Insulin Signaling Differentially Regulates the Trafficking of Insulin and Amyloid Beta Peptides at the Blood-Brain Barrier.

The blood-brain barrier (BBB) is instrumental in clearing toxic metabolites from the brain, such as amyloid-ß (Aß) peptides, and in delivering essential nutrients to the brain, like insulin. In Alzheimer's disease (AD) brain, increased Aß levels are paralleled by decreased insulin levels, which are accompanied by insulin signaling deficits at the BBB. Thus, we investigated the impact of insulin-like growth factor and insulin receptor (IGF1R and IR) signaling on Aß and insulin trafficking at the BBB. Following intravenous infusion of an IGF1R/IR kinase inhibitor (AG1024) in wild-type mice, the BBB trafficking of 125I radiolabeled Aß peptides and insulin was assessed by dynamic SPECT/CT imaging. The brain efflux of [125I]iodo-Aß42 decreased upon AG1024 treatment. Additionally, the brain influx of [125I]iodoinsulin, [125I]iodo-Aß42, [125I]iodo-Aß40, and [125I]iodo-BSA (BBB integrity marker) was decreased, increased, unchanged, and unchanged, respectively, upon AG1024 treatment. Subsequent mechanistic studies were performed using an in vitro BBB cell model. The cell uptake of [125I]iodoinsulin, [125I]iodo-Aß42, and [125I]iodo-Aß40 was decreased, increased, and unchanged, respectively, upon AG1024 treatment. Further, AG1024 reduced the phosphorylation of insulin signaling kinases (Akt and Erk) and the membrane expression of Aß and insulin trafficking receptors (LRP-1 and IR-ß). These findings reveal that insulin signaling differentially regulates the BBB trafficking of Aß peptides and insulin. Moreover, deficits in IGF1R and IR signaling, as observed in the brains of type II diabetes and AD patients, are expected to increase Aß accumulation while decreasing insulin delivery to the brain, which has been linked to the progression of cognitive decline in AD.

Amyloid-Beta Peptides 40 and 42 Employ Distinct Molecular Pathways for Cell Entry and Intracellular Transit at the Blood-Brain Barrier Endothelium.

The blood-brain barrier (BBB) plays a critical role in maintaining the equilibrium between amyloid beta (Aß) levels in blood and the brain by regulating Aß transport. Our previous publications demonstrated that BBB trafficking of Aß42 and Aß40 is distinct and is disrupted under various pathophysiological conditions. However, the intracellular mechanisms that allow BBB endothelium to differentially handle Aß40 and Aß42 have not been clearly elucidated. In this study, we identified mechanisms of Aß endocytosis in polarized human cerebral microvascular endothelial cell monolayers. Our studies demonstrated that Aß peptides with fluorescent label (F-Aß) were internalized by BBB endothelial cells via energy, dynamin, and actin-dependent endocytosis. Interestingly, endocytosis of F-Aß40 but not F-Aß42 was substantially reduced by clathrin inhibition, whereas F-Aß42 but not F-Aß40 endocytosis was reduced by half after inhibiting the caveolae-mediated pathway. Following endocytosis, both isoforms were sorted by the endo-lysosomal system. Although Aß42 was shown to accumulate more in the lysosomes, which could lead to its higher degradation and/or aggregation at lower lysosomal pH, Aß40 demonstrated robust accumulation in recycling endosomes, which may facilitate its exocytosis by the endothelial cells. These results provide a mechanistic insight into the selective ability of BBB endothelium to transport Aß40 versus Aß42. This knowledge contributes to the understanding of molecular pathways underlying Aß accumulation in the BBB endothelium and associated BBB dysfunction. Moreover, it allows us to establish mechanistic rationale for altered Aß40:Aß42 ratios and anomalous amyloid deposition in the cerebral vasculature as well as brain parenchyma during Alzheimer's disease progression. SIGNIFICANCE STATEMENT: Differential interaction of Aß40 and Aß42 isoforms with the blood-brain barrier (BBB) endothelium may contribute to perturbation in Aß42:Aß40 ratio, which is associated with Alzheimer's disease (AD) progression and severity. The current study identified distinct molecular pathways by which Aß40 and Aß42 are trafficked at the BBB, which regulates equilibrium between blood and brain Aß levels. These findings provide molecular insights into mechanisms that engender BBB dysfunction and promote Aß accumulation in AD brain.

Deconvolution of Plasma Pharmacokinetics from Dynamic Heart Imaging Data Obtained by Single Positron Emission Computed Tomography/Computed Tomography Imaging.

Plasma pharmacokinetic (PK) data are required as an input function for graphical analysis of single positron emission computed tomography/computed tomography (SPECT/CT) and positron emission tomography/CT (PET/CT) data to evaluate tissue influx rate of radiotracers. Dynamic heart imaging data are often used as a surrogate of plasma PK. However, accumulation of radiolabel in the heart tissue may cause overprediction of plasma PK. Therefore, we developed a compartmental model, which involves forcing functions to describe intact and degraded radiolabeled proteins in plasma and their accumulation in heart tissue, to deconvolve plasma PK of 125I-amyloid beta 40 (125I-Aß 40) and 125I-insulin from their dynamic heart imaging data. The three-compartment model was shown to adequately describe the plasma concentration-time profile of intact/degraded proteins and the heart radioactivity time data obtained from SPECT/CT imaging for both tracers. The model was successfully applied to deconvolve the plasma PK of both tracers from their naïve datasets of dynamic heart imaging. In agreement with our previous observations made by conventional serial plasma sampling, the deconvolved plasma PK of 125I-Aß 40 and 125I-insulin in young mice exhibited lower area under the curve than aged mice. Further, Patlak plot parameters extracted using deconvolved plasma PK as input function successfully recapitulated age-dependent plasma-to-brain influx kinetics changes. Therefore, the compartment model developed in this study provides a novel approach to deconvolve plasma PK of radiotracers from their noninvasive dynamic heart imaging. This method facilitates the application of preclinical SPECT/PET imaging data to characterize distribution kinetics of tracers where simultaneous plasma sampling is not feasible. SIGNIFICANCE STATEMENT: Knowledge of plasma pharmacokinetics (PK) of a radiotracer is necessary to accurately estimate its plasma-to-brain influx. However, simultaneous plasma sampling during dynamic imaging procedures is not always feasible. In the current study, we developed approaches to deconvolve plasma PK from dynamic heart imaging data of two model radiotracers, 125I-amyloid beta 40 (125I-Aß 40) and 125I-insulin. This novel method is expected to minimize the need for conducting additional plasma PK studies and allow for accurate estimation of the brain influx rate.

Design, Synthesis, and Preliminary Evaluation of [68Ga]Ga-NOTA-Insulin as a PET Probe in an Alzheimer's Disease Mouse Model.

Aberrant insulin signaling has been considered one of the risk factors for the development of Alzheimer's disease (AD) and has drawn considerable attention from the research community to further study its role in AD pathophysiology. Herein, we describe the development of an insulin-based novel positron emission tomography (PET) probe, [68Ga]Ga-NOTA-insulin, to noninvasively study the role of insulin in AD. The developed PET probe [68Ga]Ga-NOTA-insulin showed a significantly higher uptake (0.396 ± 0.055 SUV) in the AD mouse brain compared to the normal (0.140 ± 0.027 SUV) mouse brain at 5 min post injection and also showed a similar trend at 10, 15, and 20 min post injection. In addition, [68Ga]Ga-NOTA-insulin was found to have a differential uptake in various brain regions at 30 min post injection. Among the brain regions, the cortex, thalamus, brain stem, and cerebellum showed a significantly higher standard uptake value (SUV) of [68Ga]Ga-NOTA-insulin in AD mice as compared to normal mice. The inhibition of the insulin receptor (IR) with an insulin receptor antagonist peptide (S961) in normal mice showed a similar brain uptake profile of [68Ga]Ga-NOTA-insulin as it was observed in the AD case, suggesting nonfunctional IR in AD and the presence of an alternative insulin uptake route in the absence of a functional IR. The Gjedde-Patlak graphical analysis was also performed to predict the input rate of [68Ga]Ga-NOTA-insulin into the brain using MicroPET imaging data and supported the in vivo results. The [68Ga]Ga-NOTA-insulin PET probe was successfully synthesized and evaluated in a mouse model of AD in comparison with [18F]AV1451 and [11C]PIB to noninvasively study the role of insulin in AD pathophysiology.

Distinct Uptake Kinetics of Alzheimer Disease Amyloid-ß 40 and 42 at the Blood-Brain Barrier Endothelium.

Blood-brain barrier (BBB) endothelial cells lining the cerebral microvasculature maintain dynamic equilibrium between soluble amyloid-ß (Aß) levels in the brain and plasma. The BBB dysfunction prevalent in Alzheimer disease contributes to the dysregulation of plasma and brain Aß and leads to the perturbation of the ratio between Aß42 and Aß40, the two most prevalent Aß isoforms in patients with Alzheimer disease. We hypothesize that BBB endothelium distinguishes between Aß40 and Aß42, distinctly modulates their trafficking kinetics between plasma and brain, and thereby contributes to the maintenance of healthy Aß42/Aß40 ratios. To test this hypothesis, we investigated Aß40 and Aß42 trafficking kinetics in hCMEC/D3 monolayers (human BBB cell culture model) in vitro as well as in mice in vivo. Although the rates of uptake of fluorescein-labeled Aß40 and Aß42 (F-Aß40 and F-Aß42) were not significantly different on the abluminal side, the luminal uptake rate of F-Aß42 was substantially higher than F-Aß40. Since higher plasma Aß levels were shown to aggravate BBB dysfunction and trigger cerebrovascular disease, we systematically investigated the dynamic interactions of luminal [125I]Aß peptides and their trafficking kinetics at BBB using single-photon emission computed tomography/computed tomography imaging in mice. Quantitative modeling of the dynamic imaging data thus obtained showed that the rate of uptake of toxic [125I]Aß42 and its subsequent BBB transcytosis is significantly higher than [125I]Aß40. It is likely that the molecular mechanisms underlying these kinetic differences are differentially affected in Alzheimer and cerebrovascular diseases, impact plasma and brain levels of Aß40 and Aß42, engender shifts in the Aß42/Aß40 ratio, and unleash downstream toxic effects. SIGNIFICANCE STATEMENT: Dissecting the binding and uptake kinetics of Aß40 and Aß42 at the BBB endothelium will facilitate the estimation of Aß40 versus Aß42 exposure to the BBB endothelium and allow assessment of the risk of BBB dysfunction by monitoring Aß42 and Aß40 levels in plasma. This knowledge, in turn, will aid in elucidating the role of these predominant Aß isoforms in aggravating BBB dysfunction and cerebrovascular disease.

Semimechanistic Population Pharmacokinetic Modeling to Investigate Amyloid Beta Trafficking and Accumulation at the BBB Endothelium.

Elevated exposure to toxic amyloid beta (Aß) peptides and consequent blood-brain barrier (BBB) dysfunction are believed to promote vasculopathy in Alzheimer's disease (AD). However, the accumulation kinetics of different Aß isoforms within the BBB endothelium and how it drives BBB dysfunction are not clearly characterized. Using single positron emission computed tomography (SPECT)-computed tomography (CT) dynamic imaging coupled with population pharmacokinetic modeling, we investigated the accumulation kinetics of Aß40 and Aß42 in the BBB endothelium. Brain clearance was quantified after intracerebral administration of 125I-Aß, and BBB-mediated transport was shown to account for 54% of 125I-Aß40 total clearance. A brain influx study demonstrated lower values of both maximal rate (Vmax) and Michaelis constant (Km) for 125I-Aß42 compared to 125I-Aß40. Validated by a transcytosis study in polarized human BBB endothelial cell (hCMEC/D3) monolayers, model simulations demonstrated impaired exocytosis was responsible for inefficient permeability and enhanced accumulation of Aß42 in the BBB endothelium. Further, both isoforms were shown to disrupt the exocytosis machinery of BBB endothelial cells so that a vicious cycle could be generated. The validated model was able to capture changes in Aß steady-state levels in plasma as well as the brain during AD progression and allowed us to predict the kinetics of Aß accumulation in the BBB endothelium.

COVID-19 Transmission, Current Treatment, and Future Therapeutic Strategies.

At the stroke of the New Year 2020, COVID-19, a zoonotic disease that would turn into a global pandemic, was identified in the Chinese city of Wuhan. Although unique in its transmission and virulence, COVID-19 is similar to zoonotic diseases, including other SARS variants (e.g., SARS-CoV) and MERS, in exhibiting severe flu-like symptoms and acute respiratory distress. Even at the molecular level, many parallels have been identified between SARS and COVID-19 so much so that the COVID-19 virus has been named SARS-CoV-2. These similarities have provided several opportunities to treat COVID-19 patients using clinical approaches that were proven to be effective against SARS. Importantly, the identification of similarities in how SARS-CoV and SARS-CoV-2 access the host, replicate, and trigger life-threatening pathological conditions have revealed opportunities to repurpose drugs that were proven to be effective against SARS. In this article, we first provided an overview of COVID-19 etiology vis-à-vis other zoonotic diseases, particularly SARS and MERS. Then, we summarized the characteristics of droplets/aerosols emitted by COVID-19 patients and how they aid in the transmission of the virus among people. Moreover, we discussed the molecular mechanisms that enable SARS-CoV-2 to access the host and become more contagious than other betacoronaviruses such as SARS-CoV. Further, we outlined various approaches that are currently being employed to diagnose and symptomatically treat COVID-19 in the clinic. Finally, we reviewed various approaches and technologies employed to develop vaccines against COVID-19 and summarized the attempts to repurpose various classes of drugs and novel therapeutic approaches.

High-Density Lipoprotein Mimetic Peptide 4F Efficiently Crosses the Blood-Brain Barrier and Modulates Amyloid-ß Distribution between Brain and Plasma.

Treatments to elevate high-density lipoprotein (HDL) levels in plasma have decreased cerebrovascular amyloid -ß (Aß) deposition and mitigated cognitive decline in Alzheimer disease (AD) transgenic mice. Since the major protein component of HDL particles, apolipoprotein A-I (ApoA-I), has very low permeability at the blood-brain barrier (BBB), we investigated 4F, an 18-amino-acid ApoA-I/HDL mimetic peptide, as a therapeutic alternative. Specifically, we examined the BBB permeability of 4F and its effects on [125I]Aß trafficking from brain to blood and from blood to brain. After systemic injection in mice, the BBB permeability of [125I]4F, estimated as the permeability-surface area (PS) product, ranged between 2 and 5 × 10-6 ml/g per second in various brain regions. The PS products of [125I]4F were ∼1000-fold higher compared with those determined for [125I]ApoA-I. Moreover, systemic infusion with 4F increased the brain efflux of intracerebrally injected [125I]Aß42. Conversely, 4F infusion decreased the brain influx of systemically injected [125I]Aß42. Interestingly, 4F did not significantly alter the brain influx of [125I]Aß40. To corroborate the in vivo findings, we evaluated the effects of 4F on [125I]Aß42 transcytosis across polarized human BBB endothelial cell (hCMEC/D3) monolayers. Treatment with 4F increased the abluminal-to-luminal flux and decreased the luminal-to-abluminal flux of [125I]Aß42 across the hCMEC/D3 monolayers. Additionally, 4F decreased the endothelial accumulation of fluorescein-labeled Aß42 in the hCMEC/D3 monolayers. These findings provide a mechanistic interpretation for the reductions in brain Aß burden reported in AD mice after oral 4F administration, which represents a novel strategy for treating AD and cerebral amyloid angiopathy. SIGNIFICANCE STATEMENT: The brain permeability of the ApoA-I mimetic peptide 4F was estimated to be ∼1000-fold greater than ApoA-I after systemic injection of radiolabeled peptide/protein in mice. Further, 4F treatment increased the brain efflux of amyloid -ß and also decreased its brain influx, as evaluated in mice and in blood-brain barrier cell monolayers. Thus, 4F represents a potential therapeutic strategy to mitigate brain amyloid accumulation in cerebral amyloid angiopathy and Alzheimer disease.

Amyloid-beta impairs insulin signaling by accelerating autophagy-lysosomal degradation of LRP-1 and IR-ß in blood-brain barrier endothelial cells in vitro and in 3XTg-AD mice.

Aberrant insulin signaling constitutes an early change in Alzheimer's disease (AD). Insulin receptors (IR) and low-density lipoprotein receptor-related protein-1 (LRP-1) are expressed in brain capillary endothelial cells (BCEC) forming the blood-brain barrier (BBB). There, insulin may regulate the function of LRP-1 in Aß clearance from the brain. Changes in IR-ß and LRP-1 and insulin signaling at the BBB in AD are not well understood. Herein, we identified a reduction in cerebral and cerebrovascular IR-ß levels in 9-month-old male and female 3XTg-AD (PS1M146V, APPSwe, and tauP301L) as compared to NTg mice, which is important in insulin mediated signaling responses. Reduced cerebral IR-ß levels corresponded to impaired insulin signaling and LRP-1 levels in brain. Reduced cerebral and cerebrovascular IR-ß and LRP-1 levels in 3XTg-AD mice correlated with elevated levels of autophagy marker LC3B. In both genotypes, high-fat diet (HFD) feeding decreased cerebral and hepatic LRP-1 expression and elevated cerebral Aß burden without affecting cerebrovascular LRP-1 and IR-ß levels. In vitro studies using primary porcine (p)BCEC revealed that Aß peptides 1-40 or 1-42 (240 nM) reduced cellular levels and interaction of LRP-1 and IR-ß thereby perturbing insulin-mediated signaling. Further mechanistic investigation revealed that Aß treatment accelerated the autophagy-lysosomal degradation of IR-ß and LRP-1 in pBCEC. LRP-1 silencing in pBCEC decreased IR-ß levels through post-translational pathways further deteriorating insulin-mediated responses at the BBB. Our findings indicate that LRP-1 proves important for insulin signaling at the BBB. Cerebral Aß burden in AD may accelerate LRP-1 and IR-ß degradation in BCEC thereby contributing to impaired cerebral and cerebromicrovascular insulin effects.

Determination of Rate and Extent of Scopolamine Release from Transderm Scop® Transdermal Drug Delivery Systems in Healthy Human Adults.

To estimate strength of a scopolamine transdermal delivery system (TDS) in vivo, using residual drug vs. pharmacokinetic analyses with the goal of scientifically supporting a single and robust method for use across the dosage form and ultimately facilitate the development of more consistent and clinically meaningful labeling. A two-arm, open-label, crossover pharmacokinetic study was completed in 26 volunteers. Serum samples were collected and residual scopolamine was extracted from worn TDS. Delivery extent and rate were estimated by (1) numeric deconvolution and (2) steady-state serum concentration determined from graphical and non-compartmental analyses. In residual drug analyses, mean ± SD scopolamine release rate was 0.015 ± 0.002 mg/h (11% RSD), vs. 0.016 ± 0.006 mg/h (35% RSD) from numeric deconvolution, 0.015 ± 0.005 mg/h (34% RSD) from graphical analysis, and 0.015 ± 0.007 mg/h (44% RSD) from non-compartmental analysis. In residual drug analyses, total drug released was 1.09 ± 0.11 mg (10% RSD), vs. 1.12 ± 0.40 mg (35% RSD) from numeric deconvolution, 1.07 ± 0.35 mg (33% RSD) from graphical analysis, and 1.07 ± 0.45 (42% RSD) from non-compartmental analysis. Extent and rate of scopolamine release were comparable by both approaches, but pharmacokinetic analysis demonstrated greater inter-subject variability.

Apolipoprotein A-I Crosses the Blood-Brain Barrier through Clathrin-Independent and Cholesterol-Mediated Endocytosis.

Recent studies suggest that apolipoprotein A-I (ApoA-I), the major protein constituent of high-density lipoprotein particles, plays a critical role in preserving cerebrovascular integrity and reducing Alzheimer's risk. ApoA-I present in brain is thought to be primarily derived from the peripheral circulation. Although plasma-to-brain delivery of ApoA-I is claimed to be handled by the blood-cerebrospinal fluid barrier (BCSFB), a contribution by the blood-brain barrier (BBB), which serves as a major portal for protein delivery to brain, cannot be ruled out. In this study, we assessed the permeability-surface area product (PS) of radioiodinated ApoA-I (125I-ApoA-I) in various brain regions of wild-type rats after an intravenous bolus injection. The PS value at the cortex, caudate putamen, hippocampus, thalamus, brain stem, and cerebellum was found to be 0.39, 0.28, 0.28, 0.36, 0.69, and 0.76 (ml/g per second × 10-6), respectively. Solutes delivered into brain via the BCSFB are expected to show greater accumulation in the thalamus due to its periventricular location. The modest permeability for 125I-ApoA-I into the thalamus relative to other regions suggests that BCSFB transport accounts for only a portion of total brain uptake and thus BBB transport cannot be ruled out. In addition, we show that Alexa Flour 647-labeled ApoA-I (AF647-ApoA-I) undergoes clathrin-independent and cholesterol-mediated endocytosis in transformed human cerebral microvascular endothelial cells (hCMEC/D3). Further, Z-series confocal images of the hCMEC/D3 monolayers and Western blot detection of intact ApoA-I on the abluminal side demonstrated AF647-ApoA-I transcytosis across the endothelium. These findings implicate the BBB as a significant portal for ApoA-I delivery into brain.

Cationic carrier peptide enhances cerebrovascular targeting of nanoparticles in Alzheimer's disease brain.

Accumulation of amyloid beta (Aß) peptides in the cerebral vasculature, referred to as cerebral amyloid angiopathy (CAA), is widely observed in Alzheimer's disease (AD) brain and was shown to accelerate cognitive decline. There is no effective method for detecting cerebrovascular amyloid (CVA) and treat CAA. The targeted nanoparticles developed in this study effectively migrated from the blood flow to the vascular endothelium as determined by using quartz crystal microbalance with dissipation monitoring (QCM-D) technology. We also improved the stability, and blood-brain barrier (BBB) transcytosis of targeted nanoparticles by coating them with a cationic BBB penetrating peptide (K16ApoE). The K16ApoE-Targeted nanoparticles demonstrated specific targeting of vasculotropic DutchAß40 peptide accumulated in the cerebral vasculature. Moreover, K16ApoE-Targeted nanoparticles demonstrated significantly greater uptake into brain and provided specific MRI contrast to detect brain amyloid plaques.

Design, Development, and Characterization of Imiquimod-Loaded Chitosan Films for Topical Delivery.

Aldara™ (5% w/w imiquimod) topical cream is approved by the US FDA for the treatment of superficial basal cell carcinoma. However, the cream formulation suffers from dose variability, low drug availability due to the incomplete release, and poor patient compliance. To achieve sustained and complete release of imiquimod, chitosan films were prepared by casting using propylene glycol as a plasticizer. Chitosan films had appropriate physicochemical characteristics for wound dressing and excellent content uniformity and maintained the original physical form of imiquimod. Films were capable of releasing a defined dose of imiquimod over a period of 7 days. The bioactivity of imiquimod was not affected by its entrapment in chitosan matrix as indicated by the results of in vitro growth inhibition assay. In addition, the film formulation showed significantly (p Ë‚ 0.05) higher drug accumulation in the skin when compared to commercial cream formulation.

High-density lipoprotein mimetic peptide 4F mitigates amyloid-ß-induced inhibition of apolipoprotein E secretion and lipidation in primary astrocytes and microglia.

The apolipoprotein E (apoE) ε4 allele is the primary genetic risk factor for late-onset Alzheimer's disease (AD). ApoE in the brain is produced primarily by astrocytes; once secreted from these cells, apoE binds lipids and forms high-density lipoprotein (HDL)-like particles. Accumulation of amyloid-ß protein (Aß) in the brain is a key hallmark of AD, and is thought to initiate a pathogenic cascade leading to neurodegeneration and dementia. The level and lipidation state of apoE affect Aß aggregation and clearance pathways. Elevated levels of plasma HDL are associated with lower risk and severity of AD; the underlying mechanisms, however, have not been fully elucidated. This study was designed to investigate the impact of an HDL mimetic peptide, 4F, on the secretion and lipidation of apoE. We found that 4F significantly increases apoE secretion and lipidation in primary human astrocytes as well as in primary mouse astrocytes and microglia. Aggregated Aß inhibits glial apoE secretion and lipidation, causing accumulation of intracellular apoE, an effect that is counteracted by co-treatment with 4F. Pharmacological and gene editing approaches show that 4F mediates its effects partially through the secretory pathway from the endoplasmic reticulum to the Golgi apparatus and requires the lipid transporter ATP-binding cassette transporter A1. We conclude that the HDL mimetic peptide 4F promotes glial apoE secretion and lipidation and mitigates the detrimental effects of Aß on proper cellular trafficking and functionality of apoE. These findings suggest that treatment with such an HDL mimetic peptide may provide therapeutic benefit in AD. Read the Editorial Highlight for this article on page 580.

Sensitive Determination of Fentanyl in Low-Volume Serum Samples by LC-MS/MS.

Fentanyl is a widely used drug in the management of pain. Present LC-MS/MS methods for analysis of fentanyl require a large volume of serum, but yet the sensitivity was at about 50 pg/mL. Here, we report a modified liquid-liquid extraction method for the analysis of fentanyl in serum. The method is very sensitive with a LLOQ of 5 pg/mL while using only 0.175 mL of serum for analysis. The separation was performed on a Zorbax XDB-C18 column (4.6 × 50 mm, 1.8 µm, 600 bar) using a mobile phase of water: acetonitrile (70:30 v/v) with 0.1% formic acid that was pumped isocratically at a flow rate of 0.5 mL per minute. The calibration curve was found to be linear over a range of 5-10,000 pg/mL. The inter-day and intra-day accuracy and precision were tested using low (20 pg/mL), medium (1000 pg/mL), and high (5000 pg/mL) quality control samples of fentanyl prepared in blank human serum and were within ± 15% of the nominal value. Fentanyl was also found to be stable in various storage and sample preparation conditions, including short-term bench-top storage (for 5 h), freeze-thaw cycling (three cycles), long-term frozen condition (4.5 months at - 70°C), and post-preparative storage (for 48 h).

Pericyte Control of Gene Expression in the Blood-Brain Barrier Endothelium: Implications for Alzheimer's Disease.

Background: A strong body of evidence suggests that cerebrovascular pathologies augment the onset and progression of Alzheimer's disease (AD). One distinctive aspect of this cerebrovascular dysfunction is the degeneration of brain pericytes-often overlooked supporting cells of blood-brain barrier endothelium. Objective: The current study investigates the influence of pericytes on gene and protein expressions in the blood-brain barrier endothelium, which is expected to facilitate the identification of pathophysiological pathways that are triggered by pericyte loss and lead to blood-brain barrier dysfunction in AD. Methods: Bioinformatics analysis was conducted on the RNA-Seq expression counts matrix (GSE144474), which compared solo-cultured human blood-brain barrier endothelial cells against endothelial cells co-cultured with human brain pericytes in a non-contact model. We constructed a similar cell culture model to verify protein expression using western blots. Results: The insulin resistance and ferroptosis pathways were found to be enriched. Western blots of the insulin receptor and heme oxygenase expressions were consistent with those observed in RNA-Seq data. Additionally, we observed more than 5-fold upregulation of several genes associated with neuroprotection, including insulin-like growth factor 2 and brain-derived neurotrophic factor. Conclusions: Results suggest that pericyte influence on blood-brain barrier endothelial gene expression confers protection from insulin resistance, iron accumulation, oxidative stress, and amyloid deposition. Since these are conditions associated with AD pathophysiology, they imply mechanisms by which pericyte degeneration could contribute to disease progression.

Traffic jam at the blood-brain barrier promotes greater accumulation of Alzheimer's disease amyloid-ß proteins in the cerebral vasculature.

Amyloid-ß (Aß) deposition in the brain vasculature results in cerebral amyloid angiopathy (CAA), which occurs in about 80% of Alzheimer's disease (AD) patients. While Aß42 predominates parenchymal amyloid plaques in AD brain, Aß40 is prevalent in the cerebrovascular amyloid. Dutch mutation of Aß40 (E22Q) promotes aggressive cerebrovascular accumulation and leads to severe CAA in the mutation carriers; knowledge of how DutchAß40 drives this process more efficiently than Aß40 could reveal various pathophysiological events that promote CAA. In this study we have demonstrated that DutchAß40 shows preferential accumulation in the blood-brain-barrier (BBB) endothelial cells due to its inefficient blood-to-brain transcytosis. Consequently, DutchAß40 establishes a permeation barrier in the BBB endothelium, prevents its own clearance from the brain, and promotes the formation of amyloid deposits in the cerebral microvessels. The BBB endothelial accumulation of native Aß40 is not robust enough to exercise such a significant impact on its brain clearance. Hence, the cerebrovascular accumulation of Aß40 is slow and may require other copathologies to precipitate into CAA. In conclusion, the magnitude of Aß accumulation in the BBB endothelial cells is a critical factor that promotes CAA; hence, clearing vascular endothelium of Aß proteins may halt or even reverse CAA.

Delivery of RNA to the Blood-Brain Barrier Endothelium Using Cationic Bicelles.

Blood-brain barrier (BBB) dysfunction is prevalent in Alzheimer's disease and other neurological disorders. Restoring normal BBB function through RNA therapy is a potential avenue for addressing cerebrovascular changes in these disorders that may lead to cognitive decline. Although lipid nanoparticles have been traditionally used as drug carriers for RNA, bicelles have been emerging as a better alternative because of their higher cellular uptake and superior transfection capabilities. Cationic bicelles composed of DPPC/DC7PC/DOTAP at molar ratios of 63.8/25.0/11.2 were evaluated for the delivery of RNA in polarized hCMEC/D3 monolayers, a widely used BBB cell culture model. RNA-bicelle complexes were formed at five N/P ratios (1:1 to 5:1) by a thin-film hydration method. The RNA-bicelle complexes at N/P ratios of 3:1 and 4:1 exhibited optimal particle characteristics for cellular delivery. The cellular uptake of cationic bicelles laced with 1 mol% DiI-C18 was confirmed by flow cytometry and confocal microscopy. The ability of cationic bicelles (N/P ratio 4:1) to transfect polarized hCMEC/D3 with FITC-labeled control siRNA was tested vis-a-vis commercially available Lipofectamine RNAiMAX. These studies demonstrated the higher transfection efficiency and greater potential of cationic bicelles for RNA delivery to the BBB endothelium.

Alzheimer's disease amyloid ß-protein mutations and deletions that define neuronal binding/internalization as early stage nonfibrillar/fibrillar aggregates and late stage fibrils.

Accumulation of amyloid ß-protein (Aß) in neurons has been demonstrated to precede its formation as amyloid plaques in the extracellular space in Alzheimer's disease (AD) patients. Consequently, intraneuronal Aß accumulation is thought to be a critical first step in the fatal cascade of events that leads to neuronal degeneration in AD. Understanding the structural basis of neuronal binding and uptake of Aß might lead to potential therapeutic targets that could block this binding and the subsequent neurodegeneration that leads to the pathogenesis of AD. Previously, we demonstrated that mutation of the two adjacent histidine residues of Aß40 (H13,14G) resulted in a significant decrease in its level of binding to PC12 cells and mouse cortical/hippocampal neurons. We now demonstrate that the weakened neuronal binding follows the mutation order of H13G < H14G < H13,14G, which suggests that the primary domain for neuronal binding of Aß40 involves histidine at position 13. A novel APP mutation (E693Δ) that produced a variant Aß lacking glutamate 22 (E22Δ) in Japanese pedigrees was recently identified to have AD-type dementia without amyloid plaque formation but with extensive intraneuronal Aß in transfected cells and transgenic mice expressing this deletion. Deletion of glutamate 22 of Aß40 resulted in a 6-fold enhancement of PC12 neuronal binding that was not decreased by the H13G mutation. The dose-dependent enhanced binding of E22Δ explains the high level of intraneuronal Aß seen in this pedigree. Fluorescence anisotropy experiments at room temperature showed very rapid aggregation with increased tyrosine rigidity of Aß39E22Δ, Aß41E22Δ, and Aß42 but not Aß40. This rigidity was decreased but not eliminated by prior treatment with dimethyl sulfoxide. Surprisingly, all peptides showed an aggregated state when evaluated by transmission electron microscopy, with Aß39E22Δ having early stage fibrils, which was also verified by atomic force microscopy. This aggregation was not affected by centrifugation or pretreatment with organic solvents. The enhanced neuronal binding of Aß, therefore, results from aggregate binding to neurons, which requires H13 for Aß40 but not for E22Δ or Aß42. These latter proteins display increased tyrosine rigidity that likely masks the H13 residue, or alternatively, the H13 residue is not required for neuronal binding of these proteins as it is for Aß40. Late state fibrils also showed enhanced neuronal binding for E22Δ but not Aß40 with subsequent intraneuronal accumulation in lysosomes. This suggests that there are multiple pathways of binding/internalization for the different Aß proteins and their aggregation states or fibrillar structure.