CIBIS II Cardiac Insufficiency Bisoprolol Study Biso 5 Korr CIBIS II Cardiac Insufficiency Bisoprolol Study Double-blind, placebo-controlled, randomised trial 2,647 patients included (NYHA III + IV) Bisoprolol administered on top of standard therapy (diuretic + ACE inhibitor) Bisoprolol fumarate (2:1) is a racemate and as a derivative of phenoxyaminopropa-nol it belongs to the class of therapeutic substances known as ß-blockers. Bisoprolol fumarate is very freely soluble in water and freely soluble in ethanol and chloroform. The molecular weight is 383.48, the white crystalline substance melts at 101°C. CIBIS II Investigators and Committees, Lancet 1999, 353: 9–13

CIBIS II – Cardiac Insufficiency Bisoprolol Study Objectives Biso 5 Korr CIBIS II – Cardiac Insufficiency Bisoprolol Study Objectives Primary objective All-cause mortality Secondary objectives Cardiovascular mortality Hospital admissions Cardiovascular mortality or cardiovascular hospital admissions Permanent treatment withdrawal Bisoprolol proved to be the compound with the highest ß1-selectivity in all in vitro and in vivo studies and in all animal species investigated. One measure of ß1-selectivity is the ratio of the constants of inhibition (ci) for the ß1- and ß2-receptor. The constants of inhibition of the ß-blockers bisoprolol, atenolol, betaxolol and propranolol were determined in ligand-binding studies using rat parotid gland (mainly occupied by ß1-receptors) and rat reticulocytes (mainly occupied by ß2-receptors) in human plasma: using a non-specific radiolabelled ligand, the ß1- and ß2-receptors of the rat parotid gland and reticulocytes were completely occupied. This cell suspension was then mixed with the plasma of volunteers pretreated with different ß-blockers. The non-specific radioligand was now displaced from the receptor by addition of this serum enriched with ß-blockers. Constants of inhibition of a characteristic magnitude for each ß-blocker and receptor type could be determined from these tests; the smaller the ci-value, the higher the affinity of the ß-blocker for the receptor type concerned. The ratio of ci/ß1 to ci/ß2 was 1:75 for bisoprolol, 1:35 for atenolol and betaxolol, 1:20 for metoprolol, and 1.8:1 for propranolol. Bisoprolol therefore proved to be the ß-blocker with the highest ß1-selectivity in this model as well. CIBIS II Investigators and Committees, Lancet 1999, 353: 9–13

Biso 5 Korr CIBIS II – Cardiac Insufficiency Bisoprolol Study Main inclusion criteria Ambulatory patients with stable CHF of all aetiologies NYHA functional class III or IV Stable on ACE inhibitor and diuretic Aged 18 – 80 years Left ventricular ejection fraction £ 35% Bisoprolol proved to be the compound with the highest ß1-selectivity in all in vitro and in vivo studies and in all animal species investigated. One measure of ß1-selectivity is the ratio of the constants of inhibition (ci) for the ß1- and ß2-receptor. The constants of inhibition of the ß-blockers bisoprolol, atenolol, betaxolol and propranolol were determined in ligand-binding studies using rat parotid gland (mainly occupied by ß1-receptors) and rat reticulocytes (mainly occupied by ß2-receptors) in human plasma: using a non-specific radiolabelled ligand, the ß1- and ß2-receptors of the rat parotid gland and reticulocytes were completely occupied. This cell suspension was then mixed with the plasma of volunteers pretreated with different ß-blockers. The non-specific radioligand was now displaced from the receptor by addition of this serum enriched with ß-blockers. Constants of inhibition of a characteristic magnitude for each ß-blocker and receptor type could be determined from these tests; the smaller the ci-value, the higher the affinity of the ß-blocker for the receptor type concerned. The ratio of ci/ß1 to ci/ß2 was 1:75 for bisoprolol, 1:35 for atenolol and betaxolol, 1:20 for metoprolol, and 1.8:1 for propranolol. Bisoprolol therefore proved to be the ß-blocker with the highest ß1-selectivity in this model as well. CIBIS II Investigators and Committees, Lancet 1999, 353: 9–13

CIBIS II – Cardiac Insufficiency Bisoprolol Study Design Biso 5 Korr CIBIS II – Cardiac Insufficiency Bisoprolol Study Design Bisoprolol dose (mg) 10.00 Receptor occupancy detected by an in vitro incubation of plasma samples with a ß-adrenoceptor preparation allows for a prediction of the relative extent of ß-adrenoceptor antagonistic effects in man. In this study the time course of plasma concentration after a single oral dose of 100 mg of bisoprolol – a dosage far beyond the therapeutic dose range – was compared with the ß-adrenoceptor occupancy of the two adrenoceptor subtypes in an ex-vivo/in-vitro assay. 24 hours after the administration of 100 mg bisoprolol, there was no longer any ß2-adrenoceptor occupancy detected in the plasma samples. Up to that time, plasma levels far exceeded the normal therapeutic range. Therefore an occupancy of the ß2-receptors is practically not to be expected under therapeutic conditions. 36 to 60 hours after a single oral dose of 100 mg of bisoprolol, plasma concentrations similar to a steady state condition after once daily dosage of 10 mg were found. At an 80 % occupancy of ß1-receptors, there was no occupancy of ß2-receptors seen with bisoprolol, whilst under 200 mg of atenolol (slide 4) when there was a maximum occupancy of ß1-receptors of 80%, 25% of ß2-receptors were occupied as well. Occupancy of the ß1-receptors with bisoprolol is in the range of approximately 80 – 30% for the therapeutic dose range of 5 – 10 mg. 7.50 5.00 CIBIS II Investigators and Committees, Lancet 1999, 353: 9–13 3.75 2.50 1.25 W1 W2 W3 W4 W5 W6 W7 W8 W9 W10 W11 W12 W13 W14 W15 W16 Week No run-in period Dose increased according to tolerability

CIBIS II – Cardiac Insufficiency Bisoprolol Study Characteristics (I) Biso 5 Korr CIBIS II – Cardiac Insufficiency Bisoprolol Study Characteristics (I) Placebo Bisoprolol (n=1320) (n=1327) Demographic data Mean (range) age (years) 61 (22–80) 61 (26–80) Sex (M/F) 1062 (80%) 1070 (81%) 258 (20%) 257 (19%) NYHA class III 1096 (83%) 1106 (83%) IV 224 (17%) 221 (17%) Heart failure Documented ischaemic heart disease 654 (50%) 662 (50%) Primary dilated cardiomyopathy 157 (12%) 160 (12%) Others* 509 (40%) 505 (38%) Mean (SD) left-ventricular ejection fraction 27.6 (5.5%) 27.5 (6.0%) *Coronary angiography unavailable or no history of myocardial infarction Receptor occupancy detected by an in vitro incubation of plasma samples with a ß-adrenoceptor preparation allows for a prediction of the relative extent of ß-adrenoceptor antagonistic effects in man. In this study the time course of plasma concentration after a single oral dose of 100 mg of bisoprolol – a dosage far beyond the therapeutic dose range – was compared with the ß-adrenoceptor occupancy of the two adrenoceptor subtypes in an ex-vivo/in-vitro assay. 24 hours after the administration of 100 mg bisoprolol, there was no longer any ß2-adrenoceptor occupancy detected in the plasma samples. Up to that time, plasma levels far exceeded the normal therapeutic range. Therefore an occupancy of the ß2-receptors is practically not to be expected under therapeutic conditions. 36 to 60 hours after a single oral dose of 100 mg of bisoprolol, plasma concentrations similar to a steady state condition after once daily dosage of 10 mg were found. At an 80 % occupancy of ß1-receptors, there was no occupancy of ß2-receptors seen with bisoprolol, whilst under 200 mg of atenolol (slide 4) when there was a maximum occupancy of ß1-receptors of 80%, 25% of ß2-receptors were occupied as well. Occupancy of the ß1-receptors with bisoprolol is in the range of approximately 80 – 30% for the therapeutic dose range of 5 – 10 mg. CIBIS II Investigators and Committees, Lancet 1999, 353: 9–13

CIBIS II – Cardiac Insufficiency Bisoprolol Study Characteristics (II) Biso 5 Korr CIBIS II – Cardiac Insufficiency Bisoprolol Study Characteristics (II) Placebo Bisoprolol (n=1320) (n=1327) Receptor occupancy detected by an in vitro incubation of plasma samples with a ß-adrenoceptor preparation allows for a prediction of the relative extent of ß-adrenoceptor antagonistic effects in man. In this study the time course of plasma concentration after a single oral dose of 100 mg of bisoprolol – a dosage far beyond the therapeutic dose range – was compared with the ß-adrenoceptor occupancy of the two adrenoceptor subtypes in an ex-vivo/in-vitro assay. 24 hours after the administration of 100 mg bisoprolol, there was no longer any ß2-adrenoceptor occupancy detected in the plasma samples. Up to that time, plasma levels far exceeded the normal therapeutic range. Therefore an occupancy of the ß2-receptors is practically not to be expected under therapeutic conditions. 36 to 60 hours after a single oral dose of 100 mg of bisoprolol, plasma concentrations similar to a steady state condition after once daily dosage of 10 mg were found. At an 80 % occupancy of ß1-receptors, there was no occupancy of ß2-receptors seen with bisoprolol, whilst under 200 mg of atenolol (slide 4) when there was a maximum occupancy of ß1-receptors of 80%, 25% of ß2-receptors were occupied as well. Occupancy of the ß1-receptors with bisoprolol is in the range of approximately 80 – 30% for the therapeutic dose range of 5 – 10 mg. Concomitant medication Diuretic 1310 (99%) 1305 (98%) ACE inhibitor 1274 (96%) 1273 (96%) Dihydropyridine-type calcium antagonist 23 (2%) 23 (2%) CIBIS II Investigators and Committees, Lancet 1999, 353: 9–13 Nitrate 762 (58%) 773 (58%) Digoxin 670 (51%) 697 (53%) Amiodarone 206 (16%) 185 (14%) Anticoagulant 413 (31%) 399 (30%) Antiplatelet agent 558 (42%) 537 (40%)

CIBIS II – Cardiac Insufficiency Bisoprolol Study Survival Biso 5 Korr CIBIS II – Cardiac Insufficiency Bisoprolol Study Survival 1.0 Receptor occupancy detected by an in vitro incubation of plasma samples with a ß-adrenoceptor preparation allows for a prediction of the relative extent of ß-adrenoceptor antagonistic effects in man. In this study the time course of plasma concentration after a single oral dose of 100 mg of bisoprolol – a dosage far beyond the therapeutic dose range – was compared with the ß-adrenoceptor occupancy of the two adrenoceptor subtypes in an ex-vivo/in-vitro assay. 24 hours after the administration of 100 mg bisoprolol, there was no longer any ß2-adrenoceptor occupancy detected in the plasma samples. Up to that time, plasma levels far exceeded the normal therapeutic range. Therefore an occupancy of the ß2-receptors is practically not to be expected under therapeutic conditions. 36 to 60 hours after a single oral dose of 100 mg of bisoprolol, plasma concentrations similar to a steady state condition after once daily dosage of 10 mg were found. At an 80 % occupancy of ß1-receptors, there was no occupancy of ß2-receptors seen with bisoprolol, whilst under 200 mg of atenolol (slide 4) when there was a maximum occupancy of ß1-receptors of 80%, 25% of ß2-receptors were occupied as well. Occupancy of the ß1-receptors with bisoprolol is in the range of approximately 80 – 30% for the therapeutic dose range of 5 – 10 mg. 0.8 Bisoprolol: 156 deaths (n = 1327) Placebo: 228 deaths (n = 1320) log rank test, p < 0.0001 Survival CIBIS II Investigators and Committees, Lancet 1999, 353: 9–13 0.6 200 400 600 800 Time after inclusion (days) 34% reduction in all-cause mortality with bisoprolol

Biso 5 Korr CIBIS II – Cardiac Insufficiency Bisoprolol Study Analysis of time to death Patients 100 p=0.0011 80 83 6% Receptor occupancy detected by an in vitro incubation of plasma samples with a ß-adrenoceptor preparation allows for a prediction of the relative extent of ß-adrenoceptor antagonistic effects in man. In this study the time course of plasma concentration after a single oral dose of 100 mg of bisoprolol – a dosage far beyond the therapeutic dose range – was compared with the ß-adrenoceptor occupancy of the two adrenoceptor subtypes in an ex-vivo/in-vitro assay. 24 hours after the administration of 100 mg bisoprolol, there was no longer any ß2-adrenoceptor occupancy detected in the plasma samples. Up to that time, plasma levels far exceeded the normal therapeutic range. Therefore an occupancy of the ß2-receptors is practically not to be expected under therapeutic conditions. 36 to 60 hours after a single oral dose of 100 mg of bisoprolol, plasma concentrations similar to a steady state condition after once daily dosage of 10 mg were found. At an 80 % occupancy of ß1-receptors, there was no occupancy of ß2-receptors seen with bisoprolol, whilst under 200 mg of atenolol (slide 4) when there was a maximum occupancy of ß1-receptors of 80%, 25% of ß2-receptors were occupied as well. Occupancy of the ß1-receptors with bisoprolol is in the range of approximately 80 – 30% for the therapeutic dose range of 5 – 10 mg. Bisoprolol (n = 1327) 60 Placebo (n = 1320) p=0.0012 p=0.17 48 4% 49 4% 47 4% CIBIS II Investigators and Committees, Lancet 1999, 353: 9–13 40 p=0.58 36 3% 28 2% p=0.41 20 23 2% 23 2% p=0.75 18 1% 14 1% 7 1% 8 1% Sudden Pump Myocardial Other cardio- Non-cardio- Unknown cause death failure infarction vascular deaths vascular deaths of death Hazard ratio: (95% CI) 0.56 0.74 0.85 1.17 0.75 0.45 (0.39 – 0.80) (0.48 – 1.14) (0.31 – 2.34) (0.67 – 2.03) (0.37 – 1.50) (0.27 – 0.74)

CIBIS II – Cardiac Insufficiency Bisoprolol Study Secondary endpoints Biso 5 Korr CIBIS II – Cardiac Insufficiency Bisoprolol Study Secondary endpoints Placebo Bisoprolol Hazard ratio p (log rank (n = 1320) (n = 1327) (95% Cl) test) All-cause hospital admissions 513 (39%) 440 (33%) 0.80 (0.71–0.91) 0.0006 All cardiovascular deaths 161 (12%) 119 (9%) 0.71 (0.56–0.90) 0.0049 Combined endpoint 463 (35%) 388 (29%) 0.79 (0.69–0.90) 0.0004 Permanent treatment 192 (15%) 194 (15%) 1.00 (0.82–1.22) 0.98 withdrawals Receptor occupancy detected by an in vitro incubation of plasma samples with a ß-adrenoceptor preparation allows for a prediction of the relative extent of ß-adrenoceptor antagonistic effects in man. In this study the time course of plasma concentration after a single oral dose of 100 mg of bisoprolol – a dosage far beyond the therapeutic dose range – was compared with the ß-adrenoceptor occupancy of the two adrenoceptor subtypes in an ex-vivo/in-vitro assay. 24 hours after the administration of 100 mg bisoprolol, there was no longer any ß2-adrenoceptor occupancy detected in the plasma samples. Up to that time, plasma levels far exceeded the normal therapeutic range. Therefore an occupancy of the ß2-receptors is practically not to be expected under therapeutic conditions. 36 to 60 hours after a single oral dose of 100 mg of bisoprolol, plasma concentrations similar to a steady state condition after once daily dosage of 10 mg were found. At an 80 % occupancy of ß1-receptors, there was no occupancy of ß2-receptors seen with bisoprolol, whilst under 200 mg of atenolol (slide 4) when there was a maximum occupancy of ß1-receptors of 80%, 25% of ß2-receptors were occupied as well. Occupancy of the ß1-receptors with bisoprolol is in the range of approximately 80 – 30% for the therapeutic dose range of 5 – 10 mg. CIBIS II Investigators and Committees, Lancet 1999, 353: 9–13

Biso 5 Korr CIBIS II – Cardiac Insufficiency Bisoprolol Study Mortality by baseline findings Bisoprolol Placebo n/total n/total Ischaemia 75/662 121/654 Receptor occupancy detected by an in vitro incubation of plasma samples with a ß-adrenoceptor preparation allows for a prediction of the relative extent of ß-adrenoceptor antagonistic effects in man. In this study the time course of plasma concentration after a single oral dose of 100 mg of bisoprolol – a dosage far beyond the therapeutic dose range – was compared with the ß-adrenoceptor occupancy of the two adrenoceptor subtypes in an ex-vivo/in-vitro assay. 24 hours after the administration of 100 mg bisoprolol, there was no longer any ß2-adrenoceptor occupancy detected in the plasma samples. Up to that time, plasma levels far exceeded the normal therapeutic range. Therefore an occupancy of the ß2-receptors is practically not to be expected under therapeutic conditions. 36 to 60 hours after a single oral dose of 100 mg of bisoprolol, plasma concentrations similar to a steady state condition after once daily dosage of 10 mg were found. At an 80 % occupancy of ß1-receptors, there was no occupancy of ß2-receptors seen with bisoprolol, whilst under 200 mg of atenolol (slide 4) when there was a maximum occupancy of ß1-receptors of 80%, 25% of ß2-receptors were occupied as well. Occupancy of the ß1-receptors with bisoprolol is in the range of approximately 80 – 30% for the therapeutic dose range of 5 – 10 mg. Primary DCM 13/160 15/157 Undefined 68/505 92/509 NYHA III 116/1106 173/1096 CIBIS II Investigators and Committees, Lancet 1999, 353: 9–13 NYHA IV 40/221 55/224 Total Relative risk: 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 Mortality did not differ significantly between groups whatever the aetiology or severity of heart failure

Biso 5 Korr CIBIS II – Cardiac Insufficiency Bisoprolol Study Main results at a glance In the bisoprolol-treated group of patients there was a reduction in All-cause mortality (independent of aetiology) by 34% (p<0.0001) Sudden death by 44% (p<0.0011) All-cause hospital admissions by 20% (p<0.0006) Hospital admissions due to worsening heart failure by 36% (p<0.0001) Receptor occupancy detected by an in vitro incubation of plasma samples with a ß-adrenoceptor preparation allows for a prediction of the relative extent of ß-adrenoceptor antagonistic effects in man. In this study the time course of plasma concentration after a single oral dose of 100 mg of bisoprolol – a dosage far beyond the therapeutic dose range – was compared with the ß-adrenoceptor occupancy of the two adrenoceptor subtypes in an ex-vivo/in-vitro assay. 24 hours after the administration of 100 mg bisoprolol, there was no longer any ß2-adrenoceptor occupancy detected in the plasma samples. Up to that time, plasma levels far exceeded the normal therapeutic range. Therefore an occupancy of the ß2-receptors is practically not to be expected under therapeutic conditions. 36 to 60 hours after a single oral dose of 100 mg of bisoprolol, plasma concentrations similar to a steady state condition after once daily dosage of 10 mg were found. At an 80 % occupancy of ß1-receptors, there was no occupancy of ß2-receptors seen with bisoprolol, whilst under 200 mg of atenolol (slide 4) when there was a maximum occupancy of ß1-receptors of 80%, 25% of ß2-receptors were occupied as well. Occupancy of the ß1-receptors with bisoprolol is in the range of approximately 80 – 30% for the therapeutic dose range of 5 – 10 mg. CIBIS II Investigators and Committees, Lancet 1999, 353: 9–13

CIBIS II – Cardiac Insufficiency Bisoprolol Study Conclusions Biso 5 Korr CIBIS II – Cardiac Insufficiency Bisoprolol Study Conclusions CIBIS II successfully demonstrated that ß1-selective bisoprolol – given in addition to standard therapy – reduces significantly all-cause mortality and all-cause hospitalisation in CHF patients. Bisoprolol is the first ß-blocker which has proven its efficacy in a single large-scale CHF study with all-cause mortality as primary objective. Bisoprolol was as well tolerated as placebo with a permanent treatment withdrawal rate of 15% in both groups. Bisoprolol fumarate (2:1) is a racemate and as a derivative of phenoxyaminopropa-nol it belongs to the class of therapeutic substances known as ß-blockers. Bisoprolol fumarate is very freely soluble in water and freely soluble in ethanol and chloroform. The molecular weight is 383.48, the white crystalline substance melts at 101°C.