Synthesis and characterization of fluorine-18, fluorine-19, rhenium and technetium-99m-labelled insulin: New tracers for PET and SPECT molecular imaging studies

Two new strategies for the regioselective derivatization of insulin with 18F and 99mTc were developed. The resulting tracers can be used in PET and SPECT studies of insulin distribution and metabolism. Specifically, the synthetic methodologies towards both tracers involved conjugating prosthetic groups to human insulin previously derivatized at the PheB1 residue with a short spacer chain (6-aminohexanoic acid (AHx)) on a di- tert-butyloxycarbonyl (Boc) protected form of the hormone (DBI, 2.5). The prosthetic groups include the active esters of 4- 18F-fluorobenzoic acid (3.6), technetium-99m(triscarbonyl)(bis(2-pyridylmethyl)amino)pentanoic acid (4.7) and technetium-99m(triscarbonyl)(bis(2-naphthylmethyl)amino)pentanoic acid (4.13b). To confirm the identity of the desired insulin derivatives, non-radioactive standards were prepared and characterized by HPLC, mass spectrometry, and enzymatic digestion experiments.;An insulin receptor (IR) phosphorylation assay using CHO-hIR cells over-expressing recombinant human insulin receptors indicated no statistical difference in the extent of autophosphorylation stimulated by 2.19 as compared to that for human insulin (EC50 values of 0.82 nM and 1.0 nM, respectively). The stimulation of 2-deoxyglucose uptake in 3T3-L1 mouse adipocytes utilizing 2.19 versus unmodified human insulin gave similar EC50 values of 0.68 nM and 0.41 nM, respectively. The IC50 values for 2.19 versus that of native insulin for the displacement of 125I-insulin from HEK-293 cells were well correlated (2.6 nM for 2.19 versus 2.4 nM for unmodified human insulin).;For the experiments involving the technetium-99m labelled derivative, the active esters of technetium-99m(triscarbonyl)(bis(2-pyridylmethyl)amino)pentanoic acid (4.7) and technetium-99m(triscarbonyl)(bis(2-naphthylmethyl)amino)pentanoic acid (4.13b) were routinely synthesized in 72% and 48% yields, respectively. These sythons were subsequently conjugated to AHx-DBI ( 2.17). Following deprotection, the final products 99mTcDipy(4M)-AHx-Insulin (4.12) and 99mTcQuin(4M)-AHx-Insulin (4.16b ) were synthesized in 24 ± 5% (n = 4) radiochemical yield for 4.12 and 10 ± 8% (n = 3) for 4.16b. All 99mTc compounds were characterized by comparing retention times in radio-HPLC chromatograms with that (UV-HPLC) of their Re-analogues (ReDipy(4M)-AHx-Insulin, 4.5 and ReQuin(4M)-AHx-Insulin, 4.16a).;An attempt to shorten the overall synthesis of an 18F-insulin derivative was also made. A1,B29-Di-( tert-butyloxycarbonyl)-B1-(hydrazinonicotinoyl(6-aminohexanoyl)) insulin (HYNIC-AHx-DBI, 5.5) was conjugated to human insulin at the PheB1 site of insulin (5.5) using an Fmoc-HYNIC active ester (5.7). The product was reacted with 18FBALD (5.2) in hopes of forming the corresponding hydrazone. Despite successful reactions between HYNIC (5.12) and 18BFBALD (5.2), the insulin derivative (5.5) would not react with 5.2 at the tracer level.;In the case of the radio fluorinated tracer, the succinimidyl 4- 18F-fluorobenzoate (18FSB, 3.7) prosthetic group (83 ± 8% (n = 10) radiochemical yield) was conjugated to AHx-DBI (2.17) ultimately yielding 18FB-AHx-Insulin ( 3.10). 18FB-AHx-Insulin was repeatedly prepared in significant quantities (10-20 mCi, 370-740 MBq) in reasonable radiochemical yield (9 ± 5%, n = 9) following TFA mediated removal of the Boc protecting groups. The final product was characterized by radio-TLC, and by comparing radio-HPLC data to the UV-HPLC chromatogram of the 19F-analogue ( 19FB-AHx-Insulin, 2.19). Analysis of a 19-carrier added synthesis of 3.10 by mass spectrometry confirmed the presence of the desired insulin derivative. Dithiothreitol and endoproteinase Glu-C digestion experiments on 2.19 confirmed that the prosthetic group was in fact conjugated to the PheB1 residue.