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Application of QWBA and Short-lived Isotopes in the Drug Development Paradigm


Andrea Knapp Brian Knapp William Cherup Erik Laroco Dustin Kentala Jacob Y. Hesterman Kate Sokolnicki

MPI Research Corporate Headquarters 54943 North Main Street Mattawan, MI 49071 USA

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Application of QWBA and Short-lived Isotopes in the Drug Development Paradigm


Andrea Knapp1, Brian Knapp1, William Cherup1, Erik Laroco1, Dustin Kentala1, Jacob Y. Hesterman2, Kate Sokolnicki2
(1) MPI Research, Mattawan, MI, USA; (2) InviCRO, LLC, Boston, MA, USA

INTRODUCTION
Quantitative Whole-Body Autoradiography (QWBA) is a high-resolution tissue distribution assessment commonly used in preclinical drug development. QWBA is typically conducted in one animal per time point. The process begins with an animal euthanized at a specified time point and then frozen in a hexane/dry ice bath. After freezing, the carcass is embedded in a carboxymethylcellulose (CMC) solution and then sectioned using a cyromacrotome. Freezedried sections are mounted and exposed in preparation for scanning. Standard QWBA isotopes include 14C, 3H, and 125I due to their long half-lives and relative ease of incorporation into target compounds. Short-lived isotopes provide a solution to contemporize QWBA with multiple imaging platforms, such as PET and SPECT within a single study design, maximizing data generation, and reducing animal usage in preclinical research. MPI Research has explored and expanded its QWBA capabilities to optimize these isotopes for use. In vivo study:

Results

Results Continued
In vitro study: Figure 4. Isotope-spiked Carboxymethylcellulose (CMC) standards: g) 111In spiked CMC standards after 2-hour exposure; h) 111In spiked CMC standards after 24-hour exposure

Figure 1. 123I at 4 hours post-dose via a) QWBA and b) SPECT Uptake of 123I found in thyroid and stomach and eliminated via the urine

Over the 96-hour exposure period of each isotope-spiked standard the intensity of signal increased over time as did the signal of the background. For each isotope there is a point of diminishing returns between the energy of the isotope, decay due to half-life, and signal of the background. As noted in Figure 1, the background is higher after the 24-hour exposure as compared to the 2-hour exposure. However, too much radioactivity can saturate the phosphor screen and result in flare. As demonstrated in Figure 1, the energy of the 111In begins to expand or flare outside of the sample circle as all of the sample circles displayed are of uniform size. In vivo study: Figure 5. Comparison of 64Cu concentrations via QWBA and PET: i) Liver ; j) Kidneys

OBJECTIVES
The purpose of this work was to optimize the use of short-lived isotopes for the QWBA technique and provide a link between QWBA and common molecular imaging modalities. To this effort, three common molecular imaging short-lived isotopes were developed for optimal phosphor screen exposure time: balancing background, intensity of signal, and lower limit of quantitation. To account for the significantly shorter half-lives of these common molecular imaging isotopes, the QWBA process was optimized by eliminating time constraints.

Figure 2. In at 1 hour post-dose via c) QWBA and d) SPECT


111

METHODOLOGY
In vitro study: Eight CMC spiked standards were prepared at target concentrations of radioactivity per isotope based on half-life and maximum energy. Isotope Copper-64 Iodine-123 Indium-111 Half-life (Days) 0.529 0.547 2.8 Energy (keV) 511, 7-8 159, 27 245, 171
www.nchps.org

Wide distribution was noted throughout the body, with a large quantity of radioactivity found in the blood and kidneys

Spiked standards were sectioned at 20m and 40m. Sectioned standards were exposed to phosphor screens for durations ranging from two to 96 hours. Optimization of QWBA Processing: Frozen carcasses were immediately embedded at -70C for at least two hours. Embedded specimens were sectioned at 20m, sections freeze dried in approximately 24 hours as opposed to 36 to 48 hours for 40m sections. In vivo study: Rats were dosed with 123I, 111In or 64Cu in buffer and imaged via PET or SPECT, as appropriate, and processed for QWBA at 1, 4, 8, and 24 hours post-dose.

Figure 3. 64Cu at 1 hour post-dose via e) QWBA and f ) PET


64

Cu distributed primarily to the kidneys, liver, and gastrointestinal tract and eliminated via the feces

The amount of radioactivity present, as determined via the QWBA technique versus processing of the PET image from the 64Cu dose, correlated very well as demonstrated in Figure 5. The liver concentration provided a correlation of 0.9698 between the two image processing modalities and the kidneys provided a correlation of 0.9950. These calculations demonstrate that the radioactivity concentration obtained from both the QWBA and the molecular imaging technique provide similar results. These results can be used interchangeably or in parallel between the two modalities.

CONCLUSIONS
Short-lived isotopes were once considered unsuitable for QWBA. Time constraints have been the biggest challenge; however, by avoiding process delays and properly selecting isotopes based on the timeline needs of the study, these can be overcome. QWBA, in conjunction with PET or SPECT, maximizes data generation and reduces the number of animals used. Molecular imaging provides a first look, which can then be followed up with QWBA animals at specific time points for increased resolution of individual tissues. Pairing QWBA with another molecular imaging modality can provide a bigger picture into the distribution of the compound of interest.