Pharmacokinetic Characterization of [¹⁸F]3F4AP in non-human primates

Objectives: 4-aminopyridine (4AP) is a potassium channel blocker clinically approved to improve walking in people with multiple sclerosis (MS)¹. Upon demyelination, axonal K⁺ channels, which are normally located underneath the myelin, become exposed and increase in expression causing impaired axonal conduction². 4AP blocks these channels and restores conduction³. Recently, it has been shown that [¹⁸F]3-fluoro-4-aminopyridine, [¹⁸F]3F4AP, binds to K⁺ channels in demyelinated fibers and can be used to detect demyelinated lesions in a rodent model of MS⁴. The goal of this work was to further characterize [¹⁸F]3F4AP in non-human primates in preparation for a first-in-human study.

Methods: Radiochemical synthesis was performed using a previously published method⁵. Dynamic PET imaging with arterial blood sampling was performed on one rhesus monkey. The subject was imaged 4 times with different doses of cold 3F4AP to assess specific binding (0, 0.75, 1.25, 2.5 mg/kg). Blood samples were processed to measure radioactivity concentration in whole blood and plasma. Selected plasma samples were analyzed for radiometabolites using radio-HPLC. Plasma free fraction was measured by ultracentrifugation. Dynamic PET data were analyzed by compartmental modeling and Logan graphical analysis using the metabolite corrected arterial plasma input functions.

Results: Analysis of blood data showed that [¹⁸F]3F4AP is stable in vivo (>90% parent at 180 min post injection) and has a high plasma free fraction (~ 1.0). Dynamic PET imaging showed a fast entry into the brain (peak SUV ~5) followed by moderate to rapid washout. An unconstrained 2-tissue compartment (2TCM) model was found to best fit the data. V_T estimates were stable while truncating the data from 180 min down to 60 min (< 5% variability). Logan graphical method using 60, 120 or 180 min of data produced similar results as the 2TCM. Given the undesirable pharmacological effects of saturating K⁺ channels (namely fatal seizures) we only attempted blocking studies using subpharmacological doses, which showed no significant change in V_Ts across brain regions. Notably, high uptake (~30% higher V_T than the surrounding area) and distinct pharmacokinetics were observed in a focal area of the brain where the animal sustained a minor intracranial injury 3 years prior to imaging. This change could not be attributed to changes in perfusion of permeability since there was no change in K₁ or k₂. Furthermore, no abnormalities have been observed in this region when the same animal was imaged with other tracers, notably [¹⁸F]FDG.

Conclusions: This study shows that [¹⁸F]3F4AP has good properties for imaging the brain (e.g., high BBB permeability, high metabolic stability and high plasma free fraction) and that It yields reproducible results in primates. Blocking studies performed to date have not shown substantial displacement likely because the doses used were below the dose required to saturate the receptors. Nevertheless, abundant evidence exists supporting that [¹⁸F]3F4AP is binding to K⁺ channels in vivo including the enhancement of action potentials in demyelinated fibers⁴, colocalization of the PET signal with lesions in several animal models of demyelination⁴ and lack of correlation between the PET signal with other changes such BBB damage or inflammation. Finally, this study shows that a 2TCM and Logan graphical analysis produce consistent V_Ts even in the presence of low doses of cold 3F4AP. Research Support: R00EB020075 (P.B.), P41EB022544 (G.E.F.), T32EB013180 (G.E.F), S10OD0180035 (M.D.N.)