Effects of Dose, Sex, and Species Variations on Imaging Efficacy
of a Hepatocyte-Selective Contrast Agent in Normal Rodents
GN Ton1, WC Dow2, DA Bakan2, M Melchior2, JP Weichert1
1University of Wisconsin-Madison,WI and 2Alerion Biomedical Inc., San Diego, CAIntroduction

Results and Discussion

Fig 5. Normalized Signal Intensity-Time Profiles of Fenestra LC in(A) Male FVB Mice and (8) Male SD Rats vs FVB Mice (10 mL/kg)

Fig 3. MicroCT body kinetics of Fenestra LC in an anesthetized male rat
following administration of a single 10 mL/kg doseBaseline1h5 min

24 hSSAxial

4h

IVCS

CoronalFig 1. Hepatic uptake of Chylomicron Remnants and macromolecular
structure of ITG particle

KRVAB500

Normalized Signal Intensity (HU/Voxel)

High-resolution microCT systems capable of sub 20-micron isotropic spacial
resolution provide an attractive new approach for noninvasively studying models ofhuman diseases in live small animals.2,3 However, relatively long acquisition times
preclude the use of conventional water-soluble contrast media for imaging
pathological conditions in soft tissues. Exploiting the endogenous lipid metabolism
pathways that occur naturally in the body, we have developed a hypatocyte-selective
CT contrast agent consisting of a polyiodinated triglyceride (ITG) in a lipid emulsiondelivery system. The ITG vehicle mimics chylomicron remnants (CMR). CMR
particles represent a class of naturally-occurring plasma lipoproteins that selectively
shuttle lipids to hepatocytes in the liver (Fig 1). The contrast agent has been
evaluated for its ability to provide excellent hepatic opacification for prolonged
periods of time after injections in both normal and diseased model animals1,4,5.

IVC (mFVB)_10 mL/kg LC
IVC (mFVB)_15 mL/kg LC
Liver (mFVB)_10 mL/kg LC
Liver (mFVB)_15 mL/kg LC

400

mRat_IVC_10 mL/kg
mMouse_IVC_10 mL/kg
mRat_Liver_10 mL/kg
mMouse_Liver_10 mL/kg

400

300

300

200

200

100

1000GI

500

00204060

020

Post Injection Time (Hours)

3-D surface rendered images ofan anesthetized male rat 5 min
following injection of 10 mL/kg
Fenestra LC showed excellent
vascular enhancement while theliver agent was in the bloodpool
phase.

AB48CEECCMR

FattyAcidsSPHDLRVCapillaryendothelium

Hepatocyte

ITG Particle

Fatty acid-albumincomplexes

The aim of this project is to examine the effects of administration dose, sex,and species variations on the in vivo microCT imaging efficacy of the liver contrast
agent in normal mice and rats.

Materials and Methods
The liver contrast agent, which is now commercially available as FenestraTM
LC, from ART Advanced Research Technologies (Montreal, Canada) provided afinal iodine concentration and mean particle size of 50 mg I/mL and 200 nm,
respectively. Fenestra LC was administered as a single i.v. bolus dose via tail vein
injection. Three doses (2.5, 5, and 10 mL/kg) were given to both female and maleSprague-Dawley rats (n=2-3/group), and two doses (10 and 15 mL/kg) were injected
to male FVB mice (n=3). Anesthetized (2% isoflurane) animals were scanned using
a GE eXplore Locus microCT system (80 kVp, 450 µA, 200 msec/view, 5frames/view, 400 views/scan and 93 µm3 resolution) prior to and at predetermined
time intervals following administration of the hepatocyte-selective agent.
Images were reconstructed with the EVSBeam software (GE Medical System)and subsequently displayed and analyzed using both Amira 3-D visualization
software (V3.1) and an open-source MicroView program (http://Microview.sf.net).
CT values of volumetric ROIs in the inferior vena cava (IVC) and liver werenormalized to Hounsfield Units (HU). Normalized signal intensity (HU/voxel) was
obtained from the difference of enhanced SI and the baseline value.
Fig 2. Liver-enhanced microCT images of female (left) and male SD rats(right) 4 hours following IV administration of Fenestra LC at various doses.
2.5 mL/kg

F5 mL/kg

SAC

B,E-receptor

LSLPL

10 mL/kg

IVC

2.5 mL/kg

5 mL/kg

10 mL/kg

M

Ab. Aorta vena cava

Fig 3. shows time sequential Fenestra LC-enhanced 2-D and 3-D microCT
images of an anesthetized rat following intravenous administration of a single 10mL/kg dose. Owing to the liver-selective capability and prolonged in vivo residencetime, real-time body kinetics of Fenestra LC in rats can be monitored. Non-contrastaxial and coronal images of the rat showed poor soft tissue contrast in the thoracic
and abdominal cavities. Axial and coronal microCT images of the rat obtained atindicated post injection time intervals revealed the liver signal intensity peaks after 3hours following an iv injection. The IVC and hepatic vascular network became visible
after 4 hours due to signal void as a result of the removal of Fenestra LC from thecirculation and its localization in hepatocytes. Quantitiave characterization of thevascular network is possible with the use of combined 3D surface rendering and 2D
microCT images of the rat obtained during the blood-pool phase of Fenestra LC (t=5min post injection).

ASKSPAdrenal

GI

Excellent liver contrast enhancement and soft tissue identification were observed inboth female and male rats 4 hours following injection of Fenestra LC (10 mL/kg).

10 minIVC (mRats)_10 mL/kg
IVC (mRats)_5 mL/kg
IVC (mRats)_2.5 mL/kg
Liver (mRats)_10 mL/kg
Liver (mRats)_5 mL/kg
Liver (mRats)_2.5 mL/kg

400

400

300

300

200

200

10010000051015Post Injection Time (Hours)

2025

6h24 hGB

10 ml/kg

LL
S
GIbaseline

GBL

15 ml/kg

LLSScaecum

IVC (fRats)_10 mL/kg
IVC (fRats)_5 mL/kg
IVC (fRats)_2.5 mL/kg
Liver (fRats)_10 mL/kg
Liver (fRats)_5 mL/kg
Liver (fRats)_2.5 mL/kg

500

2hExcellent vascular contrast enhancement was achieved initially as the liver
contrast agent was introduced directly to the blood stream via tail vein injection,
followed by an increase in the degree of liver contrast enhancement as the hepatocyte
recognition and localization initiated thereafter. Elimination via hepatobiliary system
was evident after 24 hours as indicated by opacification in the gall bladder and GI
tract.

Conclusions
0510152025

Post Injection Time (Hours)

SLFig 6. MicroCT body kinetics of Fenestra LC in anesthetized male FVB
mice following injections of 10 or 15 mL/kg doseSB

500

60

The effect of species variation on the liver opacification activity of Fenestra LC
is clearly shown in Fig 5B. Liver uptake, as measured by an increase in the signal
intensity, was significantly faster in male SD rats than in male FVB mice following
administration of an iv bolus dose. The rate of the contrast agent was removed fromthe vasculature in rats was significantly faster than in mice. Pharmacokinetic
profiles of the two groups clearly demonstrated a direct correlation between theblood clearance and liver uptake of Fenestra LC.

Fig 4. Normalized Signal Intensity-Time Profiles of Fenestra LC in Male (A)and Female Sprague-Dawley Rats (B)Normalized Signal Intensity (HU/Voxel)

Chylomicron

40

Post Injection Time (Hours)

Normalized contrast enhancement profiles of Fenestra LC in the blood and livertissues following various administration doses in female and male SD rats are shownin Fig 4. Imaging data demonstrated that tissue contrast intensity in both rats andmice was dose dependent. Vascular signal intensity in rats decreased rapidly after 4hours and returned to background level after 24 hours following intravenous
administration of a single bolus dose. Liver signal intentsity peaked after 3 hours
followed by a gradual exponential decay owing to hepatobiliary elimination. Bothfemale and male rat groups exhibited comparable tissue enhancement profiles.

In vivo imaging results demonstrate that Fenestra LC provided excellent hepatic
contrast enhancement in the rodent models tested. Species variation strongly influenced thein vivo microCT imaging characteristics, while the effect of gender variant on theenhancement profiles was minimal. Due to its biomimetic nature, Fenestra LC provides
both superior anatomic and functional capabilities .

References[1] Weber SM, et. al, J Surgical Research (2004) 119:41-45.
[2] Paulus MJ, et. al, Lab Animals (2001) 30:1-10.
[3] Paulus MJ, et. al, Neoplasia (2000) 1:62-70.
[4] Weichert JP, et. al, Radiology, (2000) 216:865-871.
[5] Weichert JP, et. al, J Med Chem (1995) 38:636-646.

Supported in part by the UWCCC, UW-Radiology and Alerion Biomedical Inc.
gn.ton@hosp.wisc.edu, Poster #242