Extra-Virgin Olive Oil in Alzheimer's Disease: A Comprehensive Review of Cellular, Animal, and Clinical Studies.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is characterized by several pathological hallmarks, including the deposition of amyloid-ß (Aß) plaques, neurofibrillary tangles, blood-brain barrier (BBB) dysfunction, increased oxidative stress, and neuroinflammation. Current treatment options include monoclonal antibody drugs, acetylcholinesterase, and n-methyl-d-aspartate (NMDA) antagonists. Although those treatments provide some improvements in patients' quality of life, they fail to prevent or cure AD. Current research aims to identify novel targets and tools for AD prevention and modification. In this context, several studies showed the beneficial effect of the Mediterranean diet in the prevention and treatment of AD. One integral component of the Mediterranean diet is olive oil and extra-virgin olive oil (EVOO), which is high in phenolic compounds. EVOO and other olive-related phenolic compounds have been shown to reduce the risk of developing mild cognitive impairment (MCI) and AD. In this review, we discuss the mechanisms by which EVOO and phenolic compounds exert neuroprotective effects, including modulation of AD pathologies and promotion of cognitive health. Findings indicate that EVOO and its phenolic constituents influence key pathological processes of AD, such as Aß aggregation, tau phosphorylation, and neuroinflammation, while also enhancing BBB integrity and reducing oxidative stress. The human studies cited reveal a consistent trend where the consumption of olive oil is associated with cognitive benefits and a decreased risk of AD and related dementias. In conclusion, EVOO and its phenolic compounds hold promising potential for the prevention and treatment of AD, representing a significant shift towards more effective strategies against this complex neurodegenerative disorder.

Gentamicin Eluting 3D-Printed Implants for Preventing Post-Surgical Infections in Bone Fractures.

A surgically implantable device is an inevitable treatment option for millions of people worldwide suffering from diseases arising from orthopedic injuries. A global paradigm shift is currently underway to tailor and personalize replacement or reconstructive joints. Additive manufacturing (AM) has provided dynamic outflow to the customized fabrication of orthopedic implants by enabling need-based design and surface modification possibilities. Surgical grade 316L Stainless Steel (316L SS) is promising with its cost, strength, composition, and corrosion resistance to fabricate 3D implants. This work investigates the possibilities of application of the laser powder bed fusion (L-PBF) technique to fabricate 3D-printed (3DP) implants, which are functionalized with a multilayered antimicrobial coating to treat potential complications arising due to postsurgical infections (PSIs). Postsurgical implant-associated infection is a primary reason for implantation failure and is complicated mainly by bacterial colonization and biofilm formation at the installation site. PLGA (poly-d,l-lactide-co-glycolide), a biodegradable polymer, was utilized to impart multiple layers of coating using the airbrush spray technique on 3DP implant surfaces loaded with gentamicin (GEN). Various PLGA-based polymers were tested to optimize the ideal lactic acid: glycolic acid ratio and molecular weight suited for our investigation. 3D-Printed PLGA-GEN substrates sustained the release of gentamicin from the surface for approximately 6 weeks. The 3DP surface modification with PLGA-GEN facilitated cell adhesion and proliferation compared to control surfaces. The cell viability studies showed that the implants were safe for application. The 3DP PLGA-GEN substrates showed good concentration-dependent antibacterial efficacy against the common PSI pathogen Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis). The GEN-loaded substrates demonstrated antimicrobial longevity and showed significant biofilm growth inhibition compared to control. The substrates offered great versatility regarding the in vitro release rates, antimicrobial properties, and biocompatibility studies. These results radiate great potential in future human and veterinary clinical applications pertinent to complications arising from PSIs, focusing on personalized sustained antibiotic delivery.

The Blood-Brain Barrier in Health and Disease.

The blood-brain barrier (BBB) is a complex network of tightly regulated cells and transport proteins that separate the circulating blood from the brain tissue [...].

Blood-Brain Barrier Breakdown in Alzheimer's Disease: Mechanisms and Targeted Strategies.

The blood-brain barrier (BBB) is a unique and selective feature of the central nervous system's vasculature. BBB dysfunction has been observed as an early sign of Alzheimer's Disease (AD) before the onset of dementia or neurodegeneration. The intricate relationship between the BBB and the pathogenesis of AD, especially in the context of neurovascular coupling and the overlap of pathophysiology in neurodegenerative and cerebrovascular diseases, underscores the urgency to understand the BBB's role more deeply. Preserving or restoring the BBB function emerges as a potentially promising strategy for mitigating the progression and severity of AD. Molecular and genetic changes, such as the isoform ε4 of apolipoprotein E (ApoEε4), a significant genetic risk factor and a promoter of the BBB dysfunction, have been shown to mediate the BBB disruption. Additionally, receptors and transporters like the low-density lipoprotein receptor-related protein 1 (LRP1), P-glycoprotein (P-gp), and the receptor for advanced glycation end products (RAGEs) have been implicated in AD's pathogenesis. In this comprehensive review, we endeavor to shed light on the intricate pathogenic and therapeutic connections between AD and the BBB. We also delve into the latest developments and pioneering strategies targeting the BBB for therapeutic interventions, addressing its potential as a barrier and a carrier. By providing an integrative perspective, we anticipate paving the way for future research and treatments focused on exploiting the BBB's role in AD pathogenesis and therapy.

Oleocanthal Ameliorates Metabolic and Behavioral Phenotypes in a Mouse Model of Alzheimer's Disease.

Aging is a major risk factor for Alzheimer's disease (AD). AD mouse models are frequently used to assess pathology, behavior, and memory in AD research. While the pathological characteristics of AD are well established, our understanding of the changes in the metabolic phenotypes with age and pathology is limited. In this work, we used the Promethion cage systems® to monitor changes in physiological metabolic and behavioral parameters with age and pathology in wild-type and 5xFAD mouse models. Then, we assessed whether these parameters could be altered by treatment with oleocanthal, a phenolic compound with neuroprotective properties. Findings demonstrated metabolic parameters such as body weight, food and water intake, energy expenditure, dehydration, and respiratory exchange rate, and the behavioral parameters of sleep patterns and anxiety-like behavior are altered by age and pathology. However, the effect of pathology on these parameters was significantly greater than normal aging, which could be linked to amyloid-ß deposition and blood-brain barrier (BBB) disruption. In addition, and for the first time, our findings suggest an inverse correlation between sleep hours and BBB breakdown. Treatment with oleocanthal improved the assessed parameters and reduced anxiety-like behavior symptoms and sleep disturbances. In conclusion, aging and AD are associated with metabolism and behavior changes, with the changes being greater with the latter, which were rectified by oleocanthal. In addition, our findings suggest that monitoring changes in metabolic and behavioral phenotypes could provide a valuable tool to assess disease severity and treatment efficacy in AD mouse models.

Comparison of Oleocanthal-Low EVOO and Oleocanthal against Amyloid-ß and Related Pathology in a Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is characterized by several pathological hallmarks, including the deposition of amyloid-ß (Aß) plaques, neurofibrillary tangles, blood-brain barrier (BBB) dysfunction, and neuroinflammation. Growing evidence support the neuroprotective effects of extra-virgin olive oil (EVOO) and oleocanthal (OC). In this work, we aimed to evaluate and compare the beneficial effects of equivalent doses of OC-low EVOO (0.5 mg total phenolic content/kg) and OC (0.5 mg OC/kg) on Aß and related pathology and to assess their effect on neuroinflammation in a 5xFAD mouse model with advanced pathology. Homozygous 5xFAD mice were fed with refined olive oil (ROO), OC-low EVOO, or OC for 3 months starting at the age of 3 months. Our findings demonstrated that a low dose of 0.5 mg/kg EVOO-phenols and OC reduced brain Aß levels and neuroinflammation by suppressing the nuclear factor-κB (NF-κB) pathway and reducing the activation of NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasomes. On the other hand, only OC suppressed the receptor for advanced glycation endproducts/high-mobility group box 1 (RAGE/HMGB1) pathway. In conclusion, our results indicated that while OC-low EVOO demonstrated a beneficial effect against Aß-related pathology in 5xFAD mice, EVOO rich with OC could provide a higher anti-inflammatory effect by targeting multiple mechanisms. Collectively, diet supplementation with EVOO or OC could prevent, halt progression, and treat AD.

Glial Cell-Mediated Neuroinflammation in Alzheimer's Disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder; it is the most common cause of dementia and has no treatment. It is characterized by two pathological hallmarks, the extracellular deposits of amyloid beta (Aß) and the intraneuronal deposits of Neurofibrillary tangles (NFTs). Yet, those two hallmarks do not explain the full pathology seen with AD, suggesting the involvement of other mechanisms. Neuroinflammation could offer another explanation for the progression of the disease. This review provides an overview of recent advances on the role of the immune cells' microglia and astrocytes in neuroinflammation. In AD, microglia and astrocytes become reactive by several mechanisms leading to the release of proinflammatory cytokines that cause further neuronal damage. We then provide updates on neuroinflammation diagnostic markers and investigational therapeutics currently in clinical trials to target neuroinflammation.

Blood-Brain Barrier Disruption Increases Amyloid-Related Pathology in TgSwDI Mice.

In Alzheimer's disease (AD), several studies have reported blood-brain barrier (BBB) breakdown with compromised function. P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are transport proteins localized at the BBB luminal membrane and play an important role in the clearance of amyloid-ß (Aß). The purpose of this study was to investigate the effect of pharmacological inhibition of Aß efflux transporters on BBB function and Aß accumulation and related pathology. Recently, we have developed an in vitro high-throughput screening assay to screen for compounds that modulate the integrity of a cell-based BBB model, which identified elacridar as a disruptor of the monolayer integrity. Elacridar, an investigational compound known for its P-gp and BCRP inhibitory effect and widely used in cancer research. Therefore, it was used as a model compound for further evaluation in a mouse model of AD, namely TgSwDI. TgSwDI mouse is also used as a model for cerebral amyloid angiopathy (CAA). Results showed that P-gp and BCRP inhibition by elacridar disrupted the BBB integrity as measured by increased IgG extravasation and reduced expression of tight junction proteins, increased amyloid deposition due to P-gp, and BCRP downregulation and receptor for advanced glycation end products (RAGE) upregulation, increased CAA and astrogliosis. Further studies revealed the effect was mediated by activation of NF-κB pathway. In conclusion, results suggest that BBB disruption by inhibiting P-gp and BCRP exacerbates AD pathology in a mouse model of AD, and indicate that therapeutic drugs that inhibit P-gp and BCRP could increase the risk for AD.

Multi-faceted therapeutic strategy for treatment of Alzheimer's disease by concurrent administration of etodolac and α-tocopherol.

Alzheimer's disease (AD) is a complex neurodegenerative disorder with multiple dysfunctional pathways. Therefore, a sophisticated treatment strategy that simultaneously targets multiple brain cell types and disease pathways could be advantageous for effective intervention. To elucidate an effective treatment, we developed an in vitro high-throughput screening (HTS) assay to evaluate candidate drugs for their ability to enhance the integrity of the blood-brain barrier (BBB) and improve clearance of amyloid-ß (Aß) using a cell-based BBB model. Results from HTS identified etodolac and α-tocopherol as promising drugs for further investigation. Both drugs were tested separately and in combination for the purpose of targeting multiple pathways including neuroinflammation and oxidative stress. In vitro studies assessed the effects of etodolac and α-tocopherol individually and collectively for BBB integrity and Aß transport, synaptic markers and Aß production in APP-transfected neuronal cells, as well as effects on inflammation and oxidative stress in astrocytes. Transgenic 5XFAD mice were used to translate in vitro results of etodolac and α-tocopherol independently and with concurrent administration. Compared to either drug alone, the combination significantly enhanced the BBB function, decreased total Aß load correlated with increased expression of major transport proteins, promoted APP processing towards the neuroprotective and non-amyloidogenic pathway, induced synaptic markers expression, and significantly reduced neuroinflammation and oxidative stress both in vitro and in vivo. Collective findings demonstrated the combination produced mixed interaction showing additive, less than additive or synergistic effects on the evaluated markers. In conclusion, this study highlights the significance of combination therapy to simultaneously target multiple disease pathways, and suggest the repurposing and combination of etodolac and α-tocopherol as a novel therapeutic strategy against AD.

Physiologically Based Pharmacokinetic/Pharmacodynamic Model for Caffeine Disposition in Pregnancy.

Caffeine is the most consumed active stimulant. About 80% of pregnant women consume caffeine orally on a daily basis. Many reports indicated consumption of >200 mg caffeine during pregnancy could increase the likelihood of miscarriage. In this article, we developed a pregnancy physiological-based pharmacokinetic/pharmacodynamic (PBPK/PD) model for caffeine to examine association between maternal caffeine consumption during pregnancy and caffeine plasma levels at doses lower and higher than 200 mg to predict changes in caffeine concentrations across the 3 trimesters, and to predict associated changes in caffeine PD parameters. Two models were successfully developed using GastroPlus software, a nonpregnant model for validation purposes and a pregnant model for validation and prediction of maternal caffeine plasma concentrations following single and multiple dosing. Using observed and predicted data, we were able to validate and simulate PK changes of caffeine in nonpregnant women and the PD effect of caffeine on certain enzymes and catecholamines associated with caffeine intake. Furthermore, the pregnancy PBPK model successfully predicted changes in caffeine PK across the three trimesters. Caffeine increased exposure during pregnancy was related to reduced activity of caffeine metabolizing enzyme CYP1A2. The model also predicted increased levels of caffeine in the fetoplacental compartment (FPC) due to increased maternal caffeine plasma concentrations. Increased caffeine levels in maternal blood was accompanied by greater inhibition of the phosphodiesterase enzyme, higher cyclic adenosine monophosphate, and greater increase of epinephrine levels, which could increase the risk of pregnancy loss. The application of the developed PBPK model to predict the PD effect could provide a useful tool to help define potential cut-offs for caffeine intake in various stages of pregnancy.

Plasma Rich in Growth Factors (PRGF) Disrupt the Blood-Brain Barrier Integrity and Elevate Amyloid Pathology in the Brains of 5XFAD Mice.

Alzheimer's disease (AD) is the most common neurodegenerative disorder affecting 5.4 million people in the United States. Currently approved pharmacologic interventions for AD are limited to symptomatic improvement, not affecting the underlying pathology. Therefore, the search for novel therapeutic strategies is ongoing. A hallmark of AD is the compromised blood-brain barrier (BBB); thus, developing drugs that target the BBB to enhance its integrity and function could be a novel approach to prevent and/or treat AD. Previous evidence has shown the beneficial effects of growth factors in the treatment of AD pathology. Based on reported positive results obtained with the product Endoret®, the objective of this study was to investigate the effect of plasma rich in growth factors (PRGF) on the BBB integrity and function, initially in a cell-based BBB model and in 5x Familial Alzheimer's Disease (5xFAD) mice. Our results showed that while PRGF demonstrated a positive effect in the cell-based BBB model with the enhanced integrity and function of the model, the in-vivo findings showed that PRGF exacerbated amyloid pathology in 5xFAD brains. At 10 and 100% doses, PRGF increased amyloid deposition associated with increased apoptosis and neuroinflammation. In conclusion, our results suggest PRGF may not provide beneficial effects against AD and the consideration to utilize growth factors should further be investigated.

Role of P-glycoprotein in mediating rivastigmine effect on amyloid-ß brain load and related pathology in Alzheimer's disease mouse model.

Recently, we showed that rivastigmine decreased amyloid-ß (Aß) brain load in aged rats by enhancing its clearance across the blood-brain barrier (BBB) via upregulation of P-glycoprotein (P-gp) and low-density lipoprotein receptor-related protein 1 (LRP1). Here, we extend our previous work to clarify P-gp role in mediating rivastigmine effect on Aß brain levels and neuroprotection in a mouse model of Alzheimer's disease (AD) that expresses different levels of P-gp. APPSWE mice were bred with mdr1a/b knockout mice to produce littermates that were divided into three groups; APP(+)/mdr1(+/+), APP(+)/mdr1(+/-) and APP(+)/mdr1(-/-). Animals received rivastigmine treatment (0.3mg/kg/day) or vehicle for 8weeks using Alzet osmotic mini-pumps. ELISA analysis of brain homogenates for Aß showed rivastigmine treatment to significantly decrease Aß brain load in APP(+)/mdr1(+/+) by 25% and in APP(+)/mdr1(+/-) mice by 21% compared to their vehicle treated littermates, but not in APP(+)/mdr1(-/-) mice. In addition, rivastigmine reduced GFAP immunostaining of astrocytes by 50% and IL-1ß brain level by 43% in APP(+)/mdr1(+/+) mice, however its effect was less pronounced in P-gp knockout mice. Moreover, rivastigmine demonstrated a P-gp expression dependent neuroprotective effect that was highest in APP(+)/mdr1(+/+)>APP(+)/mdr1(+/-)>APP(+)/mdr1(-/-) as determined by expression of synaptic markers PSD-95 and SNAP-25 using Western blot analysis. Collectively, our results suggest that P-gp plays important role in mediating rivastigmine non-cholinergic beneficial effects, including Aß brain load reduction, neuroprotective and anti-inflammatory effects in the AD mouse models.

Development and qualification of an LC-MS/MS method for investigating the biological implications of micelle entrapped paclitaxel in cell culture and rats.

Paclitaxel is a front-line antineoplastic drug used in chemotherapeutic modalities for treatment of various types of malignancies. However, its efficacy is limited by dose-related toxicities. In this study, we have explored two important biological aspects of entrapping paclitaxel in PEG2000 -DSPE micelles. First, we evaluated the impact of this micellar delivery system on P-glycoprotein (P-gp)-paclitaxel interaction, and we investigated differences in plasma pharmacokinetics of free and micelle-entrapped paclitaxel. For quantification of paclitaxel, an LC-MS/MS method was developed. Paclitaxel was extracted from samples using a simple one-step protein precipitation. Chromatographic conditions included a C18 column with a mobile phase consisting of 0.1% formic acid in acetonitrile-water (60:40, v/v) pumped at 1 mL/min. The lower limit of quantitation in both plasma and cell lysate was 1.0 ng/mL. The quantitative linear range was 1-1000 ng/mL. In addition, P-gp efflux studies on free and micellar paclitaxel showed the proficiency of PEG2000 -DSPE micelles in evading P-gp-mediated efflux, thus increasing paclitaxel uptake. Furthermore, the micellar paclitaxel levels were maintained in the body for longer time as compared with taxol, which is desirable for increasing the efficacy of paclitaxel in cancer treatment.

Amyloid-ß and Astrocytes Interplay in Amyloid-ß Related Disorders.

Amyloid-ß (Aß) pathology is known to promote chronic inflammatory responses in the brain. It was thought previously that Aß is only associated with Alzheimer's disease and Down syndrome. However, studies have shown its involvement in many other neurological disorders. The role of astrocytes in handling the excess levels of Aß has been highlighted in the literature. Astrocytes have a distinctive function in both neuronal support and protection, thus its involvement in Aß pathological process may tip the balance toward chronic inflammation and neuronal death. In this review we describe the involvement of astrocytes in Aß related disorders including Alzheimer's disease, Down syndrome, cerebral amyloid angiopathy, and frontotemporal dementia.

Mixed oligomers and monomeric amyloid-ß disrupts endothelial cells integrity and reduces monomeric amyloid-ß transport across hCMEC/D3 cell line as an in vitro blood-brain barrier model.

Senile amyloid plaques are one of the diagnostic hallmarks of Alzheimer's disease (AD). However, the severity of clinical symptoms of AD is weakly correlated with the plaque load. AD symptoms severity is reported to be more strongly correlated with the level of soluble amyloid-ß (Aß) assemblies. Formation of soluble Aß assemblies is stimulated by monomeric Aß accumulation in the brain, which has been related to its faulty cerebral clearance. Studies tend to focus on the neurotoxicity of specific Aß species. There are relatively few studies investigating toxic effects of Aß on the endothelial cells of the blood-brain barrier (BBB). We hypothesized that a soluble Aß pool more closely resembling the in vivo situation composed of a mixture of Aß40 monomer and Aß42 oligomer would exert higher toxicity against hCMEC/D3 cells as an in vitro BBB model than either component alone. We observed that, in addition to a disruptive effect on the endothelial cells integrity due to enhancement of the paracellular permeability of the hCMEC/D3 monolayer, the Aß mixture significantly decreased monomeric Aß transport across the cell culture model. Consistent with its effect on Aß transport, Aß mixture treatment for 24h resulted in LRP1 down-regulation and RAGE up-regulation in hCMEC/D3 cells. The individual Aß species separately failed to alter Aß clearance or the cell-based BBB model integrity. Our study offers, for the first time, evidence that a mixture of soluble Aß species, at nanomolar concentrations, disrupts endothelial cells integrity and its own transport across an in vitro model of the BBB.

Synthesis and P-glycoprotein induction activity of colupulone analogs.

Brain amyloid-beta (Aß) plaques are one of the primary hallmarks associated with Alzheimer's disease (AD) pathology. Efflux pump proteins located at the blood-brain barrier (BBB) have been reported to play an important role in the clearance of brain Aß, among which the P-glycoprotein (P-gp) efflux transporter pump has been shown to play a crucial role. Thus, P-gp has been considered as a potential therapeutic target for treatment of AD. Colupulone, a prenylated phloroglucinol isolated from Humulus lupulus, is known to activate pregnane-X-receptor (PXR), which is a nuclear receptor controlling P-gp expression. In the present work, we aimed to synthesize and identify analogs of colupulone that are potent P-gp inducer(s) with an ability to enhance Aß transport across the BBB. A series of colupulone analogs were synthesized by modifications at both prenyl as well as acyl domains. All compounds were screened for P-gp induction activity using a rhodamine 123 based efflux assay in the P-gp overexpressing human adenocarcinoma LS-180 cells, wherein all compounds showed significant P-gp induction activity at 5 µM. In the western blot studies in LS-180 cells, compounds 3k and 5f were able to induce P-gp as well as LRP1 at 1 µM. The effect of compounds on the Aß uptake and transport was then evaluated. Among all tested compounds, diprenylated acyl phloroglucinol displayed a significant increase (29%) in Aß transport across bEnd3 cells grown on inserts as a BBB model. The results presented here suggest the potential of this scaffold to enhance clearance of brain Aß across the BBB and thus its promise for development as a potential anti-Alzheimer agent.

Development of a physiologically based pharmacokinetic/pharmacodynamic model to identify mechanisms contributing to entacapone low bioavailability.

Entacapone is an inhibitor of catechol-O-methyltransferase (COMT) and is being used to extend the therapeutic effect of levodopa in patients with advanced and fluctuating Parkinson's disease. Entacapone has low and variable oral bioavailability and the underlying mechanism(s) for this behavior have not been studied. To explain such behavior and to characterize the dynamic changes in the metabolism of entacapone, a physiologically based pharmacokinetic/pharmacodynamic (PBPK/PD) model was developed integrating in silico, in vitro and in vivo pharmacokinetic data. The model was developed and verified in healthy volunteers and subsequently expanded to predict the pharmacokinetic parameters of entacapone phosphate, a prodrug of entacapone, and to assess the impact of hepatic impairment on the pharmacokinetics of entacapone. Low and inter-individual variability in bioavailability could be attributed to the extensive first-pass metabolism by UGTs in the liver and, to a lesser extent, the small intestine. The predictive performance of this model was acceptable with predicted Cmax , AUC and PD parameters lying within 20% of the observed data. The model indicates that the low bioavailability could be attributed to the extensive first-pass effect of entacapone.

Tacrine sinusoidal uptake and biliary excretion in sandwich-cultured primary rat hepatocytes.

PURPOSE. The knowledge of hepatic disposition kinetics of tacrine, a first cholinesterase inhibitor was approved by FDA for the treatment of Alzheimer's disease (AD), would help to understand its hepatotoxicity, its therapeutic effect, and improve the management of patients with AD. The current study aims to characterize tacrine hepatic transport kinetics and study the role of organic cation transporters (OCTs), P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP2) in tacrine sinusoidal uptake and biliary excretion. METHODS. Modulation of tacrine hepatic uptake and efflux, biliary excretion index (BEI%), were performed in sandwich-cultured primary rat hepatocytes (SCHs) using transporters inhibitors. Conformation of the integrity of SCHs model was established by capturing images with light-contrast and fluorescence microscopy. RESULTS. Tacrine uptake in SCHs was carrier-mediated process and saturable with apparent Km of 31.5±9.6 µM and Vmax of 908±72 pmol/min/mg protein. Tetraethyl ammonium (TEA), cimetidine and verapamil significantly reduced tacrine uptake with more pronounced effect observed with verapamil which caused 3-fold reduction in tacrine uptake, indicating role for OCTs. Tacrine has a biliary excretion in SCHs with maximum BEI% value of 22.9±1.9% at 10 min of incubation. Addition of MK571 and valspodar decreased the BEI% of tacrine by 40 and 60% suggesting roles for canalicular MRP2 and P-gp, respectively. CONCLUSIONS. Our results show that in addition to metabolism, tacrine hepatic disposition is carrier-mediated process mediated by sinusoidal OCTs, and canalicular MRP2 and P-gp.

The Usnic Acid Analogue 4-FPBUA Enhances the Blood-Brain Barrier Function and Induces Autophagy in Alzheimer's Disease Mouse Models.

Preclinical and clinical studies have indicated that compromised blood-brain barrier (BBB) function contributes to Alzheimer's disease (AD) pathology. BBB breakdown ranged from mild disruption of tight junctions (TJs) with increased BBB permeability to chronic integrity loss, affecting transport across the BBB, reducing brain perfusion, and triggering inflammatory responses. We recently developed a high-throughput screening (HTS) assay to identify hit compounds that enhance the function of a cell-based BBB model. The HTS screen identified (S,E)-2-acetyl-6-[3-(4'-fluorobiphenyl-4-yl)acryloyl]-3,7,9-trihydroxy-8,9b-dimethyldibenzo-[b,d]furan-1(9bH)-one (4-FPBUA), a semisynthetic analogue of naturally occurring usnic acid, which protected the in vitro model against Aß toxicity. Usnic acid is a lichen-derived secondary metabolite with a unique dibenzofuran skeleton that is commonly found in lichenized fungi of the genera Usnea. In this study, we aimed to evaluate the effect of 4-FPBUA in vitro on the cell-based BBB model function and its in vivo ability to rectify BBB function and reduce brain Aß in two AD mouse models, namely, 5xFAD and TgSwDI. Our findings demonstrated that 4-FPBUA enhanced cell-based BBB function, increased Aß transport across the monolayer, and reversed BBB breakdown in vivo by enhancing autophagy as an mTOR inhibitor. Induced autophagy was associated with a significant reduction in Aß accumulation and related pathologies and improved memory function. These results underscore the potential of 4-FPBUA as a candidate for further preclinical exploration to better understand its mechanisms of action and to optimize dosing strategies. Continued research may also elucidate additional pathways through which 4-FPBUA contributed to the amelioration of BBB dysfunction in AD. Collectively, our findings supported the development of 4-FPBUA as a therapeutic agent against AD.

In vitro investigation of amyloid-ß hepatobiliary disposition in sandwich-cultured primary rat hepatocytes.

Failure in amyloid-ß (Aß) systemic clearance across the liver has been suggested to play a role in Aß brain accumulation and thus to contribute largely to the pathology of Alzheimer's disease (AD). The purpose of this study was to characterize in vitro the transport mechanisms of Aß40 across the liver using sandwich-cultured primary rat hepatocytes (SCHs) and to determine its biliary clearance (CL(bile)) and biliary excretion index (BEI%). ¹²5I-Aß40 BEI% was time dependent and reached steady state at 30 minutes, with an average value of 29.8% and a CL(bile) of 1.47 ml/min per kilogram of body weight. The role of low-density lipoprotein receptor-related protein-1 (LRP1) in mediating the basolateral uptake of ¹²5I-Aß40 in SCHs was assessed using receptor-associated protein (RAP, 2 µM). A significant reduction in ¹²5I-Aß40 BEI% and CL(bile) with RAP was observed, demonstrating a major contribution of LRP1 in mediating hepatic uptake of intact ¹²5I-Aß40 via transcytosis. Furthermore, activity studies suggested a lower role of receptor for advanced glycation end products (RAGE) in ¹²5I-Aß40 hepatic uptake. Verapamil (50 µM) and valspodar (20 µM) significantly reduced ¹²5I-Aß40 BEI%, indicating a role for P-glycoprotein (P-gp) in the biliary excretion of ¹²5I-Aß40 in SCHs. LRP1- and P-gp-mediated ¹²5I-Aß40 biliary excretion was inducible and increased BEI% by 26% after rifampicin pretreatment. In conclusion, our findings demonstrated that besides LRP1, P-gp and, to a lesser extent, RAGE are involved in ¹²5I-Aß40 hepatobiliary disposition and support the use of enhancement of Aß hepatic clearance via LRP1 and P-gp induction as a novel therapeutic approach for the prevention and treatment of AD.