European Union Garlic Inflammation Study (EUGIS) in Humans Martijn van Doorn Sonia Espirito Santo TNO-PH Leiden The Netherlands CHDR Leiden The Netherlands

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, i.e. lipid lowering drugs like statins)

Aim Study the effects of garlic on inflammatory biomarkers of atherosclerotic disease and on cancer biomarkers in man Primary endpoint: 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 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. 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. 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

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, i.e. aspirin or hormone replacement therapy, interfering with the study Chronic drug treatment /use of medication, i.e. aspirin or hormone replacement therapy, interfering with the study Participation in prior studie(s) in the last 3 months Participation in prior studie(s) in the last 3 months Blood donation, including this study, not according to the guidelines of the blood donation services Blood donation, including this study, not according to the guidelines of the blood donation services Inadequate use of contraceptives Inadequate use of contraceptives Pregnant or lactating women Pregnant or lactating women History of alcohol or drug abuse History of alcohol or drug abuse Positive test results for Hepatitis B, Hepatitis C or HIV Positive test results for Hepatitis B, Hepatitis 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)

Groups design 7 capsules of 300 mg (3 cap at breakfast and 4 cap at dinner) = one medium-large size fresh garlic clove EU-sponsored Printanor 2001: 2.1g/day for 12 weeks = 7 capsules of 300 mg (3 cap at breakfast and 4 cap at dinner) = one medium-large size fresh garlic clove – compliant with European Pharmacopoeia with respect to microbial impurities; for 12 weeks = 1 tablet of 40 mg at dinner) Atorvastatin: 40 mg/day for 12 weeks = 1 tablet of 40 mg at dinner) Placebo: tablets every day for 12 weeks

Measurements Clinical: Blood pressure, ECG 12 lead, Heart rate (HR), and Heart Rate Variability (HRV). Clinical: Blood pressure, ECG 12 lead, Heart rate (HR), and Heart Rate Variability (HRV). 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; Safety: Parameters of muscle (CPK) and liver function (ALAT), and haematology (haematocrit, WBC count). Safety: Parameters of muscle (CPK) and liver function (ALAT), and haematology (haematocrit, WBC count).

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  Informed subjects: 120  Informed subjects: 120  Screened subjects: 70  Screened subjects: 70  Included (randomized) subjects: 52  Included (randomized) subjects: 52  Subjects started 1 st occasion: 43  Subjects started 1 st occasion: 43 # Subjects planned for info: 20 # Subjects planned for info: 20 # Screenings planned: 10 # Screenings planned: 10 Recruitment is ongoing!

Ackowledgements Martijn van Doorn Koos Burggraaf Sonia Espirito Santo Hans Princen