1. Multiplex Biomarker Approach for Determining Drug-Induced Vascular Injury (DIVI) in Rat
Lothar Goretzki, Wei Zheng, Jehangir Mistry - EMD Millipore, Inc.
Abstract II. Intraperioteneal Drug Administration
Results Intravenous infusion of fenoldopam
Vascular injury is a common finding during the pre-clinical testing of mesylateresults in increased plasma levels of
Rat Vascular Injury Panels 1 and 2. Standard Curves. vWF and TIMP-1 Intraperioteneal injection of fenoldopam
drugs. A lack of understanding of mechanisms of drug-induced
vascular injury (DIVI) in animals, the absence of accepted specific and mesylate results in increased plasma levels
100000
sensitive biomarkers and often unknown relevance for human have A B of CTGF
Median Fluorescence Intensity (MFI)
100000
250
TIMP-1
Median Fluorescence Intesity (MFI)
vWF Figure 3. vWF
become significant barriers in the development of new therapeutic
Plasma vWF (green) and Plasma TIMP-1
Figure 7. In contrast to IV
(blue) levels shown as % of saline-
200 and TIMP-1
agents. In recent years a number of promising biomarker candidates 10000
Fenoldopam Day 7
Drug Administration, no
significantly
treated vehicle control
have been nominated with the need for further validation and vWF
10000
150 significant change between
TIMP-1 increased at day Fenoldopam 24h
evaluation. Here, we evaluated 9 biomarker candidates in drug-treated or saline-treated
1000
CTGF 1 subsequent to
multiplexed immunoassays for use as markers of DIVI in rat. As drug 100 Fenoldopam 6h animal was observed for A2M
Cav-1 treatment and
toxicant we used Fenoldopam Mesylate, a dopaminergic 1000
AGP
and sVCAM-1, but CTGF was
CINC-1 A2M 50 both markers Saline Day 7
elevated in drug-treated
vasodilator and well established vascular toxicant in rat. The group of 100
sVCAM-1 Hp were still elevated animals at day 7 as shown in
candidate biomarkers evaluated in this study comprises a reasonable 0
Saline 24h
Day 1 Day 7 Day 1 Day 7 at day 7. the bar graph.
predictive panel for assessing DIVI in rat. 100 Saline 6h
10 0.01 0.1 1 10 100 1000 10000
Days Post-Dose Injection
Note: In contrast to IV
0.1 10 1000
Evaluated Candidate Vascular Expression Concentration
(pg/ml for CINC-1 and TIMP-1,
Concentration (ng/ml)
0 50 100 150 200 250 infusion, no increase in A2M
CTGF (ng/ml)
ng/ml for others) and sVCMA-1 was observed
Biomarkers in drug-treated animals (data
Alpha-1-Acid Glyoprotein (AGP) Endothelial Cells, Macrophages, Figure 1A. Standard curves for Figure 1B. Standard curves Intravenous infusion of fenoldopam mesylate not shown).
Adipocytes Caveolin-1 (Cav-1), Cytokine- for Alpha-1-Acid-Glycoprotein results in decreased mensenteric artery tissue
Alpha-2-Macroglobulin (A2M) Endothelial Cells, Macrophages, Induced Chemoattractant-1 (AGP), Alpha-2-Macroglobulin
Hepatocytes extract levels of Cav-1
(CINC-1), Connective Tissue (A2M), and Haptoglobin (Hp).
Caveolin-1 (Cav-1) Endothelial Cells, Smooth Muscle Growth Factor (CTGF), Vascular The standards were incubated
Cells, Macrophages, Adipocytes
Intraperiotenal injection of fenoldopam mesylate results
Cell Adhesion Molecule-1 for 2 hours at room in increased plasma levels of vWF and TIMP-1
CINC-1/GRO Macrophages, Hepatocytes Figure 4. Caveolin-1,
(VCAM-1), Von Willebrand temperature. Fenoldopam Day 7
localized primarily to smooth
Connective Tissue Growth Factor Endothelial Cells, Smooth Muscle Factor (vWF), and Tissue muscle and endothelial cells,
Cells, uptake by Macrophages Inhibitor of Metalloproteinases-1 Figure 8. vWF
(CTGF) Fenoldopam Day 3 substantially decreased with 250
significantly increased
shown as % of slaine-treated vehicle
(TIMP-1). The standards were
Plams a vWF and TIMP-1 levles are
the onset of drug-induced
Hepatocytes, Adipocytes incubated for 2 hours at room Fenoldopam Day 1 vascular injury. The cav-1 loss 6h and 24h
Haptoglobin (Hp) 200
temperature. from the vessel wall could be subsequent to
Vascular Cell Adhesion Molecule -1 Endothelial Cells, Smooth Muscle released into the blood.
Cells, Macrophages Saline Day 7 150 treatment. TIMP-1 was
control
(VCAM-1) I. Intravenous Drug Administration However, Cav-1 was not
slightly increased, by
detectable in plasma or serum,
Von Willebrand Factor (vWF) Endothelial Cells Saline Day 1 100 factor 0.2 at 6h and 24h
presumably due to insufficient
TIMP-1 Endothelial Cells, Fibroblasts Intravenous infusion of fenoldopam mesylate results in sensitivity of the employed when compared to the
increased plasma levels of A2M, CTGF, and VCAM-1
0 500 1000 1500 2000 2500
50 TIMP-1
Caveolin-1 (ng/mg total protein) assay conditions. respective saline-
treated animals
Methods A Intravenous infusion of fenoldopam mesylate
0
6h 24h 6h 24h
FM Day 7 Figure 2A. A2M, an
Technology acute- phase protein results in decreased plasma levels of haptoglobin
Using the MAGPIX® system (Luminex magnetic bead –based Fenoldopam Day 3 analogous to the C-reactive
technology) we developed multiplexed immunoassays to measure protein in human, increased
protein analytes in rat serum/plasma, and blood vessel tissue Fenoldopam Day 1 more than 2-fold compared
samples. The magnetic beads multiplex immunoassays are comprised to the vehicle control at day Fenoldopam Day 7 Figure 5. As
Saline Day 7 1 subsequent to treatment, haptoglobin
of analyte-specific-specific antibodies and other optimized reagents in
reaching its peak level at is an acute-phase
a capture-sandwich format. Saline Day 1 day 3.
Fenoldopam Day 3
protein it is more likely
Summary
Fenoldopam Day 1 than not to rise upon  In the present study, a series of candidate biomarkers
Study Design for Drug-Induced Vascular Injury in Rat 0 50 100 150 200
the onset of vascular
Alpha-2-Macroglobulin (mg/ml)
of vascular injury were assessed to determine the
1) Intravenous drug administration injury. However, a
Figure 2B. In contrast to
Saline Day 7
ability to detect vascular injury.
Male Sprague-Dawley rats were employed. Rats were substantial decrease
Fenoldopam Day 7 B
anaesthetized with sodium pentobarbital (40 mg/kg i.p. to insert the acute-phase protein
Saline Day 1
was observed, most  Our emerging data suggest that A2M, Cav-1, CTGF,
the venous cannula for drug or vehicle infusion. Each animal was Fenoldopam Day 3
A2M, CTGF showed no likely due to hemolysis. TIMP-1, sVCAM-1 and VWF should be included in a
housed separately with food and water freely available during significant increase at the 0 0.5 1
Haptoglobin (mg/ml) 1.5 2
rat vascular injury biomarker panel(s).
end of day1 after
recovery. Animals were dosed with fenoldopam mesylate by Fenoldopam Day 1
treatment, however,  Many of the candidate biomarkers are conveniently
intravenous infusion at 6 mg//kg/h for 24h. The treated rats were CTGF levels were Intravenous infusion of fenoldopam mesylate measurable in plasma or serum.
sacrificed at the end of days 1, 3, and 7. Vehicle-control animals Saline Day 7
significantly elevated at resulted in small increase of serum AGP
were infused with saline and sacrificed at the end of day 1 and 7. the end of day 3 and 7  We were unable to detect CINC-1//GRO in any of the
Saline Day 1
0.5 -2 ml EDTA Plasma and Serum were collected from each subsequent to treatment. tested samples. The reason could be that it is
animal and kept frozen at -70oC until use. In addition, aorta, Figure 6. AGP, is temporarily elevated at an earlier time point post dose
0 1000 2000 3000 4000 Fenoldopam Day 7
CTGF (ng/ml) an acute phase
mesenteric arteries, and femoral arteries were harvested and snap administration than the time points we addressed in our
reactant and
frozen. Fenoldopam Day 3 functions also as a
studies.
2) Intraperioteneal drug administration Figure 2C. Plasma levels
Fenoldopam Day 7 C of sVCMA-1 tested
carrier protein for  The rat vascular injury biomarker assays deliver
Male Sprague-Dawley rats were dosed intraperioteneally steroid hormones
significantly higher in
Fenoldopam Day 1 accurate and reproducible measurements.
with 30 mg/kg fenoldopam mesylate or vehicle saline. Fenoldopam Day 3 and various drugs.
Animals were sacrificed 6h, 24h and 5 days post injection. fenoldopam-treated Here, it was not  Individually, the candidate biomarkers have strengths
Fenoldopam Day 1 animals. The time course Saline Day 7
significantly and limitations but combined they provide a set of tools
change is comparable to elevated in drug- to detect vascular injury at various stages in rat.
Saline Day 7 CTGF, i.e., no significant Saline Day 1
treated animals.
increase at the end of day 1
Saline Day 1 after treatment, but 0 20 40 60 80 100 120
Alpha-1-Acid Glycoprotein (mg/ml)
Corresponding Author: lothar.goretzki@merckgroup.com 0 100 200 300 400 500
significantly elevated levels
sVCAM-1 (ng/ml) at the end of day 3 and 7.
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