MicroCT Evaluation of a Long-Lasting Blood Pool and Hepatobiliary Contrast Agents
Following Multiple Intravenous Injections in Normal Mouse Models
GN Ton1, WC Dow2, DA Bakan2, M Melchior2, JP Weichert1
1University of Wisconsin-Madison,WI and 2Alerion Biomedical Inc., San Diego, CA

Results and Discussion

B

Chylomicron

48CE
B,E-receptor

EC

CMR

LPL

FattyAcids

AC

HDL

Capillaryendothelium

Hepatocyte
Fatty acid-albumincomplexes

The blood pool version of ITG wasdeveloped by incorporating PEG moieties
into the phospholipid monolayer shell,
resulting in delayed uptake by liver cells
and prolonged vascular residence time.
Both of contrast agents have demonstrated
their ability to provide excellent vascularand hepatobiliary contrast enhancement inboth normal and diseased animal models
following in a single iv injection 1-4.

Fig 1. Hepatic uptake of Chylomicron Remnants

Both the liver and blood poolcontrast agents have recently
become commercially available andknown as FenestraTM LC and VC,respectively. Schematic structuresof Fenestra products are shown in
Fig 2. In this study we evaluated thein vivo microCT imaging efficacyof Fenestra LC and VC following
multiple iv administrations. Inaddition injection tolerance profilesof the contrast agents wereexamined.

VCLC

Fig 2. Schematic Structure of Fenestra VC & LC

Materials and Methods

Fig 3. Fenestra VC and LC Dosing Regimes in Various Strains of Mice
15 mL/kg, 1 time/ weekly

1st Dose(day 0)

2nd Dose(day 7)

2nd Dose(day 4)

3rd Dose(day 7)400

400

10 mL/kg, 2 times/week

300

300

200

200

100

100

0

1st Week

2nd Week

3rd Week 4th Week0

1st Week

2nd Week

3rd Week

4th Week5th Dose(day 14)

10 mL/kg, 2 times/ weekly

4th Dose(day 21)

6th Dose(day 18)

7th Dose(day 21)

2nd Dose

4th Dose

CoronalView

3rd Dose

4th Dose

Fig 7. Normalized liver contrast intensities of Fenestra LC 4 hours
following repeating dosing schedules in a female Balb/c mouse
15 mL/kg weekly

200

150

100

50

0

1st Week 2nd Week 3rd Week 4th Week

Fenestra LC Dosing ScheduleL

Sagittal
View

L

Materials and Methods
IVC

Axial
t=0

t=4h

t=4h
S

t=4ht=4hSL

3rd Dose(day 14)

4th Dose(day 11)

Fig 5. LC-enhanced microCT images of anesthetized C57Bl/6 mice 4 hours
following in administration of multiple doses of Fenestra LC (15 mL/kg)
2nd Dose

1st Dose

Baseline

Quantitative analysis of liver signal intensities measured in the same female
Balb/c mouse at 4 hours following each weekly dosing cycle is shown in Fig 7. A
slightly higher level of liver intensity was observed after 2 rounds of injections at a 15mL/kg dose, suggesting possible accumulation of the contrast agent or its metabolites
in the liver. Fenestra LC was well tolerated in tested mice when given once a week at
15 mL/kg dose for up to 4 weeks.

As shown in Fig 4, normalized vascular contrast intensities achieved after 10
min post injections were directly proportional to the injected doses. FenestraVC waswell tolerated in tested mice when given according to the specified dosing schedules.
VC-enhanced microCT images of the same Balb/c female mouse received Fenestra
VC at a dose of 15 mL/kg weekly for 1 month displayed similar microCT efficacy
observed in a single dosing regime, offering flexibility and convenience to perform
longitudinal evaluations of the vascular system in the same animal using microCT(Fig 6).

1st Dose

Fig 6. VC-enhanced microCT images of an anesthetized Balb/c mouse 4 hfollowing administration of muiltiple doses of Fenestra VC (15 mL/kg)

15 mL/kg, 1 time/week

Fenestra VC Dosing ScheduleFenestra LC and VC were obtained from ART Advanced ResearchTechnologies (Montreal, Canada).The final iodine concentration of both formulations
was 50 mg I/mL. The contrast agents were administered intravenously to variousstrains of mice (C57Bl/6, Balb/c, FVB, and SCID) following the dosing scheduleshown in Fig 3. Anesthetized animals (n=3) were scanned using a GE eXplore Locus
microCT scanner (80 kVp, 450 µA, 200 ms, 400 views and 93 µm3 resolution) or a
Siemens microCAT II system (70 kVp, 500 µA, 180 msec, 720 steps and 91 µm3resolution) prior to and at predetermined time intervals following administration.
The animals were placed on a soft diet for at least 24 h prior to initiation of eachdosing cycle for minimizing chow-induced abdominal streak artifacts.
Images were visualized 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 werecalibrated to Hounsfield Units (HU). Normalized signal intensity (HU/voxel) wasobtained from the difference of enhanced SI and the baseline value. The animals
were weighed and monitored daily for signs of abnormal behavior for 5 weeks.

1st Dose(day 0)

Fig 4. Normalized vascular contrast intensities of Fenestra VC 10 min
following repeating dosing schedules in female Balb/c mice
Normalized Signal Intensity (HU/Voxel)A

Fig 4 represents normalized vascular contrast intensities measured in female
Balb/c mice at 10 minutes after administration of Fenestra VC (10 or 15 mL/kg)
according to the specified dosing regimes (Fig 3). The degree of vascular contrast
enhancement at 10 min after each cycle of injections was similar for both 10 and 15mL/kg dosing regimes.

Normalized Signal Intensity (HU/Voxel)

Introduction
Exploiting naturally-occurring endogenous lipid metabolism pathways in thebody, we have developed a hepatocyte-selective CT contrast agent consisting of apolyiodinated triglyceride (ITG) embedded in a chylomicron-remnant like delivery
vehicle. This lipomimetic vehicle thus selectively shuttles the ITG contrast agent
lipids it contains to hepatocytes in the liver (Fig 1).

IVC

Excellent liver contrast enhancement was achieved at 4 hours following eachcycle of administration of Fenestra LC at 15 mL/kg dose (Fig 5). Non-contrast axial
image of an anesthetized C57Bl/6 mouse showed poor soft tissue contrast. It isimpossible to accurately delineate the liver. Axial and sagittal microCT images of themouse obtained after 4 hours following injections displayed excellent liver
enhancement, allowing quantitative characterization of the liver to be possible. The
IVC and hepatic vascular network became visible after 4 hours due to the negative
effect produced by the removal of Fenestra LC from the circulation and its
localization in hepatocytes.

All the tested mice including Balb/c, C57Bl/6, FVB and SCID groups
underwent the repeat dosing schedule of Fenestra LC and VC as often as twice aweek (10 mL/kg) or once a week (15 mL/kg) exhibited normal behavioral patterns
and weight profiles throughout the study.

Conclusions
Acceptable injection tolerance and in vivo CT efficacious imaging profiles observedwith both Fenestra LC and VC in single and multiple dosing regimes provide
substantial flexibility and convenience for both anatomical and functional
evaluations of the liver and vascular system in the same animal using microCT.

References[1] Weber SM, et. al, J Surgical Research (2004) 119:41-45.
[2] Weichert JP, et. al, Radiology, (2000) 216:865-871.
[3] Weichert JP, et al., Academic Radiol (1998) 5:S16-19.
[4] 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 #243