Different strategies for reducing intestinal background radioactivity associated with imaging HSV1-tk expression using established radionucleoside probes

Abstract

One limitation of HSV1-tk reporter positron emission tomography (PET) with nucleoside analogues is the high background radioactivity in the intestine. We hypothesized that endogenous expression of thymidine kinase in bacterial flora could phosphorylate and trap such radiotracers, contributing to the high radioactivity levels in the bowel, and therefore explored different strategies to increase fecal elimination of radiotracer. Intestinal radioactivity was assessed by in vivo microPET imaging and ex vivo tissue sampling following intravenous injection of 18F-FEAU, 124I-FIAU, or 18F-FHBG in a germ-free mouse strain. We also explored the use of an osmotic laxative agent and/or a 100% enzymatically hydrolyzed liquid diet. No significant differences in intestinal radioactivity were observed between germ-free and normal mice. 18F-FHBG-derived intestinal radioactivity levels were higher than those of 18F-FEAU and 124I-FIAU; the intestine to blood ratio was more than 20-fold higher for 18F-FHBG than for 18F-FEAU and 124I-FIAU. The combination of Peptamen and Nulytely lowered intestinal radioactivity levels and increased (2.2-fold) the HSV1-tk transduced xenograft to intestine ratio for 18F-FEAU. Intestinal bacteria in germ-free mice do not contribute to the high intestinal levels of radioactivity following injection of radionucleoside analogues. The combination of Peptamen and Nulytely increased radiotracer elimination by increasing bowel motility without inducing dehydration.

FIGURE 1. Ex Vivo Biodistribution of ¹⁸F-FEAU, ¹⁸F-FHBG and ¹²⁴I-FIAU in germ-free and control mice

Ex vivo biodistribution (%ID/g) (left panel) and normalized to blood data (right panel) in mice following injection of (A) ¹⁸F-FEAU, (B) ¹⁸F-FHBG and (C) ¹²⁴I-FIAU in germ-free and control mice. No statistically significant difference in large intestine radioactivity (%ID/g) was observed between the germ-free and control animals. Radioactivity measured in the small intestine was slightly lower in germ-free animal than control for each radiotracer, although not statistically significant. ¹⁸F-FHBG was associated with higher large intestine radioactivity (2.9-fold compared to ¹⁸F-FEAU and 18-fold compared to ¹²⁴I-FIAU, p<0.001). The lowest levels were observed with ¹²⁴I-FIAU at 24 hours. The intestine-to-blood ratio for ¹⁸F-FHBG (201±37) was very high compared to that for ¹⁸F-FEAU (6.9±4.5) at 2 hours and ¹²⁴I-FIAU (6.4±2.3) at 24 hours.

FIGURE 2. ¹⁸F-FEAU, ¹⁸F-FHBG and ¹²⁴I-FIAU microPET imaging in germ-free and control mice

(A) Pictures of the blood agar plates where homogenized samples of the fecal pellet were placed (1:10 dilutions) to confirm the germ-free status versus control groups of animals. The red-to-brown color shift indicates the presence of metabolic activity, corresponding the growth of bacteria. Bacterial colonies were visible in the brown (control) plates, but not in the plates from the germ-free animals. (B) Coronal and axial PET images of germ-free versus control mice at 2 hours after injection of ¹⁸F-FEAU, ¹⁸F-FHBG and 24 hours after injection of ¹²⁴I-FIAU. ¹⁸F-FHBG is associated with the highest abdominal radioactivity compared to ¹⁸F-FEAU and ¹²⁴I-FIAU; note the higher threshold of the intensity bar (5% ID/g) for ¹⁸F-FHBG. No difference is observed in the PET images between germ-free and control animals in each radionucleoside group. (C) Comparison of the intestinal uptake of ¹⁸F-FEAU, ¹⁸F-FHBG and ¹²⁴I-FIAU in germ-free versus control mice, calculated from multiple intestinal ROIs drawn on the whole intestine on the axial plane. Values of the Maximum Pixel Value (Max %ID/g) and Total Activity (%ID/g) are illustrated. The highest values were observed for ¹⁸F-FHBG; Maximum pixel (%ID/g) intestinal background was ~6.7-fold higher than that for ¹⁸F-FEAU (p<0.05) and more than 50-fold higher than that for ¹²⁴I-FIAU (p<0.001).

FIGURE 3. Different strategies in increasing radionucleoside excretion and ¹⁸F-FEAU biodistribution

Biodistribution of ¹⁸F-FEAU in tumor bearing nude mice at 2 hours after injection, treated with Nulytely (36 hours), Peptamen (14 days) and a combination of Peptamen (14 days) and Nulytely (36 hours) versus control (regular diet and water ad libitum). Values are expressed in (A) %ID/g and (B) organ-to-blood normalized data. (*p<0.05;**p<0.01; ***p<0.001). Asterisks in (a) indicate the statistically significant difference between the Nulytely group and the control. The highest values (1.5-to-8 fold higher compared to control group) were observed in the Nulytely group. The normalization of tissue (organ) radioactivity to that in blood takes into consideration the hydration status of the mice. (C) Different intestinal cleansing strategies and their effect on animal weight. A 14±0.5% weight loss was observed after 36 hours of Nulytely treatment alone, due to dehydration and food deprivation. The combination of Nulytely and Peptamen resulted in only 6±1% weight loss during the same period of treatment.

FIGURE 4. Quantitative PET analysis and tissue sampling data

Comparison of microPET data (left panel) and tissue sampling data (right panel) for intestinal and tumor (HSV1-TK) ¹⁸F-FEAU – derived radioactivity (%ID/g) in nude mice bearing HSV1-TK transduced and wild type RG2 xenografts, treated with different bowel cleansing agents. In the left panel the values of Maximum (%ID/g) for (a) tumor RG2 (HSV1-TK) (b) intestine (calculated by drawing multiple intestinal on the whole intestine on the axial plane) and (c) the calculated tumor-to-large intestine ratio are graphed. Similar graphs are presented for the tissue sampling results (d), (e), (f). Significant differences between treatment groups are indicated (*p<0.05; **p<0.01; ***p<0.001).

FIGURE 5. Different strategies and their effect in mice

Coronal and axial ¹⁸F-FEAU PET images of nude mice bearing a RG2 HSV1-TK xenograft in the right shoulder (circle) and a RG2 wild-type xenografts in the left shoulder (dotted circle). Coronal images are shown through the mid-abdomen. Axial images are shown for a section through the abdomen indicated by the orange dotted line. Note that the high intestinal background observed with the laxative agent alone (Nulytely) is due to dehydration and high radioactivity retention.