Evaluation of anesthesia effects on the brain uptake and metabolism of demyelination tracer [18F]3F4AP

Introduction: [¹⁸F]3-fluoro-4-aminopyridine ([¹⁸F]3F4AP) is a PET radioligand for imaging demyelinated lesions in the central nervous system (CNS) based on the multiple sclerosis (MS) drug 4-aminopyridine (4AP). [¹⁸F]3F4AP binds to voltage-gated K⁺ channels present in demyelinated axons and has been used for PET imaging in rodent models of MS and demyelination, as well as non-human primates (NHPs). Pre-clinical evaluation in non-human primates has shown fast brain uptake (SUV > 3 at 4 min) and washout, high plasma availability, and high metabolic stability (> 90 % parent fraction at 2h post injection). However, first in-human studies presented a faster clearance rate and differences in metabolism of the radiotracer (Abstract 871). Due to this apparent incongruence in the pharmacokinetics and metabolism of [¹⁸F]3F4AP across species, we set out to examine possible causes for these observed results. A major difference in the experimental setup of pre-clinical rodent and NHP studies compared to in-human evaluation is the presence of anesthetics during the PET scan. Furthermore, it is known that 4AP is metabolized by the CYP2E1 enzyme, the predominant cytochrome P450 isoform responsible for human clinical isoflurane metabolism in vivo. Therefore, we posited the hypothesis that isoflurane anesthesia could play a role in the brain accumulation and clearance rate of the tracer. The goal of this study was to investigate the anesthesia effects on the brain uptake and metabolism of [¹⁸]3F4AP in mice.

Methods: [¹⁸F]3F4AP was synthesized according to previous reports. To study the anesthesia effects on the in vivo brain accumulation and metabolism of [¹⁸F]3F4AP two cohorts of mice were administered 50-250 µCi of radiotracer via tail vein injection. Radiotracer delivery was performed under isoflurane anesthesia for the first cohort (n = 9) and kept anesthetized for the duration of the experiment. For the second cohort (n = 16), mice were injected with [¹⁸F]3F4AP awake using a tail access rodent restrainer, and then allowed to move freely. For both groups, mice were euthanized 35 minutes-post-injection, intracardial puncture was performed to collect blood, and brain tissue was harvested. Gamma counting of whole blood and brain tissue was performed to evaluate radioactivity distribution. For a subset of animals, radiometabolite detection in plasma was evaluated. Whole blood samples were processed by centrifugation to separate plasma, collected plasma was then filtered through a 10 kDa molecular weight cutoff centrifugal filter, and the resulting filtrate was analyzed by HPLC for metabolism examination.

Results: Gamma counting of brain and blood collected 35 minutes-post-tracer administration showed that in vivo brain accumulation was affected by presence of isoflurane anesthesia. [¹⁸F]3F4AP brain uptake was 3.8-fold lower in awake mice in comparison to anesthetized mice (SUV_iso-br 1.26 ± 0.14 vs. SUV_awake-br 0.33 ± 0.03; p = 0.00008). In contrast, whole blood SUV values were comparable between the two groups (SUV_iso-WB 0.77 ± 0.08 vs. SUV_awake-WB 0.59 ± 0.05; p = 0.08). Radiometabolite analysis by HPLC in plasma samples also indicated an effect of anesthesia on the metabolism of the radiotracer. Greater metabolism was observed in the plasma samples of awake mice in comparison to anesthetized, revealed by the percent of parent fraction remaining at 35 min post-injection (%PF_awake 20 ± 4 % vs. %PF_iso 65 ± 7%).

Conclusions: The presented work establishes an effect of isoflurane anesthesia on the brain uptake and metabolism of [¹⁸F]3F4AP. In summary, anesthetized mice presented higher in vivo brain uptake and slower peripheral metabolism when compared to their unanesthetized counterparts. These results, which are consistent with the findings in anesthetized monkeys in contrast to awake humans, will inform upcoming studies with [¹⁸F]3F4AP in the clinic and are currently the basis of further evaluation to address the metabolism and distribution of aminopyridine-based PET tracers.

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