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A once every eight-week cabotegravir (CAB) long-acting parenteral is more effective than daily oral emtricitabine and tenofovir disoproxil fumarate in preventing human immunodeficiency virus type one (HIV-1) transmission. Extending CAB dosing to a yearly injectable advances efforts for the elimination of viral transmission. Here we report rigor, reproducibility and mechanistic insights for a year-long CAB injectable. Pharmacokinetic (PK) profiles of this nanoformulated CAB prodrug (NM2CAB) are affirmed at three independent research laboratories. PK profiles in mice and rats show plasma CAB levels at or above the protein-adjusted 90% inhibitory concentration for a year after a single dose. Sustained native and prodrug concentrations are at the muscle injection site and in lymphoid tissues. The results parallel NM2CAB uptake and retention in human macrophages. NM2CAB nanocrystals are stable in blood and tissue homogenates. The long apparent drug half-life follows pH-dependent prodrug hydrolysis upon slow prodrug nanocrystal dissolution and absorption. In contrast, solubilized prodrug is hydrolyzed in hours in plasma and tissues from multiple mammalian species. No toxicities are observed in animals. These results affirm the pharmacological properties and extended apparent half-life for a nanoformulated CAB prodrug. The report serves to support the mechanistic design for drug formulation safety, rigor and reproducibility., Here, the authors provide a mechanism for an improved version of a nanoformulated myristoylated prodrug of cabotegravir (CAB), named NM2CAB, and its bioavailability, stability and pharmacokinetics in mice and rats performed in independent academic and a contracted research labs, suggesting that the extended half-life of the prodrug is not a property of enzymatic hydrolysis but rather release or dissolution of the prodrug from the nanocrystal.

Long-acting antiretrovirals can improve therapy and prevention for HIV-1 infection. Current long-acting cabotegravir (CAB LAP) can be administered every other month. Previously, we demonstrated that a myristoylated CAB prodrug encased in poloxamer 407 provided extended plasma drug concentrations. We now demonstrate that this first-generation nanoformulated prodrug can sustain plasma CAB concentrations above the protein-adjusted 90% inhibitory concentration for 4 months in rhesus macaques. A 2.5-fold extension in CAB half-life and a 1.6-fold increase in area under the concentration-time curve were observed compared with CAB LAP.

Long-acting cabotegravir (CAB) extends antiretroviral drug administration from daily to monthly. However, dosing volumes, injection site reactions and health-care oversight are obstacles towards a broad usage. The creation of poloxamer-coated hydrophobic and lipophilic CAB prodrugs with controlled hydrolysis and tissue penetrance can overcome these obstacles. To such ends, fatty acid ester CAB nanocrystal prodrugs with 14, 18 and 22 added carbon chains were encased in biocompatible surfactants named NMCAB, NM2CAB and NM3CAB and tested for drug release, activation, cytotoxicity, antiretroviral activities, pharmacokinetics and biodistribution. Pharmacokinetics studies, performed in mice and rhesus macaques, with the lead 18-carbon ester chain NM2CAB, showed plasma CAB levels above the protein-adjusted 90% inhibitory concentration for up to a year. NM2CAB, compared with NMCAB and NM3CAB, demonstrated a prolonged drug release, plasma circulation time and tissue drug concentrations after a single 45 mg per kg body weight intramuscular injection. These prodrug modifications could substantially improve CAB’s effectiveness. Nanoformulated long-acting cabotegravir prodrugs are shown to be capable of extending the native drug’s antiretroviral activity, biodistribution and pharmacokinetics for up to 12 months in mice and rhesus macaques.

Long-acting parenteral (LAP) antiretroviral drugs have generated considerable interest for treatment and prevention of HIV-1 infection. One new LAP is cabotegravir (CAB), a highly potent integrase inhibitor, with a half-life of up to 54 days, allowing for every other month parenteral administrations. Despite this excellent profile, high volume dosing, injection site reactions and low body fluid drug concentrations affect broad use for virus infected and susceptible people. To improve the drug delivery profile, we created a myristoylated CAB prodrug (MCAB). MCAB formed crystals that were formulated into nanoparticles (NMCAB) of stable size and shape facilitating avid monocyte-macrophage entry, retention and reticuloendothelial system depot formulation. Drug release kinetics paralleled sustained protection against HIV-1 challenge. After a single 45 mg/kg intramuscular injection to BALB/cJ mice, the NMCAB pharmacokinetic profiles was 4-times greater than that recorded for CAB LAP. These observations paralleled replicate measurements in rhesus macaques. The results coupled with improved viral restriction in human adult lymphocyte reconstituted NOD/SCID/IL2Rγc−/− mice led us to conclude that NMCAB can improve biodistribution and viral clearance profiles upon current CAB LAP formulations.

Antiretroviral therapy requires lifelong daily dosing to attain viral suppression, restore immune function, and improve quality of life. As a treatment alternative, long-acting (LA) antiretrovirals can sustain therapeutic drug concentrations in blood for prolonged time periods. The success of recent clinical trials for LA parenteral cabotegravir and rilpivirine highlight the emergence of these new therapeutic options. Further optimization can improve dosing frequency, lower injection volumes, and facilitate drug-tissue distributions. To this end, we report the synthesis of a library of RPV prodrugs designed to sustain drug plasma concentrations and improved tissue biodistribution. The lead prodrug M3RPV was nanoformulated into the stable LA injectable NM3RPV. NM3RPV treatment led to RPV plasma concentrations above the protein-adjusted 90% inhibitory concentration for 25 weeks with substantial tissue depots after a single intramuscular injection in BALB/cJ mice. NM3RPV elicited 13- and 26-fold increases in the RPV apparent half-life and mean residence time compared to native drug formulation. Taken together, proof-of-concept is provided that nanoformulated RPV prodrugs can extend the apparent drug’s half-life and improve tissue biodistribution. These results warrant further development for human use.

Although the combination of highly active antiretroviral therapy (cART) can remarkably control human immunodeficiency virus type-1 (HIV-1) replication, it fails to cure HIV/AIDS disease. It is attributed to the incapability of cART to eliminate persistent HIV-1 contained in latent reservoirs in the central nervous system (CNS) and other tissue organs. Thus, withdrawal of cART causes rebound viral replication and resurgent of HIV/AIDS. The lack of success on non-ART approaches for elimination of HIV-1 include the targeted molecules not reaching the CNS, not adjusting well with drug-resistant mutants, or unable to eliminate all components of viral life cycle. Here, we show that our newly discovered Drug-S can effectively inhibit HIV-1 infection and persistence at the low concentration without causing any toxicity to neuroimmune cells. Our results suggest that Drug-S may have a direct effect on viral structure, prevent rebounding of HIV-1 infection, and arrest progression into acquired immunodeficiency syndrome. We also observed that Drug-S is capable of crossing the blood-brain barrier, suggesting a potential antiretroviral drug for elimination of CNS viral reservoirs and self-renewal of residual HIV-1. These results outlined the possible mechanism(s) of action of Drug-S as a novel antiretroviral drug for elimination of HIV-1 replication by interfering the virion structure.

Wernicke's encephalopathy, a common neurological disease, is caused by thiamine (vitamin B1) deficiency. Neuropathy resulting from thiamine deficiency is a hallmark of Wernicke-Korsakoff syndrome in chronic alcohol users. The underlying mechanisms of this deficiency and progression of neuropathy remain to be understood. To uncover the unknown mechanisms of thiamine deficiency in alcohol abuse, we used chronic alcohol consumption or thiamine deficiency diet ingestion in animal models. Observations from animal models were validated in primary human neuronal culture for neurodegenerative process. We employed radio-labeled bio-distribution of thiamine, qualitative and quantitative analyses of the various biomarkers and neurodegenerative process. In the present studies, we established that disruption of thiamine transport across the intestinal gut blood-brain barrier axis as the cause of thiamine deficiency in the brain for neurodegeneration. We found that reduction in thiamine transport across these interfaces was the cause of reduction in the synthesis of thiamine pyrophosphate (TPP), an active cofactor for pyruvate dehydrogenase E1α (PDHE1α). Our findings revealed that decrease in the levels of PDHE1α cofactors switched on the activation of PD kinase (PDK) in the brain, thereby triggering the neuronal phosphorylation of PDHE1α (p-PDHE1α). Dysfunctional phosphorylated PDHE1α causes the reduction of mitochondrial aerobic respiration that led to neurodegeneration. We concluded that impairment of thiamine transport across the gut-BBB-axis that led to insufficient TPP synthesis was critical to Wernicke-neuropathy, which could be effectively prevented by stabilizing the thiamine transporters.

Evidence shows that alcohol intake causes oxidative neuronal injury and neurocognitive deficits that are distinct from the classical Wernicke-Korsakoff neuropathy. Our previous findings indicated that alcohol-elicited blood-brain barrier (BBB) damage leads to neuroinflammation and neuronal loss. The dynamic function of the BBB requires a constant supply and utilization of glucose. Here we examined whether interference of glucose uptake and transport at the endothelium by alcohol leads to BBB dysfunction and neuronal degeneration.We tested the hypothesis in cell culture of human brain endothelial cells, neurons and alcohol intake in animal by immunofluorescence, Western blotting and glucose uptake assay methods.We found that decrease in glucose uptake correlates the reduction of glucose transporter protein 1 (GLUT1) in cell culture after 50 mM ethanol exposure. Decrease in GLUT1 protein levels was regulated at the translation process. In animal, chronic alcohol intake suppresses the transport of glucose into the frontal and occipital regions of the brain. This finding is validated by a marked decrease in GLUT1 protein expression in brain microvessel (the BBB). In parallel, alcohol intake impairs the BBB tight junction proteins occludin, zonula occludens-1, and claudin-5 in the brain microvessel. Permeability of sodium fluorescein and Evans Blue confirms the leakiness of the BBB. Further, depletion of trans-endothelial electrical resistance of the cell monolayer supports the disruption of BBB integrity. Administration of acetyl-L: -carnitine (a neuroprotective agent) significantly prevents the adverse effects of alcohol on glucose uptake, BBB damage and neuronal degeneration.These findings suggest that alcohol-elicited inhibition of glucose transport at the blood-brain interface leads to BBB malfunction and neurological complications.

The studies presented here demonstrate the protective effect of acetyl-L-carnitine (ALC) against alcohol-induced oxidative neuroinflammation, neuronal degeneration, and impaired neurotransmission. Our findings reveal the cellular and biochemical mechanisms of alcohol-induced oxidative damage in various types of brain cells. Chronic ethanol administration to mice caused an increase in inducible nitric oxide synthase (iNOS) and 3-nitrotyrosine adduct formation in frontal cortical neurons but not in astrocytes from brains of these animals. Interestingly, alcohol administration caused a rather selective activation of NADPH oxidase (NOX), which, in turn, enhanced levels of reactive oxygen species (ROS) and 4-hydroxynonenal, but these were predominantly localized in astrocytes and microglia. Oxidative damage in glial cells was accompanied by their pronounced activation (astrogliosis) and coincident neuronal loss, suggesting that inflammation in glial cells caused neuronal degeneration. Immunohistochemistry studies indicated that alcohol consumption induced different oxidative mediators in different brain cell types. Thus, nitric oxide was mostly detected in iNOS-expressing neurons, whereas ROS were predominantly generated in NOX-expressing glial cells after alcohol ingestion. Assessment of neuronal activity in ex vivo frontal cortical brain tissue slices from ethanol-fed mice showed a reduction in long-term potentiation synaptic transmission compared with slices from controls. Coadministration of ALC with alcohol showed a significant reduction in oxidative damage and neuronal loss and a restoration of synaptic neurotransmission in this brain region, suggesting that ALC protects brain cells from ethanol-induced oxidative injury. These findings suggest the potential clinical utility of ALC as a neuroprotective agent that prevents alcohol-induced brain damage and development of neurological disorders.

Computer-aided tomography is commonly used for biomedical imaging in medicine and research. Most systems utilize some form of energized probe to either visualize tissues and/or localize a labeled compound. Common to all forms of tomographic imaging is the acquisition of cross-sectional images in a 360° rotation. Computer-interfaced software is used to reconstruct two-dimensional images into a three-dimensional representation. This allows selected two-dimensional images to be analyzed in any axis. In computed tomography (CT), also called computer-aided tomography or computerized axial tomography, X-rays produced by a cathode ray tube provide an external source of radiation that passes through the subject and are collected by a detector array. Density variations of different organs, tissues, and structures allow different amounts of radiation to penetrate and reach the detector to create an anatomical image.

Current research into neurodegenerative disorders relies heavily on animal models that recapitulate human disease. This chapter focuses on genetic and toxin-based animal models of Parkinson’s disease (PD) and amyotrophic lateral sclerosis (ALS). Early models failed to duplicate many of the hallmarks of these diseases, such as the loss of dopaminergic neurons in PD and motor neuron loss in ALS, and were often dismissed as newer models were developed. Nevertheless, even current models may not fully mimic all facets of disease etiology, progression, or pathology, necessitating continued research into developing models that more closely replicate the clinical manifestations of the disease. For both PD and ALS, genetic models have been generated based on the clinical reports of familial forms of the disease. For PD, models have been created using genes coding for α-synuclein, parkin, LRRK2, DJ-1, and PINK1, while mutations in genes coding for SOD1, FUS/TLS, and TDP-43 have been used for ALS models. For either disease, fewer than 10 % of all cases are linked to genetic causes, whereas the majority of cases are termed sporadic and are likely caused by environmental factors or a combination of environmental factors and genetically imposed susceptibilities. Therefore, a large number of animal models are toxin-based, utilizing chemicals or metals that have been correlated with human disease. As a whole, current animal models serve as adequate substitutes to their human counterparts, providing valuable tools for translational and basic science research in PD and ALS, with the caveat that most models typically do not recapitulate the totality of the human disease. Therefore, this chapter intends to describe the more commonly used animal models for these diseases and relate their limitations.

Objective— Blood-brain barrier (BBB) dysfunction caused by activation of matrix metalloproteinases (MMPs) is a pathological feature in vascular/neurological disease. We describe the mechanisms of BBB dysfunction and neuroinflammation as a result of MMP-3/9 activation and disruption of vascular endothelial growth factor (VEGF)-A/VEGFR-2 interaction, impairing effective angiogenesis. Methods and Results— We investigate the hypothesis in human brain endothelial cells and animal model of chronic alcohol ingestion. Proteome array analysis, zymography, immunofluorescence, and Western blotting techniques detected the activation, expression, and levels of MMP-3 and MMP-9. We found that degradation of VEGFR-2 and BBB proteins, for example, occludin, claudin-5, and ZO-1 by MMP-3/9, causes rupture of capillary endothelium and BBB leakiness. Impairment of BBB integrity was demonstrated by increased permeability of dye tracers and Fluo-3/calcein-AM–labeled monocyte adhesion or infiltration and decrease in transendothelial electric resistance. Alcohol-induced degradation of endothelial VEGFR-2 by MMP-3/9 led to a subsequent elevation of cellular/serum VEGF-A level. The decrease in VEGFR-2 with subsequent increase in VEGF-A level led to apoptosis and neuroinflammation via the activation of caspase-1 and IL-1β release. The use of MMPs, VEGFR-2, and caspase-1 inhibitors helped to dissect the underlying mechanisms. Conclusion— Alcohol-induced MMPs activation is a key mechanism for dysfunction of BBB via degradation of VEGFR-2 protein and activation of caspase-1 or IL-1β release. Targeting VEGF-induced MMP-3/9 activation can be a novel preventive approach to vascular inflammatory disease in alcohol abuse.

Background Methamphetamine (METH), an addictive psycho-stimulant drug with euphoric effect is known to cause neurotoxicity due to oxidative stress, dopamine accumulation and glial cell activation. Here we hypothesized that METH-induced interference of glucose uptake and transport at the endothelium can disrupt the energy requirement of the blood-brain barrier (BBB) function and integrity. We undertake this study because there is no report of METH effects on glucose uptake and transport across the blood-brain barrier (BBB) to date. Results In this study, we demonstrate that METH-induced disruption of glucose uptake by endothelium lead to BBB dysfunction. Our data indicate that a low concentration of METH (20 μM) increased the expression of glucose transporter protein-1 (GLUT1) in primary human brain endothelial cell (hBEC, main component of BBB) without affecting the glucose uptake. A high concentration of 200 μM of METH decreased both the glucose uptake and GLUT1 protein levels in hBEC culture. Transcription process appeared to regulate the changes in METH-induced GLUT1 expression. METH-induced decrease in GLUT1 protein level was associated with reduction in BBB tight junction protein occludin and zonula occludens-1. Functional assessment of the trans-endothelial electrical resistance of the cell monolayers and permeability of dye tracers in animal model validated the pharmacokinetics and molecular findings that inhibition of glucose uptake by GLUT1 inhibitor cytochalasin B (CB) aggravated the METH-induced disruption of the BBB integrity. Application of acetyl-L-carnitine suppressed the effects of METH on glucose uptake and BBB function. Conclusion Our findings suggest that impairment of GLUT1 at the brain endothelium by METH may contribute to energy-associated disruption of tight junction assembly and loss of BBB integrity.

Alcohol consumption causes neurocognitive deficits, neuronal injury, and neurodegeneration. At the cellular level, alcohol abuse causes oxidative damage to mitochondria and cellular proteins and interlink with the progression of neuroinflammation and neurological disorders. We previously reported that alcohol inhibits glucose transport across the blood-brain barrier (BBB), leading to BBB dysfunction and neurodegeneration. In this study, we hypothesized that ethanol (EtOH)-mediated disruption in glucose uptake would deprive energy for human astrocytes and neurons inducing neurotoxicity and neuronal degeneration. EtOH may also have a direct effect on glucose uptake in neurons and astrocytes, which has not been previously described. Our results indicate that ethanol exposure decreases the uptake of D-(2-(3)H)-glucose by human astrocytes and neurons. Inhibition of glucose uptake correlates with a reduction in glucose transporter protein expression (GLUT1 in astrocytes and GLUT3 in neurons). Acetyl-L-carnitine (ALC), a neuroprotective agent, suppresses the effects of alcohol on glucose uptake and GLUT levels, thus reducing neurotoxicity and neuronal degeneration. These findings suggest that deprivation of glucose in brain cells contributes to neurotoxicity in alcohol abusers, and highlights ALC as a potential therapeutic agent to prevent the deleterious health conditions caused by alcohol abuse.

Microvessels, the main components of the blood-brain barrier (BBB) are vulnerable to oxidative damage during alcohol-induced stress. Alcohol produces oxidative damage within the vessels and in the brain. Using our animal model of catheter implant into the common carotid artery (CCA), we trace the footprints of alcohol-induced oxidative damage and inflammatory process at the BBB and into the brain. The uniqueness of the finding is that ethanol causes oxidative damage in all neurovascular components by activating NADPH oxidase and inducible nitric oxide synthase in the brain. It is not the oxidants but the ethanol that traverses through the BBB because we found that the highly reactive peroxynitrite does not cross the BBB. Thus, oxidative damage is caused at the site of oxidant production in the microvessels and in the brain. Our data indicate that acetaldehyde (the primary metabolite of ethanol) is the inducer/activator of these enzymes that generate oxidants in brain neurovascular cells. Evidence for alcohol-induced BBB damage is indicated by the alterations of the tight junction protein occludin in intact microvessels. Importantly, we demonstrate that the site of BBB oxidative damage is also the site of immune cells aggregation in the microvessels, which paves the path for inflammatory footprints. These findings reveal the underlying mechanisms that ethanol-elicited BBB oxidative damage initiates the brain vascular inflammatory process, which ultimately leads to neuroinflammation.

Methamphetamine (METH), an addictive psycho-stimulant drug exerts euphoric effects on users and abusers. It is also known to cause cognitive impairment and neurotoxicity. Here, we hypothesized that METH exposure impairs the glucose uptake and metabolism in human neurons and astrocytes. Deprivation of glucose is expected to cause neurotoxicity and neuronal degeneration due to depletion of energy. We found that METH exposure inhibited the glucose uptake by neurons and astrocytes, in which neurons were more sensitive to METH than astrocytes in primary culture. Adaptability of these cells to fatty acid oxidation as an alternative source of energy during glucose limitation appeared to regulate this differential sensitivity. Decrease in neuronal glucose uptake by METH was associated with reduction of glucose transporter protein-3 (GLUT3). Surprisingly, METH exposure showed biphasic effects on astrocytic glucose uptake, in which 20 µM increased the uptake while 200 µM inhibited glucose uptake. Dual effects of METH on glucose uptake were paralleled to changes in the expression of astrocytic glucose transporter protein-1 (GLUT1). The adaptive nature of astrocyte to mitochondrial β-oxidation of fatty acid appeared to contribute the survival of astrocytes during METH-induced glucose deprivation. This differential adaptive nature of neurons and astrocytes also governed the differential sensitivity to the toxicity of METH in these brain cells. The effect of acetyl-L-carnitine for enhanced production of ATP from fatty oxidation in glucose-free culture condition validated the adaptive nature of neurons and astrocytes. These findings suggest that deprivation of glucose-derived energy may contribute to neurotoxicity of METH abusers.