for the following CRF issues without notification to site staff: - misspellings that do not change the meaning of the word (excluding adverse events and medications) - location of data recorded on an incorrect CRF (eg, moving lab data from general comments to the appropriate lab table) - date errors that occur into the new year - standard time to 24-hour clock - temperature unit errors (Fahrenheit vs Centigrade) - weight unit errors (pounds vs kilograms) if a baseline weight has been established - height unit errors (in. vs cm) - administrative data (eg, event names for unscheduled visits or retests)

66 Date: 17 April 2008 Page 65 of 96 - clarifying other, specify if data are provided (eg, race, physical exam) - correcting or entering either Absolute (A)/Percentage (P) on Hematologies if blank; can be determined from differential data - if both the end date and a status of continuing is indicated (eg, for adverse events, concomitant medication, hospitalization) the end date will supersede - deleting obvious duplicate data (eg, same results sent twice with the same date but different clinical planned events week 4 and early termination) - for adverse events that record action taken code of 01 (none) in addition to any other action code, the code of 01 (none) may be deleted as it is superseded by other existing data - if equivalent units or terms are recorded instead of the acceptable Amgen standard (eg, cc for ml, SQ for SC route, Not Examined for Not Done), the Amgen units or terms will be used - if the answer to a YES or NO question is blank or obviously incorrect (eg, Answers to the following questions do not reflect the data that are recorded or missing: Were there any adverse events? Concomitant medications? Hospitalizations?) - correct CRF page numbers - for concomitant medications, if doses overlap for the same medication, they will be corrected to have consecutive dates - if concomitant medication unit, route, or frequency code is not part of the code list on the CRF, it will be changed to OT = other - if a discrepancy answer indicates an addition to medical history or physical exam, then a record will be added with a code of 88 = other 13.4 Language Case report forms must be completed in English. TRADENAMES for concomitant medications may be entered in the local language.

67 Date: 17 April 2008 Page 66 of 96 All written information and other material to be used by subjects and investigative staff must use vocabulary and language that are clearly understood Publication Policy A publication committee will be formed for this trial. Authorship of any publications resulting from this study will be determined on the basis of the Uniform Requirement for Manuscripts Submitted to Biomedical Journals (International Committee of Medical Journal Editors, 1997) which states: Each author should have participated sufficiently in the work to take public responsibility for the content. Authorship credit should be based on only substantial contributions to (a) conception and design, or analysis and interpretation of data; and to (b) drafting the article or revising it critically for important intellectual content; and on (c) final approval of the version to be published. Conditions (a), (b), and (c) must all be met. All publications (eg, manuscripts, abstracts, oral/slide presentations, book chapters) based on this study must be submitted to Amgen for corporate review. The Clinical Trial Agreement among the institution, principal investigator, and Amgen will detail the procedures for, and timing of, Amgen s review of publications Compensation Subjects will be treated and/or compensated for any study-related illness/injury pursuant to the information provided in the Compensation for Injury section of the Informed Consent (Appendix F). Depending on the type of study, subjects may be compensated for other inconveniences not associated with study-related injuries (eg, child care costs).

68 Date: 17 April 2008 Page 67 of REFERENCES 1. Coresh J, Astor BC, Greene T, Eknoyan G, Levey AS. Prevalence of chronic kidney disease and decreased kidney function in the adult US population: Third National Health and Nutrition Examination Survey. American Journal of Kidney Diseases 2003;41(1): Mogensen CE. Reduced progression of diabetic nephropathy by controlling hypertension. Practical Cariology 1983;9(4): Lewis EJ, Hunsicker LG, Bain RP, Rohde RD. The effect of angiotensin-convertingenzyme inhibition on diabetic nephropathy. The Collaborative Study Group. New England Journal of Medicine 1993;329(20): Brenner BM, Cooper ME, de Zeeuw D, et al. Effects of losartan on renal and cardiovascular outcomes in patients with type 2 diabetes and nephropathy. New England Journal of Medicine 2001;345(12): Lewis EJ, Hunsicker LG, Clarke WR, et al. Renoprotective effect of the angiotensinreceptor antagonist irbesartan in patients with nephropathy due to type 2 diabetes.[comment]. New England Journal of Medicine 2001;345(12): United States Renal Data System: USRDS 2003 Annual Data Report. Bethesda, MD: National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases; Keith D, Nichols G, Gullion C, Brown J, Smith D. Mortality of chronic kidney disease (CKD) in a large HMO population. ASN abstract 2002:SU-P Collins AJ. The hemoglobin link to adverse outcomes. Advanced Studies in Medicine 2003;3(3C):S14-S7. 9. Shulman NB. Ford CE. Hall WD. Blaufox MD. Simon D. Langford HG. Schneider KA. Prognostic value of serum creatinine and effect of treatment of hypertension on renal function. Results from the hypertension detection and follow-up program. The Hypertension Detection and Follow-up Program Cooperative Group. Hypertension. 1989;13; , 1989 May. 10. Culleton BF. Larson MG. Wilson PW. Evans JC. Parfrey PS. Levy D. Cardiovascular disease and mortality in a community-based cohort with mild renal insufficiency. Kidney International. 1999;56; Levin A. Prevalence of cardiovascular damage in early renal disease. Nephrology Dialysis Transplantation. 2001;16(Suppl 2): London G. Pathophysiology of cardiovascular damage in the early renal population. Nephrology Dialysis Transplantation 2001;16(Suppl 2): Manjunath G, Tighiouart H, Ibrahim H, MacLeod B, Salem DN, Griffith JL. Coresh J. Levey AS. Sarnak MJ. Level of kidney function as a risk factor for atherosclerotic cardiovascular outcomes in the community. Journal of the American College of Cardiology. 2003;41;47-55.

69 Date: 17 April 2008 Page 68 of Greaves SC, Gamble GD, Collins JF, Whalley GA, Sharpe DN. Determinants of left ventricular hypertrophy and systolic dysfunction in chronic renal failure. American Journal of Kidney Diseases 1994;24(5): Tucker B, Fabbian F, Giles M, Thuraisingham RC, Raine AE, Baker LR. Left ventricular hypertrophy and ambulatory blood pressure monitoring in chronic renal failure.[comment]. Nephrology Dialysis Transplantation 1997;12(4): Levin A, Singer J, Thompson CR, Ross H, Lewis M. Prevalent left ventricular hypertrophy in the predialysis population: identifying opportunities for intervention. American Journal of Kidney Diseases 1996;27(3): Parfrey PS, Foley RN, Harnett JD, Kent GM, Murray DC, Barre PE. Outcome and risk factors for left ventricular disorders in chronic uraemia. Nephrology Dialysis Transplantation 1996;11(7): Silberberg JS, Barre PE, Prichard SS, Sniderman AD. Impact of left ventricular hypertrophy on survival in end-stage renal disease. Kidney International 1989;36(2): Haffner SM, Lehto S, Ronnemaa T, Pyorala K, Laakso M. Mortality from coronary heart disease in subjects with type 2 diabetes and in nondiabetic subjects with and without prior myocardial infarction. New England Journal of Medicine 1998;339(4): Kooman JP. Leunissen KM. Cardiovascular aspects in renal disease. Current Opinion in Nephrology & Hypertension.1993,2; Longenecker JC, Coresh J, Powe NR, et al. Traditional cardiovascular disease risk factors in dialysis patients compared with the general population: the CHOICE Study. Journal of the American Society of Nephrology 2002;13(7): Foley RN. Parfrey PS. Harnett JD. Kent GM. Murray DC. Barre PE. The impact of anemia on cardiomyopathy, morbidity, and mortality in end-stage renal disease. American Journal of Kidney Diseases. 1996;28(1): Mann JF. What are the short-term and long-term consequences of anaemia in CRF patients? Nephrology Dialysis Transplantation. 1999;14(Suppl 2); Levin A, Thompson CR, Ethier J, et al. Left ventricular mass index increase in early renal disease: impact of decline in hemoglobin. American Journal of Kidney Diseases 1999;34(1): Abramson JL, Jurkovitz CT, Vaccarino V, Weintraub WS, McClellan W. Chronic kidney disease, anemia, and incident stroke in a middle-aged, community-based population: the ARIC study. Kidney International 2003;64: Al-Ahmad A, Rand WM, Manjunath G, et al. Reduced kidney function and anemia as risk factors for mortality in patients with left ventricular dysfunction. Journal of the American College of Cardiology 2001;38(4): McClellan WM, Flanders WD, Langston RD, Jurkovitz C, Presley R. Anemia and renal insufficiency are independent risk factors for death among patients with

70 Date: 17 April 2008 Page 69 of 96 congestive heart failure admitted to community hospitals: a population-based study. Journal of the American Society of Nephrology 2002;13(7): Silverberg DS, Wexler D, Blum M, et al. The correction of anemia in severe resistant heart failure with erythropoietin and intravenous iron prevents the progression of both the heart and the renal failure and markedly reduces hospitalization. Clinical Nephrology 2002;58(Suppl 1):S Silverberg DS, Wexler D, Blum M, et al. The effect of correction of anaemia in diabetics and non-diabetics with severe resistant congestive heart failure and chronic renal failure by subcutaneous erythropoietin and intravenous iron. Nephrology Dialysis Transplantation 2003;18(1): Darbepoetin alfa Investigator s Brochure, version 4. Thousand Oaks, CA: Amgen Inc.; April Gouva C, Nikolopoulos P, Ioannidis JP, Siamopoulos KC. Treating anemia early in renal failure patients slows the decline of renal function: a randomized controlled trial. Kidney International 2004; 66: Rodby RA, Rohde RD, Clarke WR, et al. The Irbesartan Type II Diabetic Nephropathy Trial: study design and baseline patient characteristics. Nephrology Dialysis Transplantation 2000;15: Medical Archive Retrieval System (MARS) Database, University of Pittsburgh Medical Center. 34. Shih JH. Sample size calculation for complex clinical trials with survival endpoints. Control Clin Trials 1995; 16: Mancini, Donna M. Effect of erythropoietin on exercise capacity in patients with moderate to severe heart failure. Circulation, 2003; 107: O'Brien P.C., Fleming T. R. A Multiple testing procedure for clinical trials. Biometrics. 1979;35: Cox D. R. Regression models and life-tables (with discussion). Journal of Royal Statistical Society. 1972;B, 34: Schwartz GG, Oliver MF, Ezekowitz MD, et. al. Rationale and design of the myocardial ischemia reduction with aggressive cholesterol lowering (MIRACL) study that evaluates Atorvastatin in unstable angina pectoris and non-w-wave acute myocardial infarction. Am. J. Cardiol. 1998;81: Obrador GT, Roberts T, St. Peter WL, Frazier E, Periera BJ, Collins A. Trends in anemia at initiation of dialysis in the United States. Kidney International. 2001;60: Hsu C-Y, McCullough C.E., Curhan G.C. Epidemiology of anemia associated with chronic renal insufficiency among adults in the United States: Results from the third national health and nutrition examination survey. J Am. Soc. Nephrol. 2002;13:

71 Date: 17 April 2008 Page 70 of APPENDICES Appendix A. Schedule of Assessments *egfr and spot urine tests will not be performed on subjects who require RRT **Adverse events will be collected from the date of the first dose through one week (7 days) following the date of the last dose of investigational product.

72 Date: 17 April 2008 Page 71 of 96 Appendix A. Schedule of Assessments (continued) *egfr and spot urine tests will not be performed on subjects who require RRT **Adverse events will be collected from the date of the first dose through one week (7 days) following the date of the last dose of investigational product.

73 Date: 17 April 2008 Page 72 of 96 Appendix B. Adverse Event Assessments Is there a reasonable possibility that the event may have been caused by investigational product? No Yes The descriptions provided below will help guide the principal investigator in making the decision to choose either yes or no : No = There is no reasonable possibility that the event may have been caused by investigational product. The adverse event: may be judged to be due to extraneous causes such as disease or environment or toxic factors may be judged to be due to the subject s clinical state or other therapy being administered is not biologically plausible does not reappear or worsen when investigational product is readministered does not follow a temporal sequence from administration of investigational product Yes = There is a reasonable possibility that the event may have been caused by investigational product. The adverse event: follows a temporal sequence from administration of investigational product is a known response to the investigational product based on clinical or preclinical data could not be explained by the known characteristics of the subject s clinical state, environmental or toxic factors, or other therapy administered to the subject disappears or decreases upon cessation or reduction of dose of investigational product reappears or worsens when investigational product is readministered

74 Date: 17 April 2008 Page 73 of 96 Adverse Events Standard Grading Score 1 = MILD aware of sign or symptom, but easily tolerated 2 = MODERATE discomfort enough to cause interference with usual activity 3 = SEVERE incapacitating with inability to work or do usual activity 4 = LIFE-THREATENING refers to an event in which the patient was, in the view of the investigator, at risk of death at the time of the event. It does not refer to an event which hypothetically might have caused death if it were more severe. 5 = FATAL

75 Date: 17 April 2008 Page 74 of 96 Appendix C. Sample Serious Adverse Event Form