Using intracerebral microdialysis to probe the efficacy of repurposed drugs in Alzheimer′s disease pathology

All disease-targeting drug trials completed to date have fallen short of meeting the clinical endpoint of significantly slowing cognitive decline in Alzheimer′s disease patients. Even the recently approved drug Aducanumab, has proven effective in removing amyloid-β, but does not reduce cognitive decline. This emphasizes the urgent need for novel therapeutic approaches that could reduce several AD neuropathologies simultaneously, eventually leading to improved cognitive performance. To validate whether our mouse model replicates AD neuropathology as observed in patients, we characterized the 3xTg AD mouse model to avoid premature translation of successful results. In this study we have repurposed two FDA-approved drugs, Fasudil and Lonafarnib, targeting the Wnt signaling and endosomal-lysosomal pathway respectively, to test their potential to attenuate AD pathology. Using intracerebral microdialysis, we simultaneously infused these disease-targeting drugs between 1-2 weeks, separately and also in combination, while collecting cerebrospinal fluid. We found that Fasudil reduces intracellular amyloid-β in young, and amyloid plaques in old animals, and overall cerebrospinal fluid amyloid-β. Lonafarnib reduces tau neuropathology and cerebrospinal fluid tau biomarkers in young and old animals. Co-infusion of both drugs was more effective in reducing intracellular amyloid-β than either drug alone, and appeared to improve contextual memory performance. However, an unexpected finding was that Lonafarnib treatment increased amyloid plaque size, suggesting that activating the endosomal-lysosomal system may inadvertently increase amyloid-β pathology if administered too late in the AD continuum. Taken together, these findings lend support to the application of repurposed drugs to attenuate AD neuropathology at various therapeutic time windows.

EXTH-52. SYSTEMIC AND BRAIN PHARMACOKINETICS OF THE AMP-ACTIVATED PROTEIN KINASE SELECTIVE INHIBITOR SBI-0206965 AS A POTENTIAL THERAPEUTIC AGENT FOR THE TREATMENT OF GLIOBLASTOMA MULTIFORME

PURPOSE AMP-activated protein kinase (AMPK) is a molecular hub for cellular metabolic control. Recent evidence suggests that AMPK is a “druggable” novel target for the treatment of Glioblastoma Multiforme (GBM). However, AMPK-inhibitory compounds are largely limited to compound C, which has a poor selectivity profile. SBI-0206965 is a diaminopyrimidine derivative that directly inhibits AMPK with 40-fold greater potency and markedly lower kinase promiscuity than compound C. The current studies provide insights into systemic pharmacokinetics and plasma to brain partitioning of SBI-0206965. METHODS We conducted an intracerebral microdialysis study employing jugular vein-cannulated Sprague Dawley rats (males, 6- 8 weeks). Serial brain extracellular fluid (ECF) and venous blood samples were collected up to 10 hrs following intraperitoneal administration of SBI-0206965 (25 mg/kg). These samples were then quantitated for SBI-0206965 levels using a LC/MS method (Thermo Scientific LTQ-FT™, Ionization: Electrospray Ionization; positive ion). PK analysis was performed using the Non–Compartmental Analysis (Phoenix® WinNonlin 8.2 Certara USA, Inc.). RESULTS Plasma and ECF peak concentrations (Cmax) were 7.15 µM and 0.68 µM, whereas the time to peak (Tmax) were 0.5 and 1 hr, respectively. The plasma and brain ECF elimination half-lives were 1.5 and 3 hours, respectively. Plasma protein binding of SBI-0206965 was 82%. A comparison of the brain ECF Cmax and area under the curve (AUC) to corresponding plasma values suggested that the brain partitioning of the compound was 10-18%. When corrected for unbound fraction in plasma the AUC ratio was 0.86. Thus, these studies show that SBI-0206965 has adequate brain penetration. Further studies are now in progress to assess selectivity of SBI-0206965 for AMPK expressing cell lines, efficacy against patient-derived GBM and PK in tumor-bearing mice. CONCLUSION Results from this study will help to design optimal dosing regimen of SBI-0206965 in our efforts to explore AMPK as a GBM-specific drug target.

In Vivo Microdialysis in Mice Captures Changes in Alzheimer’s Disease Cerebrospinal Fluid Biomarkers Consistent with Developing Pathology

Background: Preclinical models of Alzheimer’s disease (AD) can provide valuable insights into the onset and progression of the disease, such as changes in concentrations of amyloid-β (Aβ) and tau in cerebrospinal fluid (CSF). However, such models are currently underutilized due to limited advancement in techniques that allow for longitudinal CSF monitoring. Objective: An elegant way to understand the biochemical environment in the diseased brain is intracerebral microdialysis, a method that has until now been limited to short-term observations, or snapshots, of the brain microenvironment. Here we draw upon patient-based findings to characterize CSF biomarkers in a commonly used preclinical mouse model for AD. Methods: Our modified push-pull microdialysis method was first validated ex vivo with human CSF samples, and then in vivo in an AD mouse model, permitting assessment of dynamic changes of CSF Aβ and tau and allowing for better translational understanding of CSF biomarkers. Results: We demonstrate that CSF biomarker changes in preclinical models capture what is observed in the brain; with a decrease in CSF Aβ observed when plaques are deposited, and an increase in CSF tau once tau pathology is present in the brain parenchyma. We found that a high molecular weight cut-off membrane allowed for simultaneous sampling of Aβ and tau, comparable to CSF collection by lumbar puncture in patients. Conclusion: Our approach can further advance AD and other neurodegenerative research by following evolving neuropathology along the disease cascade via consecutive sampling from the same animal and can additionally be used to administer pharmaceutical compounds and assess their efficacy (Bjorkli, unpublished data).

Dorsal striatum and the temporal expectancy of an aversive event in Pavlovian odor fear learning

Interval timing, the ability to encode and retrieve the memory of intervals from seconds to minutes, guides fundamental animal behaviors across the phylogenetic tree. In Pavlovian fear conditioning, an initially neutral stimulus (conditioned stimulus, CS) predicts the arrival of an aversive unconditioned stimulus (US, generally a mild foot-shock) at a fixed time interval. Although some studies showed that temporal relations between CS and US events are learned from the outset of conditioning, the question of the memory of time and its underlying neural network in fear conditioning is still poorly understood. The aim of the present study was to investigate the role of the dorsal striatum in timing intervals in odor fear conditioning in male rats. To assess the animal's interval timing ability in this paradigm, we used the respiratory frequency. This enabled us to detect the emergence of temporal patterns related to the odor-shock time interval from the early stage of learning, confirming that rats are able to encode the odor-shock time interval after few training trials. We carried out reversible inactivation of the dorsal striatum before the acquisition session and before a shift in the learned time interval, and measured the effects of this treatment on the temporal pattern of the respiratory rate. In addition, using intracerebral microdialysis, we monitored extracellular dopamine level in the dorsal striatum throughout odor-shock conditioning and in response to a shift of the odor-shock time interval. Contrary to our initial predictions based on the existing literature on interval timing, we found evidence suggesting that transient inactivation of the dorsal striatum may favor a more precocious buildup of the respiratory frequency's temporal pattern during the odor-shock interval in a manner that reflected the duration of the interval. Our data further suggest that the conditioning and the learning of a novel time interval were associated with a decrease in dopamine level in the dorsal striatum, but not in the nucleus accumbens. These findings prompt a reassessment of the role of the striatum and striatal dopamine in interval timing, at least when considering Pavlovian aversive conditioning.

Tapping Into the Aging Brain: in Vivo Microdialysis Reveals Mirroring Pathology Between Preclinical Models and Patients With Alzheimer’s Disease

BackgroundPreclinical models of Alzheimer’s disease (AD) can provide valuable insights into the onset and progression of the disease, such as changes in concentrations of amyloid-β (Aβ) and tau in cerebrospinal fluid (CSF). However, such models are currently underutilized due to limited advancement in techniques that allow for longitudinal monitoring of CSF akin to methods employed in AD patients. An elegant way to understand the biochemical environment in the diseased brain is intracerebral microdialysis, a method that has until now been limited to short-term observations, or snapshots, of the brain microenvironment. ResultsOur novel push-pull microdialysis method in AD mice permits in vivo longitudinal monitoring of dynamic changes of Aβ and tau in CSF and allows for better translational understanding of CSF biomarkers. Specifically, we demonstrate that CSF concentrations of Aβ and tau along disease progression in transgenic mice mirror what is observed in patients, with a decrease in CSF Aβ observed when plaques are deposited, and an increase in CSF tau once tau pathology is present in the brain. We found that a high molecular weight cut-off membrane allowed for simultaneous sampling of Aβ and tau, comparable to lumbar puncture CSF collection in patients. We furthermore provide specific recommendations for optimal application of our novel microdialysis method, such as achieving optimal recovery of analytes, which depends heavily on the flow rate of perfusion, probe properties and perfusate composition. ConclusionsOur approach can further advance AD research by following evolving neuropathology along the disease cascade via consecutive sampling from the same animal, and can additionally be used to administer pharmaceutical compounds and assess their efficacy in treating AD.

Tapping into the aging brain: In vivo microdialysis reveals mirroring pathology between preclinical models and patients with Alzheimer’s disease

SUMMARYPreclinical models of Alzheimer’s disease (AD) can provide valuable insights into the onset and progression of the disease, such as changes in concentrations of amyloid-β (Aβ) and tau in cerebrospinal fluid (CSF). However, such models are currently underutilized due to limited advancement in techniques that allow for longitudinal monitoring of CSF akin to methods employed in AD patients. An elegant way to understand the biochemical environment in the diseased brain is intracerebral microdialysis, a method that has until now been limited to short-term observations, or snapshots, of the brain microenvironment. Our novel push-pull microdialysis method in AD mice permits in vivo longitudinal monitoring of dynamic changes of Aβ and tau in CSF and allows for better translational understanding of CSF biomarkers. Specifically, we demonstrate that CSF concentrations of Aβ and tau along disease progression in transgenic mice mirror what is observed in patients, with a decrease in CSF Aβ observed when plaques are deposited, and an increase in CSF tau once tau pathology is present in the brain. We found that a high molecular weight cut-off membrane allowed for simultaneous sampling of Aβ and tau, comparable to lumbar puncture CSF collection in patients. We furthermore provide specific recommendations for optimal application of our novel microdialysis method, such as achieving optimal recovery of analytes, which depends heavily on the flow rate of perfusion, probe properties and perfusate composition. Our approach can further advance AD research by following evolving neuropathology along the disease cascade via consecutive sampling from the same animal, and can additionally be used to administer pharmaceutical compounds and assess their efficacy in treating AD.

Moderately prolonged permissive hypotension results in reversible metabolic perturbation evaluated by intracerebral microdialysis - an experimental animal study

Background Damage control resuscitation (DCR) and damage control surgery (DCS) is the main strategy in patients with uncontrollable hemorrhagic shock. One aspect of DCR is permissive hypotension. However, the duration of hypotension that can be tolerated without affecting the brain is unknown. In the present study we investigate the effect of 60 min severe hypotension on the brain’s energy metabolism and seek to verify earlier findings that venous cerebral blood can be used as a marker of global cerebral energy state. Material and methods Ten pigs were anaesthetized, and vital parameters recorded. Microdialysis catheters were placed in the left parietal lobe, femoral artery, and superior sagittal sinus for analysis of lactate, pyruvate, glucose, glycerol, and glutamate. Hemorrhagic shock was induced by bleeding the animal until mean arterial pressure (MAP) of 40 mmHg was achieved. After 60 min the pigs were resuscitated with autologous blood and observed for 3 h. Results At baseline the lactate to pyruvate ratios (LP ratio) in the hemisphere, artery, and sagittal sinus were (median (interquartile range)) 13 (8–16), 21 (18–24), and 9 (6–22), respectively. After induction of hemorrhagic shock, the LP ratio from the left hemisphere in 9 pigs increased to levels indicating a reversible perturbation of cerebral energy metabolism 19 (12–30). The same pattern was seen in LP measurements from the femoral artery 28 (20–35) and sagittal sinus 22 (19–26). At the end of the experiment hemisphere, artery and sinus LP ratios were 16 (10–23), 17 (15–25), and 17 (10–27), respectively. Although hemisphere and sinus LP ratios decreased, they did not reach baseline levels (p < 0.05). In one pig hemisphere LP ratio increased to a level indicating irreversible metabolic perturbation (LP ratio > 200). Conclusion During 60 min of severe hypotension intracerebral microdialysis shows signs of perturbations of cerebral energy metabolism, and these changes trend towards baseline values after resuscitation. Sagittal sinus microdialysis values followed hemisphere values but were not distinguishable from systemic arterial values. Venous (jugular bulb) microdialysis might have a place in monitoring conditions where global cerebral ischemia is a risk.