Evidence Implicating Blood-Brain Barrier Impairment in the Pathogenesis of Acquired Epilepsy following Acute Organophosphate Intoxication.

Organophosphate (OP) poisoning can trigger cholinergic crisis, a life-threatening toxidrome that includes seizures and status epilepticus. These acute toxic responses are associated with persistent neuroinflammation and spontaneous recurrent seizures (SRS), also known as acquired epilepsy. Blood-brain barrier (BBB) impairment has recently been proposed as a pathogenic mechanism linking acute OP intoxication to chronic adverse neurologic outcomes. In this review, we briefly describe the cellular and molecular components of the BBB, review evidence of altered BBB integrity following acute OP intoxication, and discuss potential mechanisms by which acute OP intoxication may promote BBB dysfunction. We highlight the complex interplay between neuroinflammation and BBB dysfunction that suggests a positive feedforward interaction. Lastly, we examine research from diverse models and disease states that suggest mechanisms by which loss of BBB integrity may contribute to epileptogenic processes. Collectively, the literature identifies BBB impairment as a convergent mechanism of neurologic disease and justifies further mechanistic research into how acute OP intoxication causes BBB impairment and its role in the pathogenesis of SRS and potentially other long-term neurologic sequelae. Such research is critical for evaluating BBB stabilization as a neuroprotective strategy for mitigating OP-induced epilepsy and possibly seizure disorders of other etiologies. SIGNIFICANCE STATEMENT: Clinical and preclinical studies support a link between blood-brain barrier (BBB) dysfunction and epileptogenesis; however, a causal relationship has been difficult to prove. Mechanistic studies to delineate relationships between BBB dysfunction and epilepsy may provide novel insights into BBB stabilization as a neuroprotective strategy for mitigating epilepsy resulting from acute organophosphate (OP) intoxication and non-OP causes and potentially other adverse neurological conditions associated with acute OP intoxication, such as cognitive impairment.

Radiosynthesis and initial preclinical evaluation of [11C]AZD1283 as a potential P2Y12R PET radiotracer.

INTRO: Chronic neuroinflammation and microglial dysfunction are key features of many neurological diseases, including Alzheimer's Disease and multiple sclerosis. While there is unfortunately a dearth of highly selective molecular imaging biomarkers/probes for studying microglia in vivo, P2Y12R has emerged as an attractive candidate PET biomarker being explored for this purpose. Importantly, P2Y12R is selectively expressed on microglia in the CNS and undergoes dynamic changes in expression according to inflammatory context (e.g., toxic versus beneficial/healing states), thus having the potential to reveal functional information about microglia in living subjects. Herein, we identified a high affinity, small molecule P2Y12R antagonist (AZD1283) to radiolabel and assess as a candidate radiotracer through in vitro assays and in vivo positron emission tomography (PET) imaging of both wild-type and total knockout mice and a non-human primate. METHODS: First, we evaluated the metabolic stability and passive permeability of non-radioactive AZD1283 in vitro. Next, we radiolabeled [11C]AZD1283 with radioactive precursor [11C]NH4CN and determined stability in formulation and human plasma. Finally, we investigated the in vivo stability and kinetics of [11C]AZD1283 via dynamic PET imaging of naïve wild-type mice, P2Y12R knockout mouse, and a rhesus macaque. RESULTS: We determined the half-life of AZD1283 in mouse and human liver microsomes to be 37 and > 160 min, respectively, and predicted passive CNS uptake with a small amount of active efflux, using a Caco-2 assay. Our radiolabeling efforts afforded [11C]AZD1283 in an activity of 12.69 ± 10.64 mCi with high chemical and radiochemical purity (>99%) and molar activity of 1142.84 ± 504.73 mCi/µmol (average of n = 3). Of note, we found [11C]AZD1283 to be highly stable in vitro, with >99% intact tracer present after 90 min of incubation in formulation and 60 min of incubation in human serum. PET imaging revealed negligible brain signal in healthy wild-type mice (n = 3) and a P2Y12 knockout mouse (0.55 ± 0.37%ID/g at 5 min post injection). Strikingly, high signal was detected in the liver of all mice within the first 20 min of administration (peak uptake = 58.28 ± 18.75%ID/g at 5 min post injection) and persisted for the remaining duration of the scan. Ex vivo gamma counting of mouse tissues at 60 min post-injection mirrored in vivo data with a mean %ID/g of 0.9% ± 0.40, 0.02% ± 0.01, and 106 ± 29.70% in the blood, brain, and liver, respectively (n = 4). High performance liquid chromatography (HPLC) analysis of murine blood and liver metabolite samples revealed a single radioactive peak (relative area under peak: 100%), representing intact tracer. Finally, PET imaging of a rhesus macaque also revealed negligible CNS uptake/binding in monkey brain (peak uptake = 0.37 Standard Uptake Values (SUV)). CONCLUSION: Despite our initial encouraging liver microsome and Caco-2 monolayer data, in addition to the observed high stability of [11C]AZD1283 in formulation and human serum, in vivo brain uptake was negligible and rapid accumulation was observed in the liver of both naïve wildtype and P2Y12R knockout mice. Liver signal appeared to be independent of both metabolism and P2Y12R expression due to the confirmation of intact tracer in this tissue for both wildtype and P2Y12R knockout mice. In Rhesus Macaque, negligible uptake of [11C]AZD1283 brain indicates a lack of potential for translation or its further investigation in vivo. P2Y12R is an extremely promising potential PET biomarker, and the data presented here suggests encouraging metabolic stability for this scaffold; however, the mechanism of liver uptake in mice should be elucidated prior to further analogue development.