Trans-Cyclooctene Isomerization Catalyzed by Thiamine Degradation Products in Cell Culture Media

PET imaging of intrathecally dosed ASO neurology drugs is challenging due to the long time needed to achieve steady-state brain distribution, making the use of a simple ¹⁸F tag impossible with its short radioactive half-life. To overcome this challenge, a pretargeted imaging solution has been developed in which an ASO tagged with a tetrazine is dosed intrathecally, and after 24 h a reactive trans-cyclooctene (TCO) tagged with ¹⁸F is dosed intravenously. The two molecules form a click-chemistry adduct, allowing for PET imaging scans immediately following ¹⁸F-TCO administration. Although it has been demonstrated that TCOs can be relatively stable in vivo, they rapidly isomerize to cis-cyclooctenes (CCOs) in cell culture media and "aged" plasma, making many DMPK experiments challenging to interpret and not representative of the in vivo stability. The predominant cause of isomerization was determined to be thiamine degradation product(s) in media such as DMEM. Several techniques to overcome the challenges of in vitro and ex vivo isomerization during analytical experiments are herein proposed, such as the use of custom media and/or fresh plasma, adding antioxidants, using surrogate molecules, and using TCO trapping agents. These findings and techniques may also be relevant to other applications in which TCOs are incubated in thiamine-containing cell culture media.