Evaluation of strained alkynes for Cu-free click reaction in live mice

We report on our evaluation of the strain-promoted cyclooctyne-azide cycloaddition reaction for use in tumor pretargeting, comprising a side-by-side comparison of probes 1-3 bearing three distinct cyclooctyne moieties based respectively on the 1st and 2nd generation difluorinated cyclooctyne and the 1st generation dibenzocyclooctyne.

The probes were synthesized and labeled with (177)Lu with high yields. The probe stability and reactivity towards azides were evaluated in PBS and mouse serum, and their blood clearance, biodistribution and in vivo reactivity were evaluated in tumor-free mice.

In serum the three probes exhibited sufficient stability for a pretargeting application with half-lives of 12-19h. In PBS, probes 2 and 3 were more reactive towards azido-conjugated Rituximab (Rtx-N3) than 1, but in contrast to 1, their reactivity decreased in mouse serum and mouse serum albumin solutions, as a result of covalent and non-covalent interactions with albumin. Biodistribution data confirmed the interactions with serum proteins in circulation: (177)Lu-1 showed a fast elimination from blood (t1/2,β = 0.31h), while (177)Lu-2 and (177)Lu-3 were retained in blood for longer periods of time (t1/2,β = 1.08 and 3.58h, respectively). Dual isotope biodistribution experiments assessing the reaction between (125)I-Rtx-N3 and (177)Lu-1-3 in circulation in mice showed a very limited retention of 2 and 3 in blood rich organs, indicating a minimal reactivity, while no such retention was observed for 1.

The low reactivity of the studied cyclooctynes, and their serum interactions preclude their use at the low in vivo concentrations typical for pretargeting applications.