EUGIS European Union Garlic Inflammation Study in Humans

Background No clear indications that ‘garlic’ influences lipid metabolism Indications from cell and animal studies that ‘garlic’ may influence inflammation parameters Cardiovascular disease (atherosclerosis) shares common features with inflammation Current treatment strategies for atherosclerosis also focus on inflammation

Aim Study the effects of garlic on inflammatory biomarkers of atherosclerotic disease and on cancer biomarkers in man : changes in C-reactive protein ( CRP) Primary endpoint: changes in C-reactive protein ( CRP) Secondary endpoints: Secondary endpoints: – Indicators of lipid metabolism – Biochemical markers of inflammation – Blood pressure / heart rate variability – Biomarkers for anti-carcinogenic effects

Inclusion criteria Able and willing to give informed consent Able and willing to give informed consent 90 subjects of either gender* 90 subjects of either gender* Aged between years Aged between years Smoking≥ 10 cigarettes/day and BMI≥ 24.5 kg/m 2 Smoking≥ 10 cigarettes/day and BMI≥ 24.5 kg/m 2 * Power calculation to find a reduction of 30% in plasma CRP levels with  = 0.05 and a power of 80% about 30 subjects per group are needed

Exclusion criteria Any major clinical significant abnormality detected by a general health questionnaire or by blood sampling Any major clinical significant abnormality detected by a general health questionnaire or by blood sampling Chronic drug treatment / use of medication Chronic drug treatment / use of medication Participation in prior study in the last 3 months or blood donation Participation in prior study in the last 3 months or blood donation Inadequate use of contraceptives / pregnancy / lactating women Inadequate use of contraceptives / pregnancy / lactating women History of alcohol / drug abuse History of alcohol / drug abuse Positive test results for HepB/C or HIV Positive test results for HepB/C or HIV Dislike of garlic Dislike of garlic

Experimental design I.C. / screening Intervention period weeks Blood and urine collection -2 Double dummy placebo-controlled trial 3 parallel groups (n=30)

12-weeks treatment 1. Garlic (from EU-sponsored Printanor 2001) Lichtwer capsule preparation (300 mg) (3 cap - breakfast & 4 cap at dinner) 2.1g daily (3 cap - breakfast & 4 cap at dinner) 2. Atorvastatin: at dinner 40 mg/day daily at dinner 3. Placebo: garlic and statin placebo daily

Measurements Safety: Safety: – Parameters of muscle and liver function – Haematology Clinical: Clinical: – Blood pressure – 12 lead ECG (HR, conduction) – Heart Rate Variability (HRV)

Measurements Biochemical: Biochemical: – Cholesterol, HDL, and triglycerides; – CRP, vWF, Fbg, and cytokines after whole blood stimulation without or with LPS (TNF-α, IL-10); – Sensitivity of leukocytes to inflammatory stimulus; – Cancer biomarkers: – Cancer biomarkers: anti-oxidant enzymes (GPX, SOD), DNA damage (Comet assay), anti-mutagenic properties of urine (Ames test); – Metabolites of garlic compounds in plasma and urine;

Additional measurements suggested in the EU proposal SAA, and PAI-1 s-VCAM / s-ICAM / s-Selectine MCP-1 and other chemokines Endothelin 1 and 3 Plasma ox-LDL Urine isoprostanes

Current study status  Subjects at information sessions: >148  Subjects at information sessions: >148  Subjects signing IC: 142  Subjects signing IC: 142  Subjects randomised : 92  Subjects randomised : 92 –  Subjects completed: 84 –  Subjects withdrawn: 8 (6 non-related AEs) Entire clinical study period: < 5 months! Entire clinical study period: < 5 months! – last samples at Jan 15 th

Study population - baseline Female Female – n = 47 – age: yrs – BMI: 29.6 (SD: 4.1) kg/m 2 – BP: 124 (18) / 76 (12) mm Hg Male Male – n = 37 – age: yrs – BMI: 29.3 (SD: 3.2) kg/m 2 – BP: 130 (11) / 82 (10) mm Hg

Baseline lipids Female Female – Total Cholesterol:5.60 (1.01) mmol/L ( ) – HDL-Cholesterol:1.35 (0.32) mmol/L (>1.16) – LDL-Cholesterol:3.52 (0.95) mmol/L – Triglycerides:1.63 (0.95) mmol/L ( ) Male Male – Total Cholesterol:5.71 (1.04) mmol/L ( ) – HDL-Cholesterol:1.11 (0.32) mmol/L (>0.90) – LDL-Cholesterol:3.54 (0.99) mmol/L – Triglycerides:2.35 (1.32) mmol/L ( )

Adverse Events AE’s AE’s – Headache: 52 episodes in 41 subjects 52 episodes in 41 subjects – Abdominal discomfort 36 episodes in 26 subjects 36 episodes in 26 subjects – Other 60 various events (URT infection, ‘pains’, intercurrents) 60 various events (URT infection, ‘pains’, intercurrents)

PLAN All samples have been / are being shipped All samples have been / are being shipped Unblinding next week Unblinding next week Primary parameter laboratory analysis completed at Jan 31 st Primary parameter laboratory analysis completed at Jan 31 st Stats analysis time allocated Stats analysis time allocated Main report around March 1 st 2004 Main report around March 1 st 2004 Additional reports: to be discussed Additional reports: to be discussed

THANKS TNO-PG TNO-PG P Meijer R Kret R S treunding CHDR CHDR M van Doorn I Kamerling Nursing staff LUMC pharmacy LUMC pharmacy R Press Lichtwer Lichtwer S Striegl T Haffner Participants who tolerated 2.1 gram garlic/day Participants who tolerated 2.1 gram garlic/day