Biology subjects

Brugarolas, P.

Publications and source records attributed to Brugarolas, P..

13 recordsLinked to original sources

bioRxiv2026

4-methyl-3-aminopyridine: A novel active blocker of voltage-gated potassium ion channels in the central nervous system.

Aminopyridines, including 4-aminopyridine (4AP), 3,4-diaminopyridine, and [18F]3-fluoro-4-aminopyridine, are voltage-gated potassium (KV) channel blockers used clinically to enhance conduction in neurological disorders and to image demyelination by PET. Developing new aminopyridines may yield improved therapeutics or imaging agents. Here, we characterized the physicochemical properties (pKa, log D), KV channel-blocking activity, toxicity (LD50), and pharmacokinetics of a novel compound, 4-methyl-3-aminopyridine (4Me3AP). 4Me3AP was less basic and more lipophilic than 4AP and showed greater blocking potency across multiple KV channels expressed in Xenopus oocytes. In mice, 4Me3AP exhibited lower acute toxicity (LD50= 29.3 mg/kg) than 4AP (LD50= 12.7 mg/kg) and a longer plasma half-life. These findings indicate that 4Me3AP is a potent KV channel blocker with favorable pharmacological properties, supporting its potential for symptomatic treatment of demyelinating diseases.

biochemistry↗

bioRxiv2025

Imaging traumatic brain injuries in mice with potassium channel PET tracer 3F4AP

ObjectiveTraumatic brain injury (TBI) can lead to secondary injury, including axon and myelin damage, which contributes to long-term neurological deficits. The PET tracer [18F]3F4AP, a fluorinated derivative of the FDA-approved drug 4-aminopyridine, selectively binds to voltage-gated potassium (KV) channels, offering a novel approach to assess TBI-related node of Ranvier disruption and demyelination. This study evaluates [18F]3F4AP PET in penetrating and non-penetrating TBI models. MethodsEither controlled cortical impact (CCI, penetrating) or concussive (non-penetrating) TBI models were used to induce TBI in mice. Dynamic PET imaging with [18F]3F4AP was performed at time points of 0, 3, 7, 14, and/or 31 days post-injury (dpi), with quantitative analyses comparing tracer uptake in injured versus control regions. Luxol fast blue (LFB) staining was conducted to evaluate histological myelin loss. ResultsIn the CCI model, [18F]3F4AP PET imaging demonstrated a 34% increase in tracer uptake at the injury site at 7 dpi, correlating with histological evidence of demyelination. Tracer uptake gradually declined over time, reflecting potential remyelination. The concussive TBI model showed a smaller and more diffuse increase in uptake at 7 dpi compared to CCI. Conclusion[18F]3F4AP PET imaging effectively detects demyelination following TBI, with very high sensitivity in penetrating injuries. These findings highlight the potential of [18F]3F4AP as a valuable imaging biomarker for assessing TBI progression and/or therapeutic response. Further studies are warranted to explore its clinical applicability and comparison with other imaging modalities.

neuroscience↗

bioRxiv2024

Imaging demyelinated axons after spinal cord injuries with PET tracer 3F4AP

Spinal cord injuries (SCI) often lead to lifelong disability. Among the various types of injuries, incomplete and discomplete injuries, where some axons remain intact, offer potential for recovery. However, demyelination of these spared axons can worsen disability. Demyelination is a reversible phenomenon, and drugs like 4-aminopyridine (4AP), which target K+ channels in demyelinated axons, show that conduction can be restored. Yet, accurately assessing and monitoring demyelination post-SCI remains challenging due to the lack of suitable imaging methods. In this study, we introduce a novel approach utilizing the positron emission tomography (PET) tracer, [18F]3F4AP, specifically targeting K+ channels in demyelinated axons for SCI imaging. Rats with incomplete contusion injuries were imaged up to one month post-injury, revealing [18F]3F4APs exceptional sensitivity to injury and its ability to detect temporal changes. Further validation through autoradiography and immunohistochemistry confirmed [18F]3F4APs targeting of demyelinated axons. In a proof-of-concept study involving human subjects, [18F]3F4AP differentiated between a severe and a largely recovered incomplete injury, indicating axonal loss and demyelination, respectively. Moreover, alterations in tracer delivery were evident on dynamic PET images, suggestive of differences in spinal cord blood flow between the injuries. In conclusion, [18F]3F4AP demonstrates efficacy in detecting incomplete SCI in both animal models and humans. The potential for monitoring post-SCI demyelination changes and response to therapy underscores the utility of [18F]3F4AP in advancing our understanding and management of spinal cord injuries.

neuroscience↗

bioRxiv2023

Common anesthetic used in preclinical PET imaging inhibits metabolism of the PET tracer 3F4AP

PET imaging studies in laboratory animals are almost always performed under isoflurane anesthesia to ensure that the subject stays still during the image acquisition. Isoflurane is effective, safe, and easy to use, and it is generally assumed to not have an impact on the imaging results. Motivated by marked differences observed in [18F]3F4AP brain uptake and metabolism between human and nonhuman primate studies, this study investigates the possible effect of isoflurane on [18F]3F4AP metabolism and brain uptake. Isoflurane was found to largely abolish tracer metabolism in mice resulting in a 3.3-fold higher brain uptake in anesthetized mice at 35 min post radiotracer administration, which replicated the observed effect in unanesthetized humans and anesthetized monkeys. This effect is attributed to isofluranes interference in the CYP2E1-mediated breakdown of [18F]3F4AP, which was confirmed by reproducing a higher brain uptake and metabolic stability upon treatment with the known CYP2E1 inhibitor disulfiram. These findings underscore the critical need to examine the effect of isoflurane in PET imaging studies before translating tracers to humans that will be scanned without anesthesia.

neuroscience↗

bioRxiv2023

Evaluation of trans- and cis-4-fluorogabapentin for brain PET imaging

Gabapentin, a selective ligand for the 2{delta} subunit of voltage-dependent calcium channels, is an anticonvulsant medication used in the treatment of neuropathic pain, epilepsy and other neurological conditions. We recently described two radiofluorinated derivatives of gabapentin (trans-4-[18F]fluorogabapentin, [18F]tGBP4F, and cis-4-[18F]fluorogabapentin, [18F]cGBP4F) and showed that these compounds accumulate in the injured nerves in a rodent model of neuropathic pain. Given the use of gabapentin in brain diseases, here we investigate whether these radiofluorinated derivatives of gabapentin can be used for imaging 2{delta} receptors in the brain. Specifically, we developed automated radiosynthesis methods for [18F]tGBP4F and [18F]cGBP4F and conducted dynamic PET imaging in adult rhesus macaques with and without preadministration of pharmacological doses of gabapentin. Both radiotracers showed very high metabolic stability, negligible plasma protein binding and slow accumulation in the brain. [18F]tGBP4F, the isomer with higher binding affinity, showed low brain uptake and could not be displaced whereas [18F]cGBP4F showed moderate brain uptake and could be partially displaced. Kinetic modeling of brain regional time-activity curves using a metabolite-corrected arterial input function shows that a 1-tissue compartment model accurately fits the data. Graphical analysis using Logan or multilinear analysis 1 produced similar results as compartmental modeling indicating robust quantification. This study advances our understanding of how gabapentinoids work and provides an important advancement towards imaging 2{delta} receptors in the brain.

neuroscience↗

bioRxiv2023

Chemical and biophysical characterization of novel potassium channel blocker 3-fluoro-5-methylpyridin-4-amine

4-aminopyridine (4AP) is a potassium (K+) channel blocker used clinically to improve walking in people with multiple sclerosis (MS). 4AP binds to exposed K+ channels in demyelinated axons, reducing the leakage of intracellular K+ and enhancing impulse conduction. Multiple derivatives of 4AP capable of blocking K+ channels have been reported including three radiolabeled with positron emitting isotopes for imaging demyelinated lesions using positron emission tomography (PET). Here, we describe 3-fluoro-5-methylpyridin-4-amine (5Me3F4AP), a novel K+ channel blocker with potential application in PET. 5Me3F4AP has comparable potency to 4AP and the PET tracer 3-fluoro-4-aminopyridine (3F4AP). Compared to 3F4AP, 5Me3F4AP is more lipophilic (logD = 0.664 {+/-} 0.005 vs. 0.414 {+/-} 0.002) and slightly more basic (pKa = 7.46 {+/-} 0.01 vs. 7.37 {+/-} 0.07). In addition, 5Me3F4AP appears to be more permeable to an artificial brain membrane and more stable towards oxidation by the cytochrome P450 enzyme family 2 subfamily E member 1 (CYP2E1), responsible for the metabolism of 4AP and 3F4AP. Taken together, 5Me3F4AP has promising properties for PET imaging warranting additional investigation. Significance StatementThe PET tracer [18F]3-fluoro-4-aminopyridine ([18F]3F4AP) binds to K+ channels in demyelinated axons and has shown promise for imaging demyelinated lesions in animal models. However, its use in humans may be compromised due to rapid metabolism. Thus, a novel 3F4AP derivative amenable to labeling with fluorine-18 was designed and evaluated in vitro. The results indicate that 5-methyl-3F4AP exhibits high binding affinity, good physicochemical properties and slower oxidation by CYP2E1 than 3F4AP, making it a promising candidate for further PET studies.

pharmacology and toxicology↗

bioRxiv2023

Radiosynthesis automation, non-human primate biodistribution and dosimetry of K+ channel tracer 3MeO4AP

Purpose4-Aminopyridine (4AP) is a medication for the symptomatic treatment of multiple sclerosis. Several 4AP-based PET tracers have been developed for imaging demyelination. In preclinical studies, [11C]3MeO4AP has shown promise due to its high brain permeability, high metabolic stability, high plasma availability, and high in vivo binding affinity. To prepare for the translation to human studies, we developed a cGMP-compliant automated radiosynthesis protocol and evaluated the whole-body biodistribution and radiation dosimetry of [11C]3MeO4AP in non-human primates (NHPs). MethodsAutomated radiosynthesis was carried out using a GE TRACERlab FX-C Pro synthesis module. One male and one female adult rhesus macaques were used in the study. A high-resolution CT from cranial vertex to knee was acquired. PET data were collected using a dynamic acquisition protocol with 4 bed positions and 13 passes over a total scan time of [~]150 minutes. Based on the CT and PET images, volumes of interest (VOIs) were manually drawn for selected organs. Non-decay corrected time-activity curves (TACs) were extracted for each VOI. Radiation dosimetry and effective dose were calculated from the integrated TACs using OLINDA software. ResultsFully automated radiosynthesis of [11C]3MeO4AP was achieved with 7.3 {+/-} 1.2 % (n = 4) of non-decay corrected radiochemical yield within 38 min of synthesis and purification time. [11C]3MeO4AP distributed quickly throughout the body and into the brain. The organs with highest dose were the kidneys. The average effective dose of [11C]3MeO4AP was 4.27 {+/-} 0.57 Sv/MBq. No significant changes in vital signs were observed during the scan. ConclusionThe cGMP compliant automated radiosynthesis of [11C]3MeO4AP was developed. The whole-body biodistribution and radiation dosimetry of [11C]3MeO4AP was successfully evaluated in NHPs. [11C]3MeO4AP shows lower average effective dose than [18F]3F4AP and similar average effective dose as other carbon-11 tracers.

neuroscience↗

bioRxiv2022

Metabolic Stability of the Demyelination PET tracer 3F4AP and Identification of its Metabolites

[18F]3-fluoro-4-aminopyridine ([18F]3F4AP) is a PET tracer for imaging demyelination based on the multiple sclerosis drug 4-aminopyridine (4AP, dalfampridine). This radiotracer was found to be stable in rodents and nonhuman primates imaged under isoflurane anesthesia. However, recent findings indicate that its stability is greatly decreased in awake humans and mice. Since both 4AP and isoflurane are metabolized primarily by cytochrome P450 enzymes, particularly CYP2E1, we postulated that this enzyme may be responsible for the metabolism of 3F4AP. Here, we investigated the metabolism of [18F]3F4AP by CYP2E1 and identified its metabolites. We also investigated whether deuteration, a common approach to increase the stability of drugs, could improve its stability. Our results demonstrate that CYP2E1 readily metabolizes 3F4AP and its deuterated analogues and that the primary metabolites are 5-hydroxy-3-fluoro-4-aminopyridine and 3-fluoro-4-aminopyridine N-oxide. Although deuteration did not decrease the rate of the CYP2E1 mediated oxidation, our findings explain the diminished in vivo stability of 3F4AP compared to 4AP and further our understanding of when deuteration may improve the metabolic stability of drugs and PET ligands. Significance StatementUnderstanding the metabolic stability of PET tracers is paramount to its application in humans as metabolism, which varies from person to person, can affect the target-to-background signal. This study identified the predominant enzyme that metabolizes the demyelination PET tracer [18F]3F4AP and its metabolites. These findings may allow assessment of whether the radiometabolites can get into the brain and potentially lead to tracers with enhanced stability. Furthermore, this study furthers our understanding of when deuteration can improve metabolic stability.

biochemistry↗

bioRxiv2022

Radiochemical Synthesis and Evaluation of 3-methyl-4-aminopyridine in Rodents and Non-Human Primates for Imaging Potassium Channels in the CNS

Demyelination, the loss of the insulating sheath of neurons, causes failed or slowed neuronal conduction and contributes to the neurological symptoms in multiple sclerosis, traumatic brain and spinal cord injuries, stroke, and dementia. In demyelinated neurons, the axonal potassium channels Kv1.1 and Kv1.2, generally under the myelin sheath, become exposed and upregulated. Therefore, imaging these channels using positron emission tomography can provide valuable information for disease diagnosis and monitoring. Here, we describe the novel tracer for Kv1 channels [11C]3-methyl-4-aminopyridine ([11C]3Me4AP). [11C]3Me4AP was efficiently synthesized via Pd(0)-Cu(I) co-mediated Stille cross-coupling of a stannyl precursor containing a free amino group. Evaluation of its imaging properties in rats and nonhuman primates showed that [11C]3Me4AP has a moderate brain permeability and slow kinetics. Additional evaluation in monkeys showed that the tracer is metabolically stable and that a 1-tissue compartment model can accurately model the regional brain time-activity curves. Compared to the related tracers [18F]3-fluoro-4-aminopyridine ([18F]3F4AP) and [11C]3-methoxy-4-aminopyridine ([11C]3MeO4AP), [11C]3Me4AP shows lower initial brain uptake, which indicates reduced permeability to the blood-brain-barrier and slower kinetics, suggesting higher binding affinity consistent with in vitro studies. While the slow kinetics and strong binding affinity resulted in a tracer with less favorable properties for imaging the brain than its predecessors, these properties may make 3Me4AP useful as a therapeutic.

neuroscience↗

bioRxiv2022

Development of a PET radioligand for α2δ-1 subunit of calcium channels for imaging neuropathic pain

Neuropathic pain affects 7-10% of the adult population. Being able to accurately monitor biological changes underlying neuropathic pain will improve our understanding of neuropathic pain mechanisms and facilitate the development of novel therapeutics. Positron emission tomography (PET) is a noninvasive molecular imaging technique that can provide quantitative information of biochemical changes at the whole-body level by using radiolabeled ligands. One important biological change underlying the development of neuropathic pain is the overexpression of 2{delta}-1 subunit of voltage-dependent calcium channels (the target of gabapentin). Thus, we hypothesized that a radiolabeled form of gabapentin may allow imaging changes in 2{delta}-1 for monitoring the underlying pathophysiology of neuropathic pain. Here, we report the development of two 18F-labeled derivatives of gabapentin (trans-4-[18F]fluorogabapentin and cis-4-[18F]fluorogabapentin) and their evaluation in healthy rats and a rat model of neuropathic pain (spinal nerve ligation model). Both isomers were found to selectively bind to the 2{delta}-1 receptor with trans-4-[18F]fluorogabapentin having a higher affinity. Both tracers displayed around 1.5- to 2-fold increased uptake in injured nerves over the contralateral uninjured nerves when measured by gamma counting ex vivo. Although the small size of the nerves and the signal from surrounding muscle prevented visualizing these changes using PET, this work demonstrates that fluorinated derivatives of gabapentin retain binding to 2{delta}-1 and that their radiolabeled forms can be used to detect pathological changes in vitro and ex vivo. Furthermore, this work confirms that 2{delta}-1 is a promising target for imaging specific features of neuropathic pain.

neuroscience↗

bioRxiv2020

Radiochemical synthesis and evaluation in nonhuman primates of 3-methoxy-4-aminopyridine: a novel PET tracer for imaging potassium channels in the CNS

Demyelination, the loss of the protecting sheath of neurons, contributes to disability in many neurological diseases. In order to fully understand its role in different diseases and to monitor treatments aiming at reversing this process, it would be valuable to have PET radiotracers that can detect and quantify molecular changes involved in demyelination such as the uncovering and upregulation of the axonal potassium channels Kv1.1 and Kv1.2. Carbon-11 labeled radiotracers present the advantage of allowing for multiple scans on the same subject in the same day. Here, we describe [11C|3MeO4AP, a novel 11C-labeled version of the K+ channel tracer [18F]3F4AP, and characterize its imaging properties in two nonhuman primates including a monkey with a focal brain injury sustained during a surgical procedure three years prior to imaging. Our findings show that [11C]3MeO4AP is brain permeable, metabolically stable and has high plasma availability. When compared with [18F]3F4AP, [11C]3MeO4AP shows very high correlation in volumes of distribution (VT) confirming a common target. [11C]3MeO4AP shows slower washout than [18F]3F4AP suggesting stronger binding. Finally, similar to [18F]3F4AP, [11C]3MeO4AP is highly sensitive to the focal brain injury. All these features make it a promising radioligand for imaging demyelinated lesions.

neuroscience↗

bioRxiv2020

Evaluation of the potassium channel tracer 3F4AP in rhesus macaques

Demyelination causes slowed or failed neuronal conduction and is a driver of disability in multiple sclerosis and other neurological diseases. Currently, the gold standard for imaging demyelination is MRI, but despite its high spatial resolution and sensitivity to demyelinated lesions, it remains challenging to obtain specific and quantitative measures of demyelination. To understand the contribution of demyelination in different diseases and to assess the efficacy of myelin-repair therapies, it is critical to develop new in vivo imaging tools sensitive to changes induced by demyelination. Upon demyelination, axonal K+ channels, normally located underneath the myelin sheath, become exposed and increase in expression, causing impaired conduction. Here, we investigate the properties of the K+ channel PET tracer [18F]3F4AP in primates and its sensitivity to a focal brain injury that occurred three years prior to imaging. [18F]3F4AP exhibited favorable properties for brain imaging including high brain penetration, high metabolic stability, high plasma availability, high reproducibility, high specificity, and fast kinetics. [18F]3F4AP showed preferential binding in areas of low myelin content as well as in the previously injured area. Sensitivity of [18F]3F4AP for the focal brain injury was higher than [18F]FDG, [11C]PiB and [11C]PBR28, and compared favorably to currently used MRI methods.

neuroscience↗

bioRxiv2019

Structure-activity relationship studies of three novel 4-aminopyridine K+ channel blockers

4-Aminopyridine (4AP) is a specific blocker of voltage-gated potassium channels (KV1 family) clinically approved for the symptomatic treatment of patients with multiple sclerosis (MS). It has recently been shown that [18F]3F4AP, a radiofluorinated analog of 4AP, also binds to KV1 channels and can be used as a PET tracer for the detection of demyelinated lesions in rodent models of MS. Here, we investigate three novel 4AP derivatives containing methyl (-CH3), methoxy (-OCH3) and trifluoromethyl (-CF3) in the 3 position as potential candidates for PET imaging and/or therapy. We characterized the physicochemical properties of these compounds (pKa and logD) and analyzed their ability to block Shaker K+ channel under different voltage and pH conditions. Our results demonstrate that all three derivatives are able to block voltage-gated potassium channels. Specifically, 3-methyl-4-aminopyridine (3Me4AP) was found to be approximately 7-fold more potent than 4AP, whereas the methoxy (3MeO4AP) and trifluoromethyl (3CF34AP) containing compounds were about 3- to 4-fold less potent than 4AP, respectively. These results suggest that these novel derivatives are potential candidates for therapy and imaging.

biophysics↗