Abstract
486
Objectives: 4-aminopyridine (4AP) is a potassium channel blocker clinically approved for symptomatic treatment in multiple sclerosis (MS)1. In MS, demyelination causes axonal K+ channels to become exposed and increase in expression, which hampers the conduction of electrical impulses2. 4AP blocks these channels and enhances impulse 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 evaluate a carbon-11 form of 4AP, [11C]3-methoxy-4-aminopyridine, [11C]3MeO4AP, and compare it with [18F]3F4AP.
Methods: LogD and pKa were measured using standard protocols. Binding to Shaker K+ channel was evaluated using an in vitro electrophysiological assay. In vivo potency was evaluated in mice. [11C]Methyl iodide was used to label the hydroxypyridine precursor. 0-90 min dynamic PET imaging with arterial blood sampling and pharmacokinetic modeling was performed (n = 2).
Results: Physicochemical characterization indicated that 3MeO4AP is more basic than 3F4AP and less than 4AP (pKa 9.1, 9.6 and 7.7, respectively). 3MeO4AP is more lipophilic than 4AP but less than 3F4AP. In vitro, 3MeO4AP is about 2-fold less potent than 3F4AP and, in vivo, it appears to be 4-5 times more potent than 3F4AP. 11C-methylation followed by SEP-pak purification was performed in 20 min and afforded the radiotracer with a ~10% non-decay corrected radiochemical yield (n = 6), high radiochemical purity (>95%) and high molar activity (4-6 Ci/umol at EoS). Analysis of blood samples showed that [11C]3MeO4AP is stable in vivo (>70% parent at 90 min post injection) and has a high and stable plasma free fraction (PFF ~0.95). Dynamic PET imaging in one rhesus monkey showed fast entry into the brain (peak SUV ~2.5 at 20 min post injection) followed by slow washout. Regional VTs calculated using a 2-tissue compartment model showed heterogenous distribution of the tracer. The distribution was very similar to [18F]3F4AP suggesting a common target (Pearson R > 0.9). Interestingly, the subject, which had a mild intracranial injury from a craniotomy procedure 3 years prior to imaging, showed high uptake in the lesioned area (28% mean increase in VT). Preliminary pharmacokinetic analysis suggests that increase in signal was not due to increased perfusion consistent with presence of demyelination (a well-documented aspect in traumatic brain injuries). Furthermore, no focal uptake was seen with other tracers including [18F]FDG.
Conclusions: Carbon-11 tracers have the advantage that they provide the opportunity to do multiple studies on the same subject and the same day. Here, we show that a 11C version of 4-aminopyridine binds to K+ channels in vitro and has good properties for imaging the brain in vivo. Furthermore, the findings on a monkey, which had a chronic brain injury suggest that this compound, similar to its radiofluorinated counterpart, may be useful for imaging demyelinating lesions in the brain. Research Support: R00EB020075 (P.B.), T32EB013180 (G.E.F), S10OD0180035 (M.D.N.)