Determination of dihydropyrimidine dehydrogenase and thymidylate synthase activity in human peripheral blood mononuclear cells in healthy volunteers.

5-Fluorouracil (5-FU), the lead compound of the fluoropyrimidines, together
with its prodrug capecitabine are two of the most frequently prescribed
chemotherapeutic agents for the treatment of various malignant solid tumors,
such as, breast and colorectal cancer. In order to exert its cytotoxic effect,
5-FU must first be metabolised to its active metabolite 5-fluoro-2*-
deoxyuridine monophosphate (FdUMP). FdUMP inhibits thymidylate synthase which
ultimately results in the inhibition of DNA synthesis. The degradation of 5-FU
is initialized by the polymorphically expressed enzyme dihydropyrimidine
dehydrogenase (DPD). This rate-limiting step in the catabolism pathway
catalyses the conversion of 5-FU into 5,6-dihydro-5-fluorouracil (FUH2). Since
DPD is responsible for catabolising more than 80% of administered 5-FU,
patients with a complete or partial DPD deficiency are at high risk for
developing severe and sometimes fatal fluoropyrimidine-induced toxicity. An
estimated 3-5% of the Caucasian population has a decreased DPD enzyme activity,
which may be caused by amongst others mutations in DPYD, the gene encoding for
dihydropyrimidine dehydrogenase. Moreover, a known DPD-deficiency is a
contraindication in the treatment with 5-FU and 5-FU analogues.

The enzyme thymidylate synthase (TS) is one of the key targets of
fluoropyrimidine based chemotherapy, i.e. 5-fluorouracil (5-FU). The active
metabolite 5-fluoro-2-deoxyuridine-5-monophosphate (5-FdUMP) inhibits TS, which
is normally turns deoxyuridine monophosphate (dUMP) to deoxythymidine
monophosphate (dTMP). Consequently, dTMP is phosphorylated to deoxythymidine
triphosphate (dTTP) which is used in DNA synthesis and repair. In other words,
FdUMP inhibits TS and thereby de novo thymidine synthesis. The inhibition of
thymidine synthesis will block synthesis of the DNA, thereby causing the
cytotoxic effect of fluoropyrimidine based chemotherapy. It is believed that TS
activity might serve as a predictive factor for 5-FU based chemotherapy. In
several studies it was found that high TS activity or high expression of the TS
protein was associated with 5-FU resistance. In addition, other studies found a
significant improvement in survival in case of low TS activity in tumor tissue.

Next to detection of genetic mutations that may lead to a DPD-deficiency,
several phenotypic assays have been described in the literature that determine
the enzymatic DPD activity. Examples of these are measurement of 5-FU plasma
clearance after a reduced test dose, [2-C13] uracil breath test and direct
evaluation of DPD activity in peripheral blood mononuclear cells PBMCs using
HPLC with on-line UV detection, tandem mass spectrometry or radioactivity
detection.
Previous studies regarding TS enzyme activity have been conducted in surgical
obtained tissue samples. These studies demonstrated a significant difference in
TS activity between tumor tissue and normal surrounding tissue. Unfortunately,
a correlation between TS activity in PBMCs and TS activity in tissue was not
previously demonstrated. Nor was the difference in TS activity between PBMCs
from healthy subjects compared to PBMCs from cancer patients.

These tests however, are currently not routinely assessed prior to start of
therapy in clinical practice for several reasons. Besides lack of dosing
guidelines for poor, intermediate and extensive 5-FU metabolizers, another
major hurdle for using a phenotypic test prior to start of therapy is the lack
of a rapid, inexpensive and non-invasive patient-friendly assay, that can be
applied on a routine basis.
The TS enzyme activity radio assay was recently designed and has not been
conducted in either healthy subjects or patients. As with the DPD assays,
rapid, cheap, non-invasive and patient-friendly assays are lacking. Moreover,
it is not known if TS activity in PBMCs and tumor tissue correlate.
Additionally, the clinical translation from TS activity to dose- or regimen
adjustments has not been made.

Within the Department of Experimental Therapy of The Netherlands Cancer
Institute - Antoni van Leeuwenhoek Hospital (NKI-AVL) a rapid and sensitive
method was developed to determine the DPD-activity in PBMCs by HPLC with
on-line radioactivity detection. Subsequently a radio assay was designed to
determine TS enzyme activity in PBMCs,
To gain insight into the DPD and TS enzyme activity in the healthy population
measured by both assays, we propose to measure the DPD and TS enzyme activity
in 20 healthy volunteers. In addition, DPYD and TYMS gene expression will be
measured from blood obtained at all time points, and observed polymorphisms in
DPYD and TYMS will be associated with the observed DPD resp. TS enzyme
activity. Also, the uracil/dihydrouracil ratio in plasma will be determined and
related to DPD enzyme activity. Furthermore, to gain insight into the
circadian rhythm of DPD and TS, we propose to perform our method with blood
drawings on 7 different time points during one day.

Primary objective:
• To determine the range and mean population DPD and TS enzyme activity in
human peripheral blood mononuclear cells in 20 healthy volunteers.

Secondary objectives:
• To determine the circadian rhythm of DPD and TS enzyme activity in PBMCs of
12 healthy volunteers (male:female 1:1);
• To correlate DPD and TS gene expression in PBMCs with DPD and TS enzyme
activity;
• To associate any observed polymorphisms in DPYD or TYMS with the observed DPD
and TS enzyme activity.

To determine the DPD and TS enzyme activity in a normal population measured by
enzyme activity assays, healthy volunteers are asked for participation in this
study. Written informed consent will be obtained from all healthy volunteers
prior to any study procedure. From every volunteer (male:female = 1:1), 48 ml
of whole blood will be drawn at 9:00 h ± 30 minutes in the morning. A volume of
2 x 16 ml will be used for the DPD and TS enzyme activity determination. A
volume of 8 ml will be used for mRNA isolation. A volume of 4 ml will be used
to test for polymorphisms in DPYD and TYMS. A volume of 4 ml is used to isolate
plasma for uracil and dihydrouracil analysis. Volunteer demographics (initials,
date of birth, gender and wake up time) will be obtained after blood donation.
In 12 volunteers (male:female = 1:1) the circadian rhythm of DPD and TS will be
determined. These subjects will undergo blood sampling 7 times within 24 hours
at 09:00 h, 13:00 h, 17:00 h, 21:00 h, 01:00 h, 05:00 h and 9:00 h the day
after (all ± 30 minutes). In order to take blood samples at night, volunteers
will be hospitalized in the Slotervaart hospital. Nurses will draw blood
samples using a venflon while the volunteers are asleep in order to limit the
disturbance of the normal day/night rhythm.

To isolate human PBMCs, 32 ml of peripheral blood will be collected in heparin
tubes for the DPD and TS activity determination, 8 ml of blood will be
collected in CPT tubes for the gene expression analysis, 4 ml of blood will be
obtained in heparing tubes for uracil/dihydrouracil analysis and 4 ml of blood
will be obtained in EDTA tubes for pharmacogenetic analysis. In 8 patients this
will be performed once and in 12 volunteers, 48 ml of blood will be collected
at 1 time point and 44 mL of blood will be collected at 6 other time points
within 24 hours by the use of one venipunture and a venflon.
The burden of this sampling includes one venipuncture which could consist of
the following side-effects: discomfort, bruising and hematoma and very rarely
infection.