Comparison between two labeled agents in mice using a coinjection-ratio approach in contrast to a conventional group approach

IntroductionThe differences between two agents often need to be accurately defined in vivo. Usually they are injected respectively into two groups of subjects. However, if the two agents do not interact with each other in vivo, a coinjection would serve the same purpose. We believe some individual differences in biodistribution may be circumvented through this approach by calculating organ level ratios.MethodsA model system of MORF/cMORF pretargeting (MORF/cMORF is a complementary pair of DNA analogues) was employed in connection with an on-going tumor therapeutic project. Human LS174T cells were implanted into the flank of severely immuno-compromised NOD-scid IL2rgnull mice. The tumor was confirmed to express TAG-72 antigens. At 16 days post tumor inoculation, mice received IV 60 μg of MORF-conjugated CC49 (an antiTAG-72 antibody), followed 2 days later by a low-mass-dose IV coinjection containing 2.5 μg of 90Y-cMORF and 2.5 μg of 99mTc-cMORF. At 3 h post radioactivity injection, the distribution of 99mTc was imaged on a SPECT/CT camera and then organs were excised and counted for 90Y and 99mTc. Because the two labeled cMORFs do not react or interact with each other in vivo, the two groups of 90Y and 99mTc data enabled a conventional group comparison. In a new effort, 90Y/99mTc ratios were calculated. Student’s t-test and retrospective power analysis were performed for both approaches. In the new approach, the ratios were set at 1 as the null hypothesis.ResultsThe Student’s t-test in the conventional group approach indicated that the two labeled cMORFs distributed similarly, but significant differences were observed in salivary gland and large intestines. The coinjection-ratio approach certainly did not subvert the results of the conventional approach but revealed subtler differences. The P values were reduced, the powers were increased in most organs, and more significant differences were observed. The increased sensitivity was due to the reduced CV%s (SD/average*100%) of the 90Y/99mTc ratios. Therefore, some individual differences were circumvented and notably the ratio approach differentiated individual differences into ratio-correctable and ratio-uncorrectable.ConclusionsAlthough the conventional approach is reliable, the coinjection-ratio approach using organ level ratios is more sensitive and therefore is recommended whenever possible. In addition, it differentiates individual differences into “coinjection correctable” and “coinjection uncorrectable”.