Work package number:
Phase:
WP 8; The human intervention study
24-36 months
Start date:
01.02.2000
Completion date:
31.01.2004
Current status:
Ongoing
Partners responsible:
P14 Dr. Hans MG Princen; TNO Leiden
P11 Prof. Dr. Rolf Gebhardt; Univ Leipzig
P12 Dr. Marie-Helene Siess; INRA Dijon
Subcontractor:
Center for Human Drug Research, Leiden, The Netherlands,
Dr. Koos Burgggraaf
In collaboration with
P4, P13, P15 and other partners.
Person months per partner and total: P11: 2; P12: 8; P14: 12; total: 22
Already devoted person months per partner and total: P14: 5 total: 5
Objectives
To study the effect of a garlic preparation in humans on surrogate markers of cancer and
atherosclerosis (anti-carcinogenic and anti-inflammatory activity) :
This task will assess in human volunteers in a placebo-controlled study whether a specific
garlic preparation obtained from the results of tasks 1-6 influences the levels of markers of
vessel wall function, inflammation and oxidative stress, as well as carcinogen-metabolising
enzymes, and DNA-damage caused by mutagens. Furthermore, the lipid-parameters in these
volunteers will be monitored. The intervention study will be performed provided that a
significant influence of garlic on adhesion molecules and on carcinogen-metabolising
enzymes and anti-mutagenic activity in animals can be shown. The analysis of sulfur
metabolites from garlic in blood or urine will allow to know which metabolites are responsible
for the observed effects.
Description of work
Atherosclerosis markers
Determination of the influence of a specific well-defined garlic preparation on levels of von
Willebrand factor (vWF), adhesion molecules (ICAM-1, VCAM-1, ELAM), C-reactive protein
(CRP) and ox-LDL in blood and 8-isoprostanes in urine in a placebo-controlled intervention
study with 2x24 mildly hyperlipidemic smoking volunteers. Numbers are based on power
calculations. The volunteers will be treated for three months after a wash out period of 6
weeks and blood and urine will be collected three times (before, during and after the
intervention). The different parameters will be determined by specific ELISA´s. Further,
plasma lipids (cholesterol, triglycerides, HDL, LDL, Lp(a)) are determined.
Cancer biomarkers
Determination of the the levels of phase II enzymes (glutathione S-transferase and quinone
reductase) in plasma and lymphocytes. Measurement of DNA damage in human lymphocytes
using the comet assay. Determination of the anti-mutagenic properties of urine from human
subjects using the Ames test. This approach will allow to study the mechanisms by which
garlic compounds or their metabolites protect against genotoxicity. Incubation of urinary
extracts with direct-acting mutagens or with indirect-acting mutagens will allow to study if
garlic compounds act by quenching ultimate carcinogen or by inhibition of cytochromes P450
which are involved in carcinogen activation respectively.
Identification and quantification of urinary and blood sulfur metabolites will allow to know
which compounds from garlic (parent compound or metabolites) could be responsible for the
observed effects.
Progress during the third reporting period
Clinical endpoints for the study were defined. The inflammation marker C-reactive protein was
chosen as primary endpoint. Secondary efficacy parameters are parameters of endothelial
function (biochemically), sensitivity of leukocytes to an inflammatory stimulus, indicators of
lipid metabolism and blood pressure, as well as anti-oxidant enzymes and markers of DNA
damage caused by carcinogens. In addition, metabolites of garlic in plasma and urine will be
tried to be identified. Inclusion criteria were defined and a choice was made for the garlic
preparation (Spanish Printanor from harvest 2001) to be used and the positive control (a
statin). A protocol for the study was prepared (see annex) which will be submitted to the
Medical Ethical Committee. Analytical testing of the garlic preparation to be used is underway.
As acronym for the human intervention study EUGIS was chosen: European Union Garlic
Inflammation Study. The study design in short is given below.
Study Design:
Parallel design with 3 groups treated with a garlic powder (Spanish Printanor 2001) versus
control. As a positive control a statin will be used.
Garlic powder contains all compounds present in fresh garlic and is processed in such a way
that sulfur-rich compounds (alliin) expected to have a beneficial effect on health are
preserved.
Planned Sample:
Ninety subjects of either gender aged between 40-75 years with known risk factors not readily
subject for therapeutic intervention for cardiovascular disease (such as overweight, smoking,
women postmenopausal (note: effect HRT on CRP) etc.) and not using medication for this
condition will be recruited for the study.
Power calculation:
The power to find a change of 30% in plasma CRP levels with 30 subjects per arm and α=
0.05 is 0.87. To find a change of 25% the power is 0.72.
Preparations and Dosages:
 Placebo
 Statin (Atorvastatin) 40 mg/d
 Garlic preparation (Spanish Printanor 2001) obtained within the EU-G&H program 2.1 g/d
Deliverables
 DH 17: Documentation for Ethical Committee and Central regulatory authoritiy as well as
local authority: definition of product and project design, specific end-points of clinical
study and definition of inclusion criteria for volunteers (P14)
Done
 DH. 21: Performing intervention study with report and analysis of study results (P14, P11,
P12)
 DH. 22: Publication of human study results (P12, P14)
 DH. 28: Final Case Report Form according to national requirements: final protocol and
amendments and product description by the supplier (P14)
Done
Milestones
Year 3 Definition of specific end-points of clinical study; Definition of inclusion criteria for
volunteers and of product design; Preparation of in vivo studies: review of the project design
and preparation of study documentation including preparation of draft protocol and CRF as
well as final documents for central regulatory and local authorities; preparation of product
information by the supplier; preparation of final documents, protocol, amendment, CRF,
randomisation and medication code envelopes; patient insurance (P11, P14, P12). All done
Year 4 Contact and selection of volunteers, preparing investigatory study file to GCP
standards; medical screening of volunteers; medical and clinical examinations for study
initiation, laboratory screening, performing clinical study including laboratory screening,
analytical testing of samples, study coordination, data entry and quality control of data,
statistical analysis, draft report of study results; final report writing (all P14). Determination of
the levels of phase II enzymes (glutathion S-transferase and quinone reductase) in human
lymphocytes and plasma before and after ingestion of a garlic preparation by measuring the
activities and by Western blot. Evaluation of the DNA alterations in human lymphocytes and
determination of the anti-mutagenic effects of urine. Identification and quantification of sulphur
compounds in blood and urine (all P12).
Exploitation and dissemination activities
No data collected. Study will start in April 2003.
Ethical aspects and safety provisions
Ethical aspects have been considered in the preparation of the human study.