The analgesic and antipyretic drug acetaminophen (APAP) is bioactivated to the reactive intermediate N-acetyl-p-benzoquinoneimine, which is scavenged by glutathione (GSH). APAP overdose can deplete GSH leading to the accumulation of APAP-protein adducts and centrilobular necrosis in the liver. N-acetylcysteine (NAC), a cysteine prodrug and GSH precursor, is often given as a treatment for APAP overdose. The rate-limiting step in GSH biosynthesis is catalyzed by glutamate cysteine ligase (GCL) a heterodimer composed of catalytic and modifier (GCLM) subunits. Previous studies have indicated that GCL activity is likely to be an important determinant of APAP toxicity. In this study, we investigated APAP toxicity, and NAC or GSH ethyl ester (GSHee)-mediated rescue in mice with normal or compromised GCLM expression. Gclm wild-type, heterozygous, and null mice were administered APAP (500 mg/kg) alone, or immediately following NAC (800 mg/kg) or GSHee (168 mg/kg), and assessed for hepatotoxicity 6 h later. APAP caused GSH depletion in all mice. Gclm null and heterozygous mice exhibited more extensive hepatic damage compared to wild-type mice as assessed by serum alanine aminotransferase activity and histopathology. Additionally, male Gclm wild-type mice demonstrated greater APAP-induced hepatotoxicity than female wild-type mice. Cotreatment with either NAC or GSHee mitigated the effects of APAP in Gclm wild-type and heterozygous mice, but not in Gclm null mice. Collectively, these data reassert the importance of GSH in protection against APAP-induced hepatotoxicity, and indicate critical roles for GCL activity and gender in APAP-induced liver damage in mice.

This study investigated the role of cellular antioxidant defense mechanisms in modulating the neurotoxicity of domoic acid (DomA), by using cerebellar granule neurons (CGNs) from mice lacking the modifier subunit of glutamate-cysteine ligase (Gclm). Glutamate-cysteine ligase (Glc) catalyzes the first and rate-limiting step in glutathione (GSH) biosynthesis. CGNs from Gclm (Ϫ/Ϫ) mice have very low levels of GSH and are 10-fold more sensitive to DomA-induced toxicity than CGNs from Gclm (ϩ/ϩ) mice. GSH ethyl ester decreased, whereas the Gcl inhibitor buthionine sulfoximine increased DomA toxicity. Antagonists of ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate receptors and of N-methyl-D-aspartate (NMDA) receptors blocked DomA toxicity, and NMDA receptors were activated by DomA-induced L-glutamate release. The differential susceptibility of CGNs to DomA toxicity was not due to a differential expression of ionotropic glutamate receptors, as evidenced by similar calcium responses and L-glutamate release in the two genotypes. A calcium chelator and several antioxidants antagonized DomA-induced toxicity. DomA caused a rapid decrease in cellular GSH, which preceded toxicity, and the decrease was primarily due to DomA-induced GSH efflux. DomA also caused an increase in oxidative stress as indicated by increases in reactive oxygen species and lipid peroxidation, which was subsequent to GSH efflux. Astrocytes from both genotypes were resistant to DomA toxicity and presented a diminished calcium response to DomA and a lack of DomA-induced L-glutamate release. Because polymorphisms in the GCLM gene in humans are associated with low GSH levels, such individuals, as well as others with genetic conditions or environmental exposures that lead to GSH deficiency, may be more susceptible to DomA-induced neurotoxicity.

Objective:The aim of the present study was to determine whether a combination of anti-HIV drugs – tenofovir (TFV), lopinavir (LPV) and ritonavir (RTV) – in a lipid-stabilized nanosuspension (called TLC-ART101) could enhance and sustain intracellular drug levels and exposures in lymph node and blood cells above those in plasma.Design:Four macaques were given a single dose of TLC-ART101 subcutaneously. Drug concentrations in plasma and mononuclear cells of the blood (PBMCs) and lymph nodes (LNMCs) were analysed using a validated combination LC-MS/MS assay.Results:For the two active drugs (TFV, LPV), plasma and PBMC intracellular drug levels persisted for over 2 weeks; PBMC drug exposures were three- to four-fold higher than those in plasma. Apparent terminal half-lives (t1/2) of TFV and LPV were 65.3 and 476.9 h in plasma, and 169.1 and 151.2 h in PBMCs. At 24 and 192 h, TFV and LPV drug levels in LNMCs were up to 79-fold higher than those in PBMCs. Analysis of PBMC intracellular TFV and its active metabolite TFV-diphosphate (TFV-DP) indicated that intracellular exposures of total TFV and TFV-DP were markedly higher and persisted longer than in humans and macaques dosed with oral TFV prodrugs, tenofovir disoproxil fumarate (TDF) or tenofovir alafenamide (TAF).Conclusions:A simple, scalable three-drug combination, lipid-stabilized nanosuspension exhibited persistent drug levels in cells of lymph nodes and the blood (HIV host cells) and in plasma. With appropriate dose adjustment, TLC-ART101 may be a useful HIV treatment with a potential to impact residual virus in lymph nodes.

Gene expression profiling is a widely used technique with data from the majority of published microarray studies being publicly available. These data are being used for meta-analyses and in silico discovery; however, the comparability of toxicogenomic data generated in multiple laboratories has not been critically evaluated. Using the power of prospective multilaboratory investigations, seven centers individually conducted a common toxicogenomics experiment designed to advance understanding of molecular pathways perturbed in liver by an acute toxic dose of N-acetyl-p-aminophenol (APAP) and to uncover reproducible genomic signatures of APAP-induced toxicity. The nonhepatotoxic APAP isomer N-acetyl-m-aminophenol was used to identify gene expression changes unique to APAP. Our data show that c-Myc is induced by APAP and that c-Myc-centered interactomes are the most significant networks of proteins associated with liver injury. Furthermore, sources of error and data variability among Centers and methods to accommodate this variability were identified by coupling gene expression with extensive toxicological evaluation of the toxic responses. We show that phenotypic anchoring of gene expression data is required for biologically meaningful analysis of toxicogenomic experiments.

Existing oral antiretroviral (ARV) agents have been shown in human studies to exhibit limited lymph node penetration and lymphatic drug insufficiency. As lymph nodes are a reservoir of HIV, it is critical to deliver and sustain effective levels of ARV combinations in these tissues. To overcome lymph node drug insufficiency of oral combination ARV therapy (cART), we developed and reported a long-acting and lymphocyte-targeting injectable that contains three ARVs-hydrophobic lopinavir (LPV) and ritonavir (RTV), and hydrophilic tenofovir (TFV)-stabilized by lipid excipients in a nanosuspension. A single subcutaneous (SC) injection of this first-generation formulation of drug combination nanoparticles (DcNPs), named TLC-ART101, provided persistent ARV levels in macaque lymph node mononuclear cells (LNMCs) for at least 1 week, and in peripheral blood mononuclear cells (PBMCs) and plasma for at least 2 weeks, demonstrating long-acting pharmacokinetics for all three drugs. In addition, the lymphocyte-targeting properties of this formulation were demonstrated by the consistently higher intracellular drug concentrations in LNMCs and PBMCs versus those in plasma. To provide insights into the complex mechanisms of absorption and disposition of TLC-ART101, we constructed novel mechanism-based pharmacokinetic (MBPK) models. Based upon plasma PK data obtained after single administration of TLC-ART101 (DcNPs) and a solution formulation of free triple-ARVs, the models feature uptake from the SC injection site that respectively routes free and nanoparticle-associated ARVs via the blood vasculature and lymphatics, and their eventual distribution into and clearance from the systemic circulation. The models provided simultaneous description of the complex long-acting plasma and lymphatic PK profiles for all three ARVs in TLC-ART101. The long-acting PK characteristics of the three drugs in TLC-ART101 were likely due to a combination of mechanisms including: (1) DcNPs undergoing preferential lymphatic uptake from the subcutaneous space, (2) retention in nodes during lymphatic first-pass, (3) subsequent slow release of ARVs into blood circulation, and (4) limited extravasation of DcNP-associated ARVs that resulted in longer persistence in the circulation.