β-galactosidase is a stable, quantitative and sensitive reporter extensively used for gene expression studies. For example, studies involving promoter function in transgenic animals as well as gene delivery studies in animal models have utilized the β-gal gene.22–24
However, these studies invariably involve sacrifice of the animal and subsequent extraction of the desired tissues for detection of expression. Consequently, such studies only provide a snapshot image of β-gal expression. The use of noninvasive tomographic imaging techniques such as PET or SPECT can provide real-time, quantitative information of biochemical or molecular processes repetitively in the same subject. Such methods when applied to gene expression imaging could therefore provide the means to follow β-gal expression longitudinally in living subjects which would be of great value both for preclinical studies and future clinical gene therapy trials.5
A wide variety of galactoside analogs that are β-gal enzyme substrates are routinely available for spectroscopic analysis of β-galactosidase activity. Enzymatic hydrolysis of these substrates leads to the formation of an intensely-colored, water-insoluble, reaction product which is selectively localized to the site of reporter gene expression and can be conveniently monitored by colorimetry. The goal of the present study was to synthesize and evaluate a suitably radioiodinated β-gal substrate that would exploit this selective trapping mechanism for in vivo imaging of β-gal expression using SPECT. For this purpose, we focused on the chromogenic β-gal substrate: 5-iodoindol-3-yl-β-D-galactopyranoside (IBDG) as a candidate for radioiodination and biological evaluation. For these initial studies, we radioiodinated IBDG with 125I instead of the standard SPECT radioisotope 123I due to its lower cost, longer half-life and its imaging capability with dedicated small animal SPECT scanners. Radiosynthesis of [125I]IBDG was achieved using a radioiododestannylation reaction followed by acetyl deprotection to afford [125I]IBDG in >75% overall radiochemical yield and >99% chemical and radiochemical purity.
Prior to conducting in vivo
studies we evaluated the specificity of [125
I]IBDG for cell uptake and retention in β-gal expressing cells using an in vitro
cell uptake assay. The cellular trapping of [125
I]IBDG was evaluated at 3 substrate concentrations (0, 0.5 and 1.0 mM). In these studies, [125
I]IBDG displayed optimum cellular trapping of radioactivity (6.5 – 7-fold increase) in β-gal expressing D54 cells (D54L
) as compared to control cells (D54
) at the 0.5 mM substrate concentration. Importantly, we did not observe a significant difference in radioactivity uptake between the two cell types at either the 24 h or 48 h time interval in the absence of carrier (0 mM). This observation can be attributed to the fact that the IBDG substrate concentration was significantly lower relative to the enzymes’ Km
value which is reported to be in the 0.1 – 4 mM range.25
Encouraged by these initial findings, we administered [125I]IBDG intravenously to CD1 mice having D54L and D54 solid tumor xenografts and conducted serial SPECT/CT imaging studies. Since our in vitro cell uptake studies demonstrated optimal trapping of [125I]IBDG at a 0.5 mM substrate concentration, radioligand doses were formulated at a similar IBDG concentration for the imaging studies. Visualization of either tumor was not possible throughout the 24 h imaging interval due to insufficient tumor uptake of radioactivity. Radioactivity clearance occurred mainly via the renal pathway as evidenced by high initial kidney uptake followed by excretion into bladder and the absence of radioactivity in liver. Negligible radioactivity levels were seen in most other organs including thyroid during the entire imaging sequence.
To confirm that the lack of tumor uptake of the radioligand was due to poor delivery to the tumor and not due to lack of β-gal enzyme recognition in vivo, SPECT imaging studies were repeated following direct intratumoral injection of [125I]IBDG to D54L and D54 tumors implanted in the same mouse on opposite flanks. Analysis of the SPECT image data revealed strikingly different kinetics of radioactivity clearance between the two tumor types. The fast clearance of radioactivity from the D54 tumor site relative to the β-gal expressing D54L tumor enabled selective SPECT visualization of the D54L tumor at >4 h post-injection. Radioactivity clearance which occurred predominantly via renal excretion as seen with the intravenous administration route resulted in high radioactivity levels in mouse bladder at early time intervals (2 h – 4 h), which, later declined to near background levels by 7 h post-injection. In addition, near background levels of radioactivity uptake were seen in most other organs including liver and thyroid, the latter indicative that [125I]IBDG is relatively stable to in vivo metabolic deiodination.
Important requirements for a successful in vivo imaging radioligand include a high uptake in target tissues in conjunction with a rapid clearance from non-target tissues. Since β-gal is localized within the cytoplasm, the radioligand must be sufficiently hydrophobic to diffuse through the cell membrane to reach its intended target. Once inside the cell, the radiolabeled product resulting from enzymatic action should also demonstrate low diffusibility to ensure its cellular retention. Our intratumoral injection imaging data confirms that [125I]IBDG (log P = 0.8) undergoes both facile cell permeation and selective intracellular trapping in D54L cells following enzymatic hydrolysis by β-gal. Furthermore, the rapid washout of radioactivity from the D54 control cell site indicates that unprocessed radioligand is being efficiently cleared out of the cell and from the circulation into the renal compartment. In this regard, our biological results underscore the advantage of using radiolabeled enzyme substrates that afford trapping of the product of a catalytic reaction over inhibitors as radioligands for imaging enzyme expression since continuous enzyme processing of such substrates affords high signal amplification at the site of enzymatic processing.
The imaging data from the intratumoral injection studies suggested to us that limited delivery of the radioligand to tumors on systemic injection, which is likely due to high renal clearance, plays a key role in the observed lack of tumor uptake. To further understand the poor tumor-targeting behavior of [125I]IBDG after systemic administration, blood metabolite analysis studies were conducted in tumor-bearing CD1 mice following intravenous injection of the radioligand. In these studies, the intact radioligand ([125I]IBDG) accounted for less than 40% of the total radioactivity present in mouse blood at 5 min post-injection. The remaining radioactivity comprised of two polar metabolites, which we confirmed were not [125I]iodide by radio-HPLC analysis. Importantly, the total blood radioactivity in a mouse at 5 min post-injection was only 13 – 15 µCi following a 600 µCi injection of [125I]IBDG. Since only renal and bladder radioactivity were apparent in the early SPECT images, this data further confirms that the majority of the systemically administered [125I]IBDG is being rapidly excreted in urine either as the intact radioligand or as a metabolite.
In summary, we synthesized and evaluated a radioiodinated β-gal substrate ([125I]IBDG) as a radioligand for in vivo SPECT imaging of tumor β-galactosidase enzyme expression. Although [125I]IBDG showed high differential uptake (6.5 – 7-fold) in β-galactosidase expressing tumor cells over control cells in in vitro studies it demonstrated insufficient uptake in β-gal expressing tumors for SPECT imaging upon systemic injection. However, SPECT imaging of CD1 mice following direct intratumoral injection of [125I]IBDG to β-gal expressing D54L tumors and control D54 tumors co-implanted in the same mouse demonstrated selective retention of radioactivity at the D54L tumor at 2 h through 7 h post-injection resulting in clear visualization of this tumor. Analysis of the imaging and blood metabolite profile data suggest that the poor tumor localization of [125I]IBDG is likely a result of high and rapid renal excretion. Thus, despite useful biological characteristics such as good cell permeability, substrate specificity and fast clearance from non-target tissues, our studies indicate that [125I]IBDG is unsuitable for the in vivo imaging of β-gal expression. We conclude that further structural modification of IBDG to retard its renal clearance and improve cell uptake or the evaluation of alternative β-gal substrates with improved pharmacokinetic properties is warranted to improve the β-gal expression imaging capability of this class of radioligands. These studies are currently underway in our laboratory.