Preparation of 111In-DTPA morpholino oligomer for low abdominal accumulation

An ability to quantitate the beta cell mass by noninvasive nuclear imaging will be very useful in the prevention, diagnosis, and treatment of diabetes. However, to be successful, radioactivity from the pancreas must not be obscured by the background radioactivity in the abdomen. Pretargeting offers the promise of achieving high target organ to normal tissue ratios. In preparation for pancreas imaging studies by pretargeting using morpholino oligomers (MORF/cMORF), it was necessary to develop a simple and efficient method to radiolabel the cMORF effector. Because we have shown that labeling the cMORF with 111In via DTPA reduces excretion into the intestines compared to labeling with 99mTc via MAG3, the conjugation of DTPA to cMORF was investigated for 111In labeling. The amine-derivatized cMORF was conjugated with DTPA using 1-ethyl-3(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) as an alternative to the conventional cyclic anhydride. The conjugation efficiency (represented by the number of DTPA groups attached per cMORF) was investigated by changing the EDC, DTPA, and cMORF molar ratios. Different open columns were considered for the purification of DTPA–cMORF. Before conjugation, each cMORF molecule was confirmed to have an amine by trinitrobenzene sulfonic acid (TNBS) assay using the ω-amino butyric acid as positive standard and the non-amine derivatized cMORF as negative standard. The average number of DTPA groups per cMORF was 0.15–0.20 following the conjugation over a cMORF/DTPA molar ratio of 0.5–5 and over a cMORF/EDC molar ratio of 20–60. The conjugation efficiency was lower than expected probably due to steric hindrance. A 1×50 cm P-4 column using ammonium acetate as eluting buffer provided an adequate separation of DTPA–cMORF from free DTPA. The 111In labeling efficiency by transchelation from acetate exceeded 95%, thus avoiding the need for postlabeling purification. Despite the lower than expected conjugation efficiency in which no more than one-fifth of the cMORF were DTPA-derivatized, a specific radioactivity of at least 300 μCi/μg or 1.90 Ci/μmol of cMORF was achieved. In conclusion, a protocol is described for 111In-DTPA–cMORF that provides the high specific activity favorable to beta cell imaging because of the low mass fraction of beta cells in pancreas (1–2%) and obviates the need for postlabeling purification.