Risk Factor Identification and Assessment in Hypertension and Diabetes (RIAHD) Study

Final version 7.12 2 June 2004 1

Risk factor Identification and Assessment in Hypertension and Diabetes (RIAHD) study.

A survey and assessment of cardiovascular risk factors in Swedish hypertensive and diabetic patients.

Study Chair:

RIAHD study organization, Thomas Hedner, Department of Clinical Pharmacology, Sahlgrenska University Hospital, Göteborg, Sweden.

Supported by: The Swedish Society of Hypertension Lennart Hansson Memorial Fund

Sponsor: Bristol-Myers Squibb Sanofi-Synthelabo Final version 7.12 2 June 2004 2

TABLE OF CONTENTS

SUMMARY...... 4

1. STUDY OBJECTIVES...... 5 Primary Objective...... 5 Secondary Objectives...... 5 Background...... 5 Hypertension...... 5 Diabetes...... 6 Hyperviscosity...... 8 Inflammartory and endothelial markers...... 8 Clinical Judgement ...... 7 Microalbuminuria/ albuminuria...... 6 Type 2 diabetes and renal protection...... 7 Diabetes, hypertension and microalbuminuria - population in Sweden...... 8 MARELD...... 8 3. STUDY DESIGN...... 10

4. SELECTION AND ENROLLMENT OF SUBJECTS...... 11 Selection of centres in RIAHD...... 11 Exclusion criteria...... 11 Inclusion criteria High risk group (Hypertension, DM and MA/A)...... 11 Inclusion criteria Low risk group (Hypertension but no DM or MA/A)...... 12 Study Enrollment Procedures...... 12 5. CLINICAL AND LABORATORY EVALUATIONS...... 12 Risk assessment...... 12 Blood sampling...... 13 Clinical Judgement...... 13 Health Care consumption...... 13 Home Blood Pressure...... 13 Schedule of Evaluations...... 14 Special Instructions and Definitions of Evaluations...... 15 6. SUBJECT SAFETY...... 17

7. STATISTICAL CONSIDERATIONS...... 17 Parameters...... 17 Sample size...... 18 Statistical methods...... 19 Interim analysis...... 19 8. STUDY DURATION...... 19 Final version 7.12 2 June 2004 3

9. STUDY ORGANIZATION...... 19

10. ETHICAL AND REGULATORY STANDARDS...... 19

11. STUDY CONDUCT...... 20 Responsibilities of the Clinic(s)...... 20 Responsibilities of the Organizers/Sponsors...... 20 Source document requirements...... 21 The use and completion of case report forms (CRFs)...... 21 12. ADMINISTRATIVE POINTS AND PROCEDURES...... 21 Record retention in investigating centre(s)...... 21 13. CONFIDENTIALITY

14. OWNERSHIP OF DATA AND USE OF THE STUDY RESULTS...... 22

15. CLINICAL STUDY REPORT...... 22

16. PUBLICATIONS...... 22

17. PROTOCOL AMENDMENTS...... 23

18. BIBLIOGRAPHY...... 23

GLOSSARY AND ABBREVIATIONS …………………………………………………… 22

APPENDICES...... 27 Study Organisation And Participating Clinical Sites...... 28 Key study individuals (names and address)...... Study sites (Investigators and study nurses)...... Study Flow...... Study organization...... RIAHD Screening (the RIAHD High Risk Group)...... RIAHD Screening (the RIAHD Low Risk Group)...... Final version 7.12 2 June 2004 4

SUMMARY

Study Title Risk factor Identification and Assessment in Hypertension and Diabetes (RIAHD) study. A survey and assessment of cardiovascular risk factors in Swedish hypertensive and diabetic patients

Objectives The primary objective of the RIAHD study is: - Assessment of novel circulating cardiovascular risk factors (RF) such as blood viscosity and inflammatory markers in high-risk patients, i.e. those with hypertension, diabetes and micro/ macroalbuminuria versus age and gender matched low risk patients, i.e. those with hypertension without diabetes or micro/ macroalbuminuria.

Secondary objectives of the RIAHD study include: - To assess objective cardiovascular risk in high and low risk patients according to European Society of Hypertension/European Society of Cardiology (ESH/ESC) Systematic Coronary Risk Evaluation system (SCORE) - Assessment of subjective expressed risk (clinical judgment) by physicians and patients - Assessment of Home Blood Pressures and Office Blood Pressures to establish cardiovascular risk in cohorts - Assessment of the potential impact of selected genetic polymorphisms of cardiovascular relevance on risk profile in terms of other risk factors, associated clinical conditions (ACC) and presence of target organ damage (TOD) - Assessment of health economic impact of disease by evaluating health care utilisation, and sick leave - Reassessment of cardiovascular and metabolic morbidity as well as total mortality after 5 years

Design and Outcomes RIAHD is a multicentre cardiovascular screening of RF, ACC and TOD in high-risk patients (hypertension, diabetes and micro/ macroalbuminuria), in comparison with low risk patients (hypertension without diabetes or micro/ macroalbuminuria)

Visits and Duration The study will include one clinic visit where risk factors will be assessed by medical history, medical examination, blood sampling, blood pressure (BP) assessments and questionnaires.

The study informed consent will also include the possibility of a five-year follow-up.

Sample Size and Population Final version 7.12 2 June 2004 5

By screening of representative cohorts of patients/subjects, 500 high-risk patients (hypertension, diabetes and micro/ macroalbuminuria) and 500 low-risk patients (hypertension without diabetes or micro/ macroalbuminuria) will be included in the protocol. 1. STUDY OBJECTIVES

Primary Objective The primary objective of the RIAHD study is: - Assessment of novel circulating cardiovascular risk factors (RF) such as blood viscosity and inflammatory markers in high-risk patients, i.e. those with hypertension, diabetes and micro/ macroalbuminuria versus age and gender matched low risk patients, i.e. those with hypertension without diabetes or micro/ macroalbuminuria.

Secondary Objectives Secondary objectives of the RIAHD study include: - To assess objective cardiovascular risk in high and low risk patients according to European Society of Hypertension/European Society of Cardiology (ESH/ESC) Systematic Coronary Risk Evaluation system (SCORE) - Assessment of subjective expressed risk (clinical judgment) by physicians and patients - Assessment of Home Blood Pressures and Office Blood Pressures to establish cardiovascular risk in cohorts - Assessment of the potential impact of selected genetic polymorphisms of cardiovascular relevance on risk profile in terms of other risk factors, associated clinical conditions (ACC) and presence of target organ damage (TOD). - Assessment of health economic impact of disease by evaluating health care utilisation and sick leave - Reassessment of cardiovascular and metabolic morbidity as well as total mortality after 5 years

Background During the next decade cardiovascular metabolic disease will rank as the number one cause of morbidity and mortality in the world. Especially, hypertension and diabetes will increase in the developing world and this will account for a dramatic increase in complications (Murray and Lopez, (A and B) 1997). Due to this evolving scenario, novel strategies are needed to improve detection, treatment and follow up. In particular, preventive measures have to be implemented in order to influence the global increase in cardiovascular and cerebrovascular disease.

Hypertension Hypertension is a major risk factor for stroke and myocardial infarction (ESH/ESC Guidelines 2003). There is an increasing prevalence of systolic hypertension in the elderly, a condition, which seems to be carrying even more risk than diastolic hypertension. Also there is an increase Final version 7.12 2 June 2004 6

in patients with masked hypertension i.e. those patients who have high blood pressure on ambulatory or home blood pressure readings compared to clinic readings.

In particular, hypertension patients with additional risk factors (RF) as well as those with associated clinical conditions (ACC) and target organ damage (TOD) are at particular risk. In such patients, blood pressure levels for initiating treatments and also target levels on treatment are lower than those in low risk patients.

Diabetes In Sweden approximately 400000 patients have diabetes mellitus, 15% type-1 (IDDM) and 85% type-2 (NIDDM). Of the NIDDM patients, approximately 35% have accompanying hypertension. Furthermore approximately 30% of all hypertensive patients with NIDDM have microalbuminuria.

Patients with hypertension, diabetes, and renal disease are at very high risk for developing cardiovascular (CV) disease. Because there is the potential to delay the progression of renal disease and reduce the risk of CV disease in this high-risk hypertensive population, it is important to identify patients with risk factors for NIDDM, renal disease, and CV disease and target them for early, aggressive anti-hypertensive therapy. (The sixth report of the JNC 1997, WHO Hypertension guidelines 1999) The pool of at-risk hypertensive patients include:  All patients with diabetes mellitus  All patients with microalbuminuria and proteinuria  All hypertensive patients with CV or renal disease or with multiple risk factors for CV or renal disease

Microalbuminuria/ albuminuria Normally functioning kidneys preserve virtually all of the protein in the circulation. Therefore, the presence of abnormal levels of protein in urine (proteinuria) is a sign of kidney damage or malfunction. Albumin represents a portion of the circulating protein and is a reliable measure of the amount of daily protein excretion in the urine. The normal daily urinary excretion of albumin is less than 30 mg/day. The urinary albumin excretion rate (UAER) of individuals who have microalbuminuria is between 30 mg/day and 300 mg/day. When the UAER is greater than 300 mg/day, the condition is called proteinuria. (The terms macroalbuminuria and proteinuria are often used interchangeably.) One of the major risk factors for development of microalbuminuria is hypertension (Bakris 1996). Microalbuminuria is thus very important, since it identifies hypertensive patients at high risk for renal and CV disease that could benefit from early intervention. Prospective studies have shown that patients with type-2 DM and microalbuminuria have nearly 2½ times greater risk for death from any cause and 2 times the risk of CV events and death (Dinneen, 1997). But, even if it is easy to test for, and is a very reliable indicator of the earliest stage of renal disease, screening for microalbuminuria is often overlooked in at-risk patients. Final version 7.12 2 June 2004 7

NIDDM and renal protection The IRMA 2 trial (Parving et al, 2001) was a double blind, placebo-controlled study conducted in 590 patients with hypertension, type 2 diabetes and microalbuminuria (early stage kidney disease). The angiotensin II receptor antagonist (AIIRA) irbesartan, 300 mg taken once daily, demonstrated a 70 percent relative risk reduction (p<0.001) in the progression from an early to a later and more serious stage of kidney disease (also called diabetic nephropathy) compared with patients who did not receive irbesartan, despite achievement of comparable levels of blood pressure reduction in all groups. Based on IRMA 2 findings, for every 10 hypertensive patients with type 2 diabetes and microalbuminuria treated with irbesartan 300 mg daily for 2 years, it is anticipated that one patient would avoid developing more advanced diabetic renal disease within the two years.

The IDNT trial (Lewis et al 2001) was a double blind, placebo-controlled study conducted in 1,715 patients with hypertension, type 2 diabetes and proteinuria (late stage kidney disease). Irbesartan demonstrated a 20 percent relative risk reduction (p=0.02) in the primary endpoint (progression to the first occurrence of: doubling of serum creatinine, end-stage renal disease or all-cause mortality) versus the control group (placebo in addition to other antihypertensive therapies). Irbesartan further demonstrated a 23 percent relative risk reduction (p=0.006) in the primary endpoint versus the antihypertensive drug amlodipine, a calcium channel blocker. Irbesartan yielded these protective effects in addition to achieving blood pressure reduction similar to that observed in the comparator groups. Although there was not a statistically significant difference among the treatment groups in the overall secondary composite endpoint of fatal and nonfatal cardiovascular events, patients in the irbesartan group had a 37 percent reduction of hospitalisations due to congestive heart failure compared to those receiving the antihypertensive amlodipine (p<0.001) and a 23 percent reduction (p=0.15) when compared to patients in the control group.

The RENAAL (Brenner et al 2001) trial was designed in a simmilar way as IDNT. It was a double-blind, placebo-controlled study conducted in 1513 patients with type 2 diabetes and proteinuria. Losartan was superior to the control group (placebo in addition to other antihypertensive therapies) and demonstrated a 16 percent relative risk reduction (ARR 3,6%, p=0.02) in the primary endpoint (progression to the first occurrence of: doubling of serum creatinine, end-stage renal disease or all-cause mortality. Patients in the losartan group also demonstrated a 32 percent reduction of hospitalizations due to congestive heart failure compared to those receiving placebo including the antihypertensive amlodipine (p<0.005).

American Diabetes Association has published a revised position statement on diabetic nephropathy (ADA recommendations 2001), incorporating recent evidence from those studies. Diabetes has become the most common cause of end-stage renal disease (ESRD) in the US and Europe. Over half of the diabetic patients starting on dialysis have type-2 diabetes and the estimated costs for this are expected to grow enormously in the near future. The referred studies above have demonstrated that the onset and course of diabetic nephropathy can be ameliorated, but interventions have their greatest impact if instituted at a point very early in the course of the disease. Final version 7.12 2 June 2004 8

ADA recommends an annual screening for microalbuminuria since a high proportion of patients with type-2 diabetes are found to have microalbuminuria or overt nephropathy shortly after the diagnosis of their diabetes. The finding of microalbuminuria is an indication for screening for possible vascular disease and for aggressive intervention to reduce all cardiovascular risk factors.

To reduce the risk and/or slow the progression of nephropathy, blood pressure control should be optimised. The primary goal of anti-hypertensive therapy among diabetic patients is to decrease blood pressure to <130 mmHg systolic and <80 mm Hg diastolic since hypertension markedly accelerates the progression of diabetic nephropathy. Among patients with type-1 diabetic nephropathy, ACE inhibitors are the initial agents of choice. Among hypertensive patients with type-2 diabetes and microalbuminuria or clinical albuminuria, AIIRAs are the initial agents of choice. These recommendations are based on the positive results of IRMA 2, IDNT and RENAAL, and they represent the first time that AIIRAs are officially recommended as initial drugs of choice for any indication.

Diabetes, hypertension and microalbuminuria - population in Sweden Table 1 shows the frequency of patients with DM, type-2 DM and the percentage with hypertension and microalbuminuria. This is an estimate of the population in Sweden from which the RIAHD high-risk study cohort will be selected.

Table 1 % n patients DM 100 400000 type-2 85 340000 HT 35 119000 U-albumin > 30mg/day 40 47600

MARELD MARELD (Micro Albuminuria and Risk Evaluation in Diabetes) is a quality assurance registry to initiate routines for measurements and evaluations of the local frequency of microalbuminuria in the diabetic population in Sweden. Over 12.000 but less than 13 000 patients with diabetes are screened for microalbuminuria. If the test is positive, a new test is performed after 3 months. If the test is still positive, the patient fulfils the criteria for microalbuminuria (Socialstyrelsen 1998). Risk factors are noted in a protocol, and in the case of hypertension, anti-hypertensive drug used is noted. The forms are then collected for evaluation. In this screening approximately 20% of the diabetic patients will fulfil the criteria for cardiovascular high-risk patient as stated in the follow up RIAHD protocol.

Hyperviscosity Epidemiological and clinical studies have demonstrated a link between classical risk factors and hemorheological markers such as fibrinogen as well as whole blood and plasma viscosity. Changes have been shown in patients with renal disease, ischaemic heart disease, or peripheral Final version 7.12 2 June 2004 9

arterial disease in addition to hypertension (De Backer et al, 2002). Viscosity is a parameter that traditionally has been used to describe the rheological properties of a fluid. Viscosity is a relationship between “shear stress” and “shear ratio” which is a measure of flow resistance in blood towards the formation. The viscosity of blood is determined by a number of “structural” factors such as hematocrit, plasma viscosity, erythrocyte aggregation and erythrocyte deformability (Jonsson et al, 2002). Whole blood viscosity is influenced by a number of factors. It tends to increase with age and also there is an influence by the lipid profile since viscosity correlates positively to increased LDL as well as VLDL cholesterol and negatively to HDL cholesterol. Hypertension is also linked to an increased whole blood viscosity and there is also an increase in viscosity in smokers. Changes in the whole blood viscosity have also been linked to a worse cardiovascular prognosis (Jonsson et al, 2002).

Today, blood viscosity is not used in risk categorization of patients with increased risk for cardiovascular complications. Furthermore there are no intervention studies since blood viscosity has not previously been a target for treatment. Therefore, blood viscosity is still a risk factor that needs to be investigated further in order to illustrate its potential in cardiovascular risk syndromes.

Inflammatory and endothelial markers Several markers of inflammation are elevated in patients with cardiovascular risk syndromes. Studies that have evaluated the relevance of high sensitive CRP (Hs-CRP, typically 1-3 mg/l), in contrast to the levels used in clinical practice (>10mg/l), have been shown to predict CV morbidity in associated clinical conditions. (Kuller et al., 1996, Ridker et al., 1997) Hs-CRP is thought to represent a low-grade inflammation in the arterial vessels wall in association with atherosclerosis (Ross 1999).

Clinical judgment Evidence-based medicine (EBM) integrates clinical experience and patient values with the best available research information. There are four steps in incorporating the best available research evidence in medical decision making: asking answerable questions; accessing the best information; appraising the information for validity and relevance; and applying the information to patient care. Applying EBM to individual patients requires drawing up a balance sheet of benefits and harms based on research and individual patient data. Over the past few years, researchers have looked at the causes of practice variation between physicians using essentially non-Brunswikian methods. The RIADH study will center on the investigation and management of cardiovascular disease in general and in metabolically compromised patients in particular. These diseases, diabetes, microalbuminuria and hypertension, their investigation and management are all associated with high morbidity and cost, and are the subject of high levels of public and health service concern. In RIAHD, we will study the causes of medical practice variation in this area and examine some reasons why physicians make treatment choices that do not appear to be based on the best available evidence. Many initiatives have taken to improve medical treatment decisions. Educational strategies for doctors have been effective in at least 50% of cases. Some reflection on one's own performance seems to be a common feature of the most effective strategies. So far, such reflections have mainly focused on the observed outcomes of the doctors' decisions, i.e. on what doctors do in Final version 7.12 2 June 2004 10

practice. In order to assess what doctors really do, and not only what they express what they do, analysis of judgment process itself may be useful (Kirwan et al 1990).

Health economics Economics is becoming increasingly important in health care, since budget constraints are becoming tougher. Increased spending in health care is due to an ageing population, technological advances and changing disease epidemiology, among other factors. The focus on economics means that the introduction of a new intervention or test should be evaluated from an economic as well as from a medical point of view. A health economic analysis will be performed as a part of RIAHD, which will cover direct as well as indirect costs for patients in each group. The aim is to evaluate whether there are differences in health care consumption and productivity loss between the two groups. Data collection will be from patients and their physicians by a questionnaire.

Questions will be asked on the normal daily occupation, on the patients' perception on how their health status influences their possibility to perform their daily tasks and on absenteeism during the last four weeks prior to filling out the questionnaire. Median wage, obtained from SCB (Statistics Sweden), will be used as a proxy for valuing the indirect cost.

Data on health care utilization, including visits to general practitioners, hospital visits, inpatient periods, and consumption of pharmaceuticals will be collected for the 12-month period prior to filling out the questionnaire. Costs for resources used will be taken from national DRG data, from county council price lists, form the official price list for pharmaceuticals, and from other sources.

3. Study Design For visual overview of the study design and organization, see Appendix.

This study is a survey of the cardiovascular risk factors in patients with of a combination of hypertension, diabetes mellitus and microalbuminuria in middle-aged to elderly patients in Sweden.

The expected number of patients/subjects to be recruited is 1 000. The participants will be recruited from outpatient units in Sweden. By screening of representative cohorts of patients/subjects, 500 high-risk patients (hypertension, diabetes and micro/ macroalbuminuria) and 500 low risk patients (hypertension without diabetes or micro/ macroalbuminuria) will be included in the protocol. High-risk patients from the MARELD registry could be included directly in the RIAHD study after verifying inclusion requirements for the high-risk group, i.e. presence of hypertension, diabetes mellitus and micro/ macroalbuminuria (Appendix).

All patients will be tested for microalbuminuria and diabetes. A form will completed for medical history, cardiovascular RF, ACC and TOD as well as any current medical treatment.

Blood pressure will be measured in the clinic as well as at home (self measurement). Final version 7.12 2 June 2004 11

Questionnaires will be finalized for risk assessment by the patient as well as the physician.

Blood sampling will be performed for indirect (calculated) blood viscosity by using total protein and hematocrit as well as by using fibrinogen and other inflammatory parameters. In a subgroup of patients, blood viscosity will be assessed by using direct viscosity measurements with the Bohlin Rheometer.

Blood, serum and plasma will also be sampled and analysed for inflammatory and endothelial markers as well as selected genetic polymorphisms. Additional markers, reflecting low-grade inflammation or endothelial damage, that are being assessed are as follows; inflammatory (TNFa, IL1a, IL1b, IL2, IL4, IL6, IL8, IL10, NCP1, EGF, VEGF and IFNg) and endothelial (ICAM, VCAM, vWF), by the Randox Biochip array (www.randox.com). In addition BNP is measured as a global marker for cardiovascular risk.

Blood will also be stored in a biobank for analysis of genetic polymorphisms related to the 5- year-follow-up of cardiovascular and metabolic morbidity and mortality as well as total mortality. No other use of the biobank will be allowed outside cardiovascular and metabolic morbidity and mortality as well as total mortality

4. SELECTION AND ENROLLMENT OF PATIENTS

Selection of centres in RIAHD

Study cohort patient selection will be based on the investigator network generated for the MARELD registry that will be able to include sufficient number of patients, e.g. 10 high-risk and 10 matched low-risk patients/ centre. Furthermore, RIAHD patient recruitment will also take into account the geographical distribution in Sweden.

Reference cohort patient selection will be based on a cohort of non-diabetic patients who are negative in screening for microalbuminuria/albuminuria (MA/A) but on treatment for hypertension based on a history of increased systolic and/or diastolic blood pressures (140/90 mmHg).

Exclusion criteria - Any condition that prevents the patient giving their informed consent such as dementia, aphasia etc - Inability to perform Home Blood pressure measurements, laboratory tests, answering study questionnaires or other study related procedures. - Active drug or alcohol use or dependence that, in the opinion of the site investigator, would interfere with adherence to study requirements. - Serious illness that prevents the patient/subject participating in the study. Final version 7.12 2 June 2004 12

- Inability or unwillingness of subject or legal guardian/representative to give written informed consent.

Inclusion criteria High risk group (Hypertension, DM and MA/A) - Male and female age > 40 years - Treated systemic hypertension (systolic and or diastolic) - Diabetes mellitus type 2 - Micro- or macroalbuminuria

Inclusion criteria Low risk group (Hypertension but no DM or MA/A) - Male and female age > 40 years - Treated systemic hypertension (systolic and or diastolic) - Absence of diabetes mellitus - Absence of micro- or macroalbuminuria

Study Enrolment Procedures Screening 1. Informed consent 2. Exclusion criteria 3. Clinic seated blood pressure measurement 4. Fasting blood/ plasma glucose 5. Microalbuminuria assessment with Micral or other MA/A test. If the test is positive or if the patients have a diagnosed MA/A, a quantitative U-creatinine/ U-protein ratio test is mandatory for the correct diagnosis of MA/A (see Appendix). 6. Inclusion criteria

If enrolled; 1. Physical examination 2. Registration of medication 3. Registration of risk factors (RF, ACC, TOD) 4. Blood sampling (viscosity, inflammatory and endothelial markers, genetics) 5. Physician questionnaire (Clinical jugement) 6. Home BP (self BP measurement) 7. Patient questionnaire (Clinical judgement and health care consumption)

5. CLINICAL AND LABORATORY EVALUATIONS

Risk assessment

Evaluation of risk factors, TOD and ACC will be performed according to the ESH/ ESC guidelines (Appendix). Criteria for diagnoses for ACC will are given in Appendix. Final version 7.12 2 June 2004 13

Blood sampling

Blood sampling will be made for evaluation of serum plasma and whole blood parameters (Appendix 7), for routine laboratory evaluation (see section below) as well as for additional evaluations of circulating inflammatory and endothelial markers (see below).

Clinical judgement The objectives of the Clinical judgment part in RIAHD study is to analyze the processes underlying the real and not only expressed clinical judgment process, give examples of its use in the medical context, and discuss its potential for improving prescribing decisions. Clinical judgments analysis can thus look behind the outcome of a decision to the underlying decision process itself. Carefully constructed or selected real or written patient case histories as well as constructed “paper patient” case material is required for this analysis. By using this technique of real and “paper patient” cases, doctors' clinical judgment and prescribing behavior will be assessed in the RIAHD study through the series of simulated (paper patients) case presentations.

Health care consumption

A health economic analysis will be performed, covering direct and indirect costs for patients in each group. The purpose of this is to see if there is a difference in health care consumption and productivity loss between the two groups. Questions are asked on the normal daily occupation, on the patients' perception on how their health status influences their possibility to perform their daily tasks and on absenteeism during the last four weeks prior to filling out the questionnaire. Median wage, obtained from SCB (Statistics Sweden), will be used as a proxy for valuing the indirect cost. Data on health care utilisation, including visits to general practitioners, hospital visits, inpatient periods, and consumption of pharmaceuticals will be collected for the 12-month period prior to filling out the questionnaire. Costs for resources used will be taken from national DRG data, from county council price lists, form the official price list for pharmaceuticals, and from other sources.

Schedule of Evaluations

Evaluation Screening Enrolment 1v Post- evaluation Informed Consent X Screening and matching X Physical Examination X Registration of medication X Registration of RF, ACC, TOD X Blood samples X Physician questionnaire (CJ) X Final version 7.12 2 June 2004 14

Home Blood Pressure X

Patient Questionnaire (CJ and HCC) X Post-eval X

Suitable patients will be screened after having been informed on and accepted the objectives of the study, the procedures, the information which could be drawn from this survey, the potential benefits for a better understanding of his disease. The physician or the nurse will provide a full explanation of the procedure for urine screening to the patient.

Fasting plasma or blood glucose measurement is performed.

The clinic blood pressure is measured after some minutes in the sitting position according to standard procedures.

A urine sample is collected, preferably by overnight urine for testing of MA/A. The diagnosis of MA/A for the high-risk patient group should be confirm by a quantitative U-creatinine/ U- proteinuria test.

The patient will be enrolled provided all inclusion and no exclusion criteria are present. Low-risk patients should be matched versus high-risk patients (Appendix).

Additional examinations, laboratory tests and questionnaires

During the patient visit, or at home, questionnaires will be finalized by the patient as well as by the physician for risk assessment.

Blood will be sampled for indirect (calculated) blood viscosity by haematocrit and total protein as well as by other methods including fibrinogen in all patients. In available patients close to Sahlgrenska University hospital, blood viscosity will also be assessed by the Bohlin Rheometer. This will provide a methodological assessment of the indirect (calculated) blood viscosity measurements.

Blood, serum and plasma will also be sampled for inflammatory and endothelial markers and for measurement of serum/plasma markers as well as selected genetic polymorphisms. A biobank will be created for analysis of genetic polymorphisms related to the 5-year-follow-up of cardiovascular and metabolic morbidity and mortality as well as total mortality. No other use of the biobank will be allowed outside cardiovascular and metabolic morbidity and mortality as well as total mortality.

Special Instructions and Definition of Evaluations

Microalbuminuria Final version 7.12 2 June 2004 15

Microalbuminuria will be assessed with reagent strips (Micral tests® Roche, see Appendix).

Physical examination Wight, length and blood pressure measurements (BPM): BPM will be performed in the sitting position according to ESH/ESC standard routines by sphygomanometry.

Registration of medication Medical treatment is defined as any current Rx or OTC pharmaceuticals. See appendix (CRF).

Registration of risk factors Medical history will be evaluated according to the questionnaire in appendix (CRF). The questions are in accordance with the cardiovascular risk factor stratification as described in the ESH/ESC Guidelines 2003. Cardiovascular and cerebrovascular disease will also be assessed for the year preceding the screening visit.

Standard Laboratory Evaluations S-Creatinine, EVF, S-protein, S-cholesterol, fS-LDL, HDL, fS-TG, fS-glucose, CRP, fibrinogen, HbA1c, and other relevant screening tests will be performed and analysed by standardised clinical chemistry methods.

Additional evaluations of circulating markers Additional markers, reflecting low-grade inflammation or endothelial damage, to be assessed are as follows; inflammatory (TNFa, IL1a, IL1b, IL2, IL4, IL6, IL8, IL10, NCP1, EGF, VEGF and IFNg) and endothelial (ICAM, VCAM, vWF), by the Randox Biochip array (www.randox.com). In addition BNP will be assessed as a global marker for cardiovascular risk.

Sampling for selected genetic polymorphisms and planned 5 year follow up Analysis of selected cardiovascular and metabolic genetic polymorphisms related to the planned 5-year-follow-up of cardiovascular and metabolic morbidity and mortality as well as total mortality.

Physician questionnaire The examining physician will perform a regular clinical status assessment. Further on, a clinical assessment by the physician of the individual cardiovascular risk of the patient will be performed. Selected questions will be put to the physician to assess their expressed as well as real judgements on cardiovascular and metabolic health risks in accordance with the questionnaire described in appendix, CRF.

Patient questionnaires The patient will perform a self-assessment of his/her own cardiovascular risk in accordance with the questionnaire described in appendix (CRF). Treatment history will be assessed for one year preceding the screening visit. All contacts with health care providers will be listed. Selected questions will be put to patients to assess their expressed as well as real judgements on cardiovascular and metabolic health risks.

Home (self) blood pressure measurement Final version 7.12 2 June 2004 16

After appropriate training of the patient, home blood pressure will twice daily be assessed during 5 consecutive days. A semi-automatic device will be used. The blood pressure are measured both in the morning, before leaving home if relevant, and in the afternoon after returning home. The patient will rest for 5 minutes and the blood pressure should be measured in the sitting position. At each occasion two measurements are registered in the patient book. (Appendix).

Definitions of history/ risk-factors

Hyperlipidemia - Treated or detected hyperlipidemia (S-cholesterol >5mmol/l and/or LDL>3mmol/l, diet or medical anti-hyperlipidemia treatment) Overweight - Abdominal circumference male>102, female >108 Smoking - Former smoker - Current smoker CRP - >1mg/ml Stroke - Rapidly occurring clinical signs of focal brain damage, obviously not caused by non- vascular mechanism. Symptoms regarded focal are: unilateral or bilateral motor disturbance unilateral or bilateral sensory disturbance, aphasia/dysphasia, hemianopsia, deviation conjugée, diplopia, dysphasia with rapid onset, apraxis with rapid onset, ataxia with rapid onset, disturbance of perception with rapid onset. - Global symptoms are accepted in a patient with subarachnoid haemorrhage or who is deeply comatose excluding those caused by failing systemic circulation (shock, Adams- Strokes syndrome or hypertensive encephalopathy). Ischemic heart disease - Angina pectoris, myocardial infarction or previous percutaneous transluminal cardiac angioplasty (PTCA) or cardiac angioplasty bypass graft (CABG) Angina pectoris - Symptomatic or treated angina pectoris: if PTCA or CABG has been performed and no symptoms remain, a check in ischemic heart disease should be preferred. Cardiac failure - Clinical history of cardiac failure or UCG with ejection fraction (EF) <45% Atrial fibrillation - Diagnosed paroxysmal atrial fibrillation, atrial fibrillation on admission to the hospital or during the observation period Claudicatio intermittens - Diagnosis of peripheral arterial disease or symptomatic claudicatio intermittens with ankle brachial index <0,9 Carotid stenosis or surgery - Known carotid stenosis of > 50% or history of carotid surgery Retinopathies - Diagnosed retinopathies Renal diseases Final version 7.12 2 June 2004 17

- Diabetic nephropathies - Severe renal disease - Albuminuria or elevated S-Creatinine TOD - LV-hypertrophy - Carotid plaque - Other cerebrovascular or cardiovascular disease

6. SUBJECT SAFETY

Monitoring Since no drug will be administered in the frame of this protocol, no serious adverse event or adverse events related to research procedures are expected. However, general medical safety concerns should be exercised in case any unexpected safety issue should occur.

Safety instructions specific to the trial In case any event would occur in the study, it will be recorded and reported (Appendix)

7. STATISTICAL CONSIDERATIONS

Parameters

The primary parameters studied in this study will be:

- Levels of viscosity and inflammatory markers between groups and in relation to different cardiovascular risks in hypertensive patients

Secondary parameters studied:

- Score of cardiovascular risk factors in high-risk patients (hypertension, diabetes and micro/ macroalbuminuria) vs. low risk patients (hypertension without diabetes or micro/ macroalbuminuria).

- Determination of blood glucose markers in relation to risk factors in patients with micro/ macroalbuminuria and hypertension.

- Assessment of inflammatory and endothelial markers.

- Assessment of “clinical judgment” in terms of physicians and patients risk assessment.

- Assessment of Home Blood pressures and Office blood pressures to establish cardiovascular risk in cohorts with established hypertension. Final version 7.12 2 June 2004 18

- Assessment of the potential impact of selected genetic polymorphisms of cardiovascular relevance on risk profile in terms of other risk factors, associated clinical conditions (ACC) and presence of target organ damage (TOD). - Determine achieved clinic and home blood pressure levels and anti-hypertensive treatment in this group of patients.

- Assessment of health economic impact of disease by evaluating health care utilisation, sick leave, direct pharmaceutical treatment cost.

- Reassessment of cardiovascular and metabolic morbidity as well as total mortality after 5 years.

Sample size In all, 500 high-risk patients (HT, DM and MA) and 500 low-risk patients (HT, no DM or MA) will be included in the study.

The primary objective of the study is to measure and compare blood viscosity parameters in two different patient populations. The power law coefficients, n and k, which are unique to each blood sample, are related with blood viscosity parameters in the form of a mathematical equation. Further on they are fixed independently of the shear rate. In a study (Hussain MA, Puniyani RR 1999) the power law coefficient, n, was (0.703 ± 0.027) in patients with a cardiovascular event (i.e. stroke) compared to n = (0.708 ± 0.025) in healthy controls. One goal of the proposed study is to test the null hypothesis that the two population means are equal. The criterion for significance (alpha) has been set at 0,050. The test is 2-tailed, which means that an effect in either direction will be interpreted. With the proposed sample size of 500 and 500 for the two groups, the study will have power of 85,9% to yield a statistically significant result.

This computation assumes that the mean difference is 0,005 (corresponding to means of 0,708 versus 0,703) and the common within-group standard deviation is 0,026 (based on SD estimates of 0,025 and 0,027).

This effect was selected as the smallest effect that would be important to detect, in the sense that any smaller effect would not be of clinical or substantive significance. It is also assumed that this effect size is reasonable, in the sense that an effect of this magnitude could be anticipated in this field of research.

On average, a study of this design would enable us to report the mean difference with a precision (95,0% confidence level) of plus/minus 0,003 points. For example, an observed difference of 0,005 would be reported with a 95,0% confidence interval of 0,002 to 0,008. The precision estimated here is the median precision. Precision will vary as a function of the observed standard deviation (as well as sample size), and in any single study it will be narrower or wider than this estimate. Final version 7.12 2 June 2004 19

Statistical methods Population characteristics (including demographics, severity of the disease, co-morbidities, current treatment or no treatment) will be summarized into count of non-missing data, mean, standard deviation, minimum, maximum, median, 5% percentile and 95% percentile for quantitative variables and count and percentage of the population for categorical data. The outcomes may be detailed by region.

Interim analysis No interim analysis is planned in this study.

8. STUDY DURATION

The total duration of the study will be determined by the rate of recruitment and presence of events in the study population. Estimated recruitment rate: Each centre will find approximately 2 high risk patients and 2 low risk patients (matched) every working week. The RIAHD study is planned to start Q4 2004 and to be completed within 3-6 month.

9. STUDY ORGANIZATION

This is a multicenter study involving approximately 50 centres (general practioners) in Sweden organized by a Steering committee. The study will be coordinated by the sponsor. CRFs will be in paper form, transformed and sent by mail for data processing. Centralized data and statistical analyses will be performed by a 3rd party statistical unit.

Centre set up The sponsor will ensure centre set up and training of the centre staff.

Data Monitoring During data entry, predefined compulsory data will be checked and supplementary information will be asked for from the investigator. On site monitoring will not be performed.

10. ETHICAL AND REGULATORY STANDARDS

Ethical principles This protocol is in accordance with the principles laid down by the 18th World Medical Assembly (Helsinki 1964) and amendments laid down by the 29th WMA General Assembly, Tokyo, Japan, October 1975, 35th WMA General Assembly, Venice, Italy, October 1983, 41st WMA General Final version 7.12 2 June 2004 20

Assembly, Hong Kong, September 1989, 48th WMA General Assembly, Somerset West, Republic of South Africa, October 1996 and the 52nd WMA General Assembly, Edinburgh, Scotland, October 2000.

Laws and regulations This protocol is also in accordance with laws and regulations in Sweden as well as relevant and applicable guidelines.

Informed consent It is the responsibility of the Clinic to obtain informed consent in compliance with national requirements from each subject prior to entering the study or, where relevant, prior to evaluating the subjects’ suitability for the study.

Ethics Review Committee The Investigator will submit this protocol to the Ethics Review Committee. The Investigator is required to forward a copy of the written approval/advice signed by the chairman to the sponsor. On the approval/advice sheet, the trial (title, protocol number and version), the documents studies (protocol, informed consent material), advertisement when applicable) and the date of the review should be clearly stated. 11. STUDY CONDUCT

Responsibilities of the Clinic(s) The Clinic(s) undertake(s) to perform the study in accordance with good clinical practice and specifically either good clinical practice for study on medicinal products in the European Community (ISBN-92 825 9563-3) or 21 CFR-part 312 subpart D and guidelines for the monitoring of clinical investigations. The Clinic is required to ensure compliance with respect to procedures required by the protocol. The Clinic agrees to provide all information requested in the Case Report Form in an accurate and legible manner according to instructions provided.

Responsibilities of the Organizers/Sponsors The Sponsors of this study have responsibilities to Health Authorities to take all reasonable steps to ensure the proper conduct of the study as regard to ethics, protocol adherence, integrity and validity of the data recorded on the case report forms. The Sahlgrenska Academy, Göteborgs University, takes the full responsibility to handle the collected data and biobank according to current regulations and to devote necessary resources and get necessary permissions for this. The sponsor has the right, but no obligations, to take part of the 5-year follow up part of the study. An inquiry should be sent to the sponsor before the planned 5-year follow up part is executed. Final version 7.12 2 June 2004 21

Source document requirements Patients’ hospital records will be source documents.

The use and completion of case report forms (CRFs) It is the responsibility of the Clinic to prepare and maintain adequate and accurate CRFs, which have been designed by the Sponsors to record all observations and other data pertinent to the clinical investigation. All CRFs should be completed in their entirety in a neat, legible manner to ensure accurate interpretation of data; a black ballpoint pen should be used to ensure the clarity of reproduced copies of all CRFs when faxed, sent by regular mail or collected on site. All Clinics are encouraged to fill in the electronic CRF from (details to be provided by the Sponsors). Should a correction be made, the information to be modified must not be overwritten. The corrected information will be transcribed next to the previous value with the reason for the correction, initialled and dated by the authorized person.

12. ADMINISTRATIVE POINTS AND PROCEDURES

Curriculum vitae An updated curriculum vitae will be obtained from all investigators.

Record retention in investigating centre(s) The Clinic must maintain all study records, patient files and other source data for the maximum period of time permitted by the hospital, institution or private practice. However national regulations should be taken into account, the longest time having to be considered.

For study performed in the European Community, the Clinic is required to arrange for the retention of the patient identification codes for at least 15 years after the completion or discontinuation of the study. Any centre will notify the sponsor before destroying any data or records.

13. CONFIDENTIALITY

All materials, information (oral or written) and unpublished documentation provided to the Investigators (or any company/institution acting on their behalf), inclusive of this protocol and the patient Case Report Forms, are the exclusive property of the Sponsor and may not be given or disclosed, either in part or in whole, by the Investigator or by any person under his/her authority to any third party without the prior express consent of the Sponsor. Final version 7.12 2 June 2004 22

However, the submission of this protocol and other necessary documentation to the Ethics Committee (IRB/IEC) is expressly permitted, the IRB/IEC members having the same obligation of confidentiality. The Investigator shall consider all information, results, discoveries, records accumulated, acquired, or deduced in the course of the study, other than that information to be disclosed by law, as confidential and shall not disclose any such results, discoveries, records to any third party without the Sponsor’s prior written consent.

14. OWNERSHIP OF DATA AND USE OF THE STUDY RESULTS The Sahlgrenska Academy, Göteborg University has the ownership of all data and results collected during this study and acts under the supervision of the Steering committee. The biobank is owned by the Sahlgrenska Academy, Göteborg University.

15. CLINICAL STUDY REPORT A Clinical Study Report will be prepared based on the results of the study.

16. PUBLICATIONS In multicentre study conducted by a Steering Committee, the responsibility for presentations and/or publications is linked to the Steering Committee. The Steering Committee must send a copy of the manuscript or abstract to the Sponsor for review at least forty-five (45) days before submission.

All the study participants (Clinics and Committee members) will make a prior delegation of responsibility for primary presentation and/or primary publication of the results to the Steering Committee. No other publication is allowed before the primary publication. Any presentation or publication by any one that participates in the study must mention the study and must be approved by the Steering Committee in advance. Moreover, it is mandatory to make reference to the primary publication.

17. PROTOCOL AMENDMENTS

It is specified that the appendices attached to this protocol and referred to in the main text of this protocol form an integral part of the protocol.

No changes or amendments to this protocol may be made by the Clinic or by the Sponsor after the protocol has been agreed to and signed by both parties unless such change(s) or amendment(s) have been fully discussed and agreed upon by the Clinic and the Sponsor. Any Final version 7.12 2 June 2004 23

change agreed upon will be recorded in writing, the written amendment will be signed by the Clinic and by the Sponsor and the signed amendment will be appended to this protocol.

Approval/advice of amendments by Ethics Review Committee or similar body (IRB, CCPPRB) is required prior to their implementation, unless there are overriding safety reasons. If the change or deviation increases risk to the study population, or adversely affects the validity of the clinical investigation or the subject’s rights, full approval/advice must be obtained prior to implementation. For changes that do not involve increased risk or affect the validity of the investigation or the subjects’ rights, approval/advice may be obtained by expedited review, where applicable.

In some instances, an amendment may require a change to a consent form. The Clinic must receive approval/advice of the revised consent form prior to implementation of the change. In addition, changes to the Case Report Forms, if required, will be incorporated in the amendment.

Prior to initiating the changes, protocol amendment must be submitted to the Ethics committee(s) involved.

18. BIBLIOGRAPHY

Murray CJ and Lopez AD. (A) Global mortality, disability, and the contribution of risk factors: Global Burden of Disease Study. Lancet 1997; 349: 1436-42.

Murray CJ and Lopez AD. (B) Regional patterns of disability-free life expectancy and disability- adjusted life expectancy: Global Burden of Disease Study. Lancet 1997; 349: 1347-52.

ESH/ESC Guidelines. J Hypertens. 2003; 21: 1011-1053.

Practice guidelines for primary care physicians: 2003 ESH/ ASC hypertension guidelines. J Hypertens. 2003; 21: 1779-1788

The sixth report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure. Arch Intern Med. 1997; 157: 2413-2446.

Guidelines Subcommittee. 1999 World Health Organization-International Society of Hypertension guidelines for the management of hypertension. J Hypertens. 1999; 17: 151-183.

De Backer TLM, De Buyzere M, Segers P, Carlier S, De Sutter J, Van de Velde C, De Backer G. The role of whole blood viscosity in premature coronary artery disease in women. Atherosclerosis 2002; 165: 367-73

Jonsson G, Fossum E, Kjeldsen S E, Høieggen A, Os I, Eide I, Westeheim A. Lower plasma noradrenaline and blood viscosity on carvedilol vs atenolol in men with recent myocardial infarction. Blood Pressure 2002; 11: 377-384. Final version 7.12 2 June 2004 24

Kuller LH, Tracy RP, Shaten J, Meilahn EN. Relation of C-reactive protein and coronary heart disease in MRFIT nested case-control study. Multiple Risk Factor Intervention Trial. Am J Epidemiol. 1996; 144: 537-547

Ridker PM, Cushman M, Stampfer MJ, Tracy RP, Hennekens CH. Inflammation, aspirin, and the risk of cardiovascular disease in apparently healthy men. N Engl J Med. 1997; 336: 973-979

Ross R. Atherosclerosis-an inflammatory disease. N Engl J Med. 1999; 340: 115-126

Bakris GL. Microalbuminuria: prognostic implications. Curr Opin Nephrol Hypertens. 1996; 5: 219-223.

Dinneen SF, Gerstein HC. The association of microalbuminuria and mortality in non-insulin- dependent diabetes mellitus: a systematic overview of the literature. Arch Intern Med. 1997; 157: 1413-1418.

Parving HH. Et al, The effect of irbesartan on the development of diabetic nephropathy in patients with type 2 diabetes. N Engl J Med. 2001; 345: 870-878

Lewis E. at al, Renoprotective effect of the angiotensin-receptor antagonist irbesartan in patients with nephropathy due to type 2 diabetes. N Engl J Med. 2001; 345: 851-860

Brenner, B M et al, Effects of losartan on renal and cardiovascular outcomes in patients with type 2 diabetes and nephropathy. N Engl J Med. 2001; 345: 861-869

ADA recommendations. Diabetes Care. 2002;25: Suppl 1

Socialstyrelsen, State of the Art; Diabetesnefropati 1998

Kjeldsen SE, Jamerson K, Julius S, Hedner T, Pehrsson N-G. Characteristics of reversed office hypertension – May treatment resistant home hypertension be explained by smoking? Abstract ESH Paris June 13-17, 2004.

Sjöholm Å, Nyström T. Kronisk inflammation kan orsaka typ-diabetes. Läkartidningen 2004; 101: 1716-1721

Hussain MA, Puniyani RR. Relationship between Power Law Coefficients and Major Blood Constituents Affecting the Whole Blood Viscosity. Journal of Biosciences 1999; 24(3): 329-337

Kirwan JR, Chaput de Saintonge M, Joyce CRB. Clinical Judgement analysis. Quaterly J Med. 1990, Series 76, No 281, pp 935-949 Final version 7.12 2 June 2004 25

GLOSSARY AND ABBREVIATIONS

RIAHD Risk Factor Assessment in Hypertension and Diabetes

ESH European Society of Hypertension

ESC European Society of Cardiology

SCORE Systematic Coronary Risk Evaluation

RF Risk Factor

ACC Associated Clinical condition

TOD Target Organ Damage

BP Blood Pressure

BPM Blood Pressure Measurement

IDDM Insulin Dependent Diabetes Mellitus

NIDDM Non Insulin Dependent Diabetes Mellitus

CV Cardiovascular

AIIRA Angiotensin II Receptor Antagonists Final version 7.12 2 June 2004 26

Appendices

Appendix 1 Study Organisation

Appendix 2 Study sites (Investigators and study nurses)

Appendix 3 Study Flow

Appendix 4 Study organisation Chart

Appendix 5 RIAHD screening (High risk and low risk groups)

Appendix 6 CRF

Appendix 7 Laboratory and blood sampling routines

Appendix 8 SAE report

Appendix 9 Patient informed consent Final version 7.12 2 June 2004 27

Appendix 1

Study Organization And Participating Clinical Sites

Telephone Facsimile e-mail Steering Committee Thomas Hedner 031-342 2974 031-419368 [email protected] (chair) Thomas Leoo 08-470 1877 08-470 1848 [email protected] Anders Niklason Ola Samuelsson Per-Anders Jansson Stanko Skrtic 031-342 29 40 031-82 67 23 [email protected] LiMing Gan Per Boström 08-470 1875 08-470 1848 [email protected]

RIADH working group Thomas Hedner Thomas Leoo Anders Niklason Per Boström

Expert reference group Leo Niskanen Sverre Kjeldsen

Statistics Final version 7.12 2 June 2004 28

Appendix 2

Study sites (Investigators and study nurses) Final version 7.12 2 June 2004 29

Appendix 3 Study Flow

RIAHD Screening

Mareld Ca. 10 000 DM 13 000 DM patients without MA Screened for MA Create routines and do Ca. 3000 DM risk assessment with HT+MA

RIAHD 500 Matched Controls 500 (Hypertensive patients, no DM or MA)

RIAHD Study

RIAHD 500 hypertensive high risk patients (DM&MA) 500 hypertensive low risk patients (no DM or MA) Risk evaluation and riskfactor identification

Screening & Visit

Patient evaluation Blood samples Standard Lab Blood viscosity Inflammatory blood markers RF, ACC and TOD according to ESH/ESC Health economics Clinical judgement physician & patient Home Blood Pressure

Planned follow-up

Patient 5 yrs follow-up for endpoint Final version 7.12 2 June 2004 30

Appendix 4

Study organisation chart

Study support Sv Hypertoniföreningen L Hanssons minnesfond Sponsors Bristol-Myers Squibb Sanofi Synthelabo

Sponsors Steering committee Study management Planning & Study secretariat

Site selection Study sites Patients Screening Home BP Site set up Enrolment Questionnaires Evaluations Monitoring Endpoint committee Endpoint evaluation

CRF LAB Only for 5 yrs follow-up

Data Management Final version 7.12 2 June 2004 31

Appendix 5

RIAHD screening The RIAHD HIGH RISK GROUP

 Informed Consent Negative->out

 Screening for Hypertension.Treated Hypertension - Positive - go on screening for Diabetes

Negative->out  Screening For Diabetes. Known treated Diabetes Positive - go on screening for microalbuminuria

 Enrolment requires two positive samples with a three month interval as in the Mareld screening and a verifying quantitative measurement

Negative ->out Negative ->out Mareld Screening Positive Negative->out New screening after 3 months Positive A verifying quantitative = MA measurement

Positive enrollment in RIAHD High risk group Final version 7.12 2 June 2004 32

Appendix 5, cont.

RIAHD Screening The RIAHD LOW RISK GROUP Negative->out

 Age and gender matched with a patient from RIADH High Risk Group Positive – go to screening

 Informed Consent

Negative->out  Screening for Hypertension. Treated Hypertension - Positive – Next step exclusion of Diabetes

Elevated->out  Exclusion of diabetes. fS glucose test Normal - Next step exclusion of microalbuminuria

Negative enrollment in RIAHD Low Risk  Exclusion of Microalbuminuria Group Positive ->out

Microalbuminuria screening procedures Reagent strips; Micral tests® Roche Packaging; Micral test sticks Clinic by the Sponsors. Storage conditions; Reagent strips should be stored at room temperature between 15-30°C (59°- 86°F). Products must not be used after expiration date and bottles must not be stored in direct sunlight. Explanation for use; Reagent test areas on the Reagent strips are ready to use upon removal from the bottle and the entire reagent strip is disposable. The strips are read visually, requiring no additional laboratory equipment. The directions must be followed exactly. The reagent strips must be kept in the bottle with the cap tightly closed to maintain reagent reactivity. To obtain optimal results, testing should be done on fresh urine. Investigational reagent accountability; Study representatives will check with each site to ensure an adequate supply of the reagents is provided to the Clinic. Final version 7.12 2 June 2004 33

Appendix 7

Laboratory and blood sampling routines

According to protocol specifications and choosen routine laboratory . Final version 7.12 2 June 2004 34

Appendix 8

SAE Report