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Ibrahim M Ibrahim,1,2 Aditya N Bade,1 Zhiyi Lin,3 Dhruvkumar Soni,3 Melinda Wojtkiewicz,1 Bhagya Laxmi Dyavar Shetty,1 Nagsen Gautam,3 JoEllyn M McMillan,1 Yazen Alnouti,3 Benson J Edagwa,1 Howard E Gendelman1,31Department of Pharmacology and Experimental Neuroscience, College of Medicine, University of Nebraska Medical Center, Omaha, NE, USA; 2Department of Pharmacology, College of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia; 3Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, NE, USAPurpose: A palmitoylated prodrug of emtricitabine (FTC) was synthesized to extend the drug’s half-life, antiretroviral activities and biodistribution.Methods: A modified FTC prodrug (MFTC) was synthesized by palmitoyl chloride esterification. MFTC’s chemical structure was evaluated by nuclear magnetic resonance. The created hydrophobic prodrug nanocrystals were encased into a poloxamer surfactant and the pharmacokinetics (PK), biodistribution and antiretroviral activities of the nanoformulation (NMFTC) were assessed. The conversion of MFTC to FTC triphosphates was evaluated.Results: MFTC coated with poloxamer formed stable nanocrystals (NMFTC). NMFTC demonstrated an average particle size, polydispersity index and zeta potential of 350 nm, 0.24 and −20 mV, respectively. Drug encapsulation efficiency was 90%. NMFTC was readily taken up by human monocyte-derived macrophages yielding readily detected intracellular FTC triphosphates and an extended PK profile.Conclusion: NMFTC shows improved antiretroviral activities over native FTC. This is coordinate with its extended apparent half-life. The work represents an incremental advance in the development of a long-acting FTC formulation.Keywords: palmitoyl chloride, viral reservoirs, long-acting antiretrovirals, human immunodeficiency virus type 1, monocyte-derived macrophage

Hardeep S Oberoi,1 Natalia V Nukolova,1,2 Frederic C Laquer,3 Larisa Y Poluektova,4 Jiangeng Huang,1 Yazen Alnouti,1 Masanao Yokohira,5 Lora L Arnold,5 Alexander V Kabanov,1,2 Samuel M Cohen,5 Tatiana K Bronich,1,21Department of Pharmaceutical Sciences and Center for Drug Delivery and Nanomedicine, College of Pharmacy, University of Nebraska Medical Center, Omaha, NE, USA; 2Department of Chemistry, MV Lomonosov Moscow State University, Leninskie Gory, Moscow, Russia; 3Department of Chemistry, University of Nebraska at Omaha, 4Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, 5Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE, USAAbstract: Polymer micelles with cross-linked ionic cores are shown here to improve the therapeutic performance of the platinum-containing anticancer compound cisplatin. Biodistribution, antitumor efficacy, and toxicity of cisplatin-loaded core cross-linked micelles of poly(ethylene glycol)-b-poly(methacrylic acid) were evaluated in a mouse ovarian cancer xenograft model. Cisplatin-loaded micelles demonstrated prolonged blood circulation, increased tumor accumulation, and reduced renal exposure. Improved antitumor response relative to free drug was seen in a mouse model. Toxicity studies with cisplatin-loaded micelles indicate a significantly improved safety profile and lack of renal abnormalities typical of free cisplatin treatment. Overall, the study supports the fundamental possibility of improving the potential of platinum therapy using polymer micelle-based drug delivery.Keywords: cross-linked micelle, cisplatin, ovarian cancer, block ionomer complex, drug delivery

Georgette D Kanmogne1, Sangya Singh1, Upal Roy1, Xinming Liu1, JoEllyn McMillan1, Santhi Gorantla1, Shantanu Balkundi1, Nathan Smith1, Yazen Alnouti2, Nagsen Gautam2, You Zhou3, Larisa Poluektova1, Alexander Kabanov2, Tatiana Bronich2, Howard E Gendelman11Departments of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, 2Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE; 3Center for Biotechnology, University of Nebraska-Lincoln, Lincoln, NE, USAAbstract: Despite the successes of antiretroviral therapy (ART), HIV-associated neurocognitive disorders remain prevalent in infected people. This is due, in part, to incomplete ART penetration across the blood–brain barrier (BBB) and lymph nodes and to the establishment of viral sanctuaries within the central nervous system. In efforts to improve ART delivery, our laboratories developed a macrophage-carriage system for nanoformulated crystalline ART (nanoART) (atazanavir, ritonavir, indinavir, and efavirenz). We demonstrate that nanoART transfer from mononuclear phagocytes (MP) to human brain microvascular endothelial cells (HBMEC) can be realized through cell-to-cell contacts, which can facilitate drug passage across the BBB. Coculturing of donor MP containing nanoART with recipient HBMEC facilitates intercellular particle transfer. NanoART uptake was observed in up to 52% of HBMEC with limited cytotoxicity. Folate coating of nanoART increased MP to HBMEC particle transfer by up to 77%. To translate the cell assays into relevant animal models of disease, ritonavir and atazanavir nanoformulations were injected into HIV-1-infected NOD/scid-γcnull mice reconstituted with human peripheral blood lymphocytes. Atazanavir and ritonavir levels in brains of mice treated with folate-coated nanoART were three- to four-fold higher than in mice treated with noncoated particles. This was associated with decreased viral load in the spleen and brain, and diminished brain CD11b-associated glial activation. We postulate that monocyte-macrophage transfer of nanoART to brain endothelial cells could facilitate drug entry into the brain.Keywords: nanoART, folate, monocyte-endothelial cell interactions, blood–brain barrier, antiretroviral therapy, nanomedicine

Lack of adherence, inaccessibility to viral reservoirs, long-term drug toxicities, and treatment failures are limitations of current antiretroviral therapy (ART). These limitations lead to increased viral loads, medicine resistance, immunocompromise, and comorbid conditions. To this end, we developed long-acting nanoformulated ART (nanoART) through modifications of existing atazanavir, ritonavir, and efavirenz suspensions in order to establish cell and tissue drug depots to achieve sustained antiretroviral responses. NanoART's abilities to affect immune and antiviral responses, before or following human immunodeficiency virus type 1 infection were tested in nonobese severe combined immune-deficient mice reconstituted with human peripheral blood lymphocytes. Weekly subcutaneous injections of drug nanoformulations at doses from 80 mg/kg to 250 mg/kg, 1 day before and/or 1 and 7 days after viral exposure, elicited drug levels that paralleled the human median effective concentration, and with limited toxicities. NanoART treatment attenuated viral replication and preserved CD4(+) Tcell numbers beyond that seen with orally administered native drugs. These investigations bring us one step closer toward using long-acting antiretrovirals in humans. [ABSTRACT FROM AUTHOR]

LD14b is an amyloid-β (Aβ) 17β-hydroxysteroid dehydrogenase type 10 (Aβ-17β-HSD10) protein-protein interaction modulator that shows promising in vitro and ex vivo activity to rescue Aβ-induced mitochondrial dysfunction, Aβ-induced toxicity, and Aβ-mediated inhibition of estradiol synthesis.The current study investigated in vitro human S9 fractions metabolic stability, apparent permeability, human and mouse plasma protein binding, in vivo pharmacokinetics, and tissue distribution in Balb/cJ mice. A fast (8-min), sensitive, reliable, and reproducible LC-MS/MS method was developed and validated over the dynamic range of 1-1000 ng/mL for the quantification of LD14b in different biological matrices (plasma, liver, kidney, brain, lungs, heart).LD14b was metabolically stable in human liver S9 fractions with 70% remaining after 90 minutes of incubation, showed intermediate apparent permeability of 3.55 × 10 -06 cm/s and 6.16 × 10 -06 cm/s for apical-to-basolateral (A-to-B) and basolateral-to-apical (B-to-A), respectively across the Caco-2 monolayer, and was medium/highly bound to human plasma proteins (84.1%), mouse plasma proteins (85.7%), and mouse brain homogenate (95.4%).LD14b showed an in vivo predicted % absorption of 52% in Balb/cJ mice and was well-distributed to the peripheral tissues (liver, kidney, lungs, and heart) including the brain.

Background: Alcohol-associated cardiomyopathy (ACM) is a cardiac muscle disease characterized by inflammation and oxidative stress. Thromboxane-prostanoid receptor (TP-R) plays an important role in the pathogenesis of cardiovascular disease. Herein, we hypothesize that TP-R mediates alcohol-induced early cardiac injury., Methods: Eight-week-old male C57BL/6 wild-type mice were fed a chronic ethanol (ET) or control diet (CON) for 10 days followed by a single binge of ethanol or maltose-dextrin through oral gavage. A cohort of ethanol-fed mice received SQ 29,548 (SQ), a TP-R antagonist. RNA sequencing, real-time PCR, and western blot analysis were performed on left ventricle to investigate alterations in genes and/or proteins mediating oxidative stress, inflammation, and cardiac remodeling. Sirius Red staining was performed to measure myocardial fibrosis., Results: RNA-sequencing analysis of myocardium from CON and ET groups identified 142 genes that were significantly altered between the two groups. In particular, the gene expression of thioredoxin-interacting protein (TXNIP), a component of NLR family pyrin domain containing 3 (NLRP3) signaling, which mediates oxidative stress and inflammatory response, was upregulated in response to ethanol exposure. The myocardial protein levels of TP-R and thromboxane A2 synthase were increased upon alcohol exposure. Ethanol increased the levels of 4-hydroxynonenal, a marker of oxidative stress, with a concomitant increase in the protein levels of TXNIP and NLRP3, and administration of SQ attenuated these effects. Additionally, ethanol increased the protein levels of pro-inflammatory mediators, including tumor necrosis factor alpha and the NLRP3 downstream product, secretory interleukin 1 beta, and SQ blunted these effects. Finally, the Sirius red staining of the myocardium revealed an increase in collagen deposition in ethanol-fed mice which was attenuated by TP-R antagonism., Conclusion: This study demonstrates that ethanol promotes the NLRP3 signaling pathway within the myocardium, leading to a pro-inflammatory milieu that potentially initiates early myocardial remodeling, and TP-R antagonism attenuates this effect., (© 2024 The Author(s). Alcohol, Clinical and Experimental Research published by Wiley Periodicals LLC on behalf of Research Society on Alcohol.)

Bile acid-CoA: amino acid N-acyltransferase (BAAT) catalyzes bile acid conjugation, the last step in bile acid synthesis. BAAT gene mutation in humans results in hypercholanemia, growth retardation, and fat-soluble vitamin insufficiency. The current study investigated the physiological function of BAAT in bile acid and lipid metabolism using Baat -/- mice. The bile acid composition and hepatic gene expression were analyzed in 10-week-old Baat -/- mice. They were also challenged with a westernized diet (WD) for additional 15 weeks to assess the role of BAAT in bile acid, lipid, and glucose metabolism. Comprehensive lab animal monitoring system and cecal 16S ribosomal RNA gene sequencing were used to evaluate the energy metabolism and microbiome structure of the mice, respectively. In Baat -/- mice, hepatic bile acids were mostly unconjugated and their levels were significantly increased compared with wild-type mice. Bile acid polyhydroxylation was markedly up-regulated to detoxify unconjugated bile acid accumulated in Baat -/- mice. Although the level of serum marker of bile acid synthesis, 7α-hydroxy-4-cholesten-3-one, was higher in Baat -/- mice, their bile acid pool size was smaller. When fed a WD, the Baat -/- mice showed a compromised body weight gain and impaired insulin secretion. The gut microbiome of Baat -/- mice showed a low level of sulfidogenic bacteria Bilophila. Conclusion: Mouse BAAT is the major taurine-conjugating enzyme. Its deletion protected the animals from diet-induced obesity, but caused glucose intolerance. The gut microbiome of the Baat -/- mice was altered to accommodate the unconjugated bile acid pool., (© 2022 The Authors. Hepatology Communications published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

Ultra-long-acting integrase strand transfer inhibitors were created by screening a library of monomeric and dimeric dolutegravir (DTG) prodrug nanoformulations. This led to an 18-carbon chain modified ester prodrug nanocrystal (coined NM2DTG) with the potential to sustain yearly dosing. Here, we show that the physiochemical and pharmacokinetic (PK) formulation properties facilitate slow drug release from tissue macrophage depot stores at the muscle injection site and adjacent lymphoid tissues following single parenteral injection. Significant plasma drug levels are recorded up to a year following injection. Tissue sites for prodrug hydrolysis are dependent on nanocrystal dissolution and prodrug release, drug-depot volume, perfusion, and cell-tissue pH. Each affect an extended NM2DTG apparent half-life recorded by PK parameters. The NM2DTG product can impact therapeutic adherence, tolerability, and access of a widely used integrase inhibitor in both resource limited and rich settings to reduce HIV-1 transmission and achieve optimal treatment outcomes., (© 2022. The Author(s).)

Background: Hepatocyte nuclear factor 4α (HNF4α) and glucocorticoid receptor (GR), master regulators of liver metabolism, are down-regulated in fatty liver diseases. The present study aimed to elucidate the role of down-regulation of HNF4α and GR in fatty liver and hyperlipidemia., Methods: Adult mice with liver-specific heterozygote (HET) and knockout (KO) of HNF4α or GR were fed a high-fat-high-sugar diet (HFHS) for 15 days. Alterations in hepatic and circulating lipids were determined with analytical kits, and changes in hepatic mRNA and protein expression in these mice were quantified by real-time PCR and Western blotting. Serum and hepatic levels of bile acids were quantified by LC-MS/MS. The roles of HNF4α and GR in regulating hepatic gene expression were determined using luciferase reporter assays., Results: Compared to HFHS-fed wildtype mice, HNF4α HET mice had down-regulation of lipid catabolic genes, induction of lipogenic genes, and increased hepatic and blood levels of lipids, whereas HNF4α KO mice had fatty liver but mild hypolipidemia, down-regulation of lipid-efflux genes, and induction of genes for uptake, synthesis, and storage of lipids. Serum levels of chenodeoxycholic acid and deoxycholic acid tended to be decreased in the HNF4α HET mice but dramatically increased in the HNF4α KO mice, which was associated with marked down-regulation of cytochrome P450 7a1, the rate-limiting enzyme for bile acid synthesis. Hepatic mRNA and protein expression of sterol-regulatory-element-binding protein-1 (SREBP-1), a master lipogenic regulator, was induced in HFHS-fed HNF4α HET mice. In reporter assays, HNF4α cooperated with the corepressor small heterodimer partner to potently inhibit the transactivation of mouse and human SREBP-1C promoter by liver X receptor. Hepatic nuclear GR proteins tended to be decreased in the HNF4α KO mice. HFHS-fed mice with liver-specific KO of GR had increased hepatic lipids and induction of SREBP-1C and PPARγ, which was associated with a marked decrease in hepatic levels of HNF4α proteins in these mice. In reporter assays, GR and HNF4α synergistically/additively induced lipid catabolic genes., Conclusions: induction of lipid catabolic genes and suppression of lipogenic genes by HNF4α and GR may mediate the early resistance to HFHS-induced fatty liver and hyperlipidemia., (© 2022. The Author(s).)

Activation of inhibitor of nuclear factor NF-κB kinase subunit-β (IKKβ), characterized by phosphorylation of activation loop serine residues 177 and 181, has been implicated in the early onset of cancer. On the other hand, tissue-specific IKKβ knockout in Kras mutation-driven mouse models stalled the disease in the precancerous stage. In this study, we used cell line models, tumor growth studies, and patient samples to assess the role of IKKβ and its activation in cancer. We also conducted a hit-to-lead optimization study that led to the identification of 39-100 as a selective mitogen-activated protein kinase kinase kinase (MAP3K) 1 inhibitor. We show that IKKβ is not required for growth of Kras mutant pancreatic cancer (PC) cells but is critical for PC tumor growth in mice. We also observed elevated basal levels of activated IKKβ in PC cell lines, PC patient-derived tumors, and liver metastases, implicating it in disease onset and progression. Optimization of an ATP noncompetitive IKKβ inhibitor resulted in the identification of 39-100, an orally bioavailable inhibitor with improved potency and pharmacokinetic properties. The compound 39-100 did not inhibit IKKβ but inhibited the IKKβ kinase MAP3K1 with low-micromolar potency. MAP3K1-mediated IKKβ phosphorylation was inhibited by 39-100, thus we termed it IKKβ activation modulator (IKAM) 1. In PC models, IKAM-1 reduced activated IKKβ levels, inhibited tumor growth, and reduced metastasis. Our findings suggests that MAP3K1-mediated IKKβ activation contributes to KRAS mutation-associated PC growth and IKAM-1 is a viable pretherapeutic lead that targets this pathway.

Hepatobiliary diseases and their complications cause the accumulation of toxic bile acids (BA) in the liver, blood, and other tissues, which may exacerbate the underlying condition and lead to unfavorable prognosis. To develop and validate prognostic biomarkers for the prediction of complications of cholestatic liver disease based on urinary BA indices, liquid chromatography-tandem mass spectrometry was used to analyze urine samples from 257 patients with cholestatic liver diseases during a 7-year follow-up period. The urinary BA profile and non-BA parameters were monitored, and logistic regression models were used to predict the prognosis of hepatobiliary disease-related complications. Urinary BA indices were applied to quantify the composition, metabolism, hydrophilicity, and toxicity of the BA profile. We have developed and validated the bile-acid liver disease complication (BALDC) model based on BA indices using logistic regression model, to predict the prognosis of cholestatic liver disease complications including ascites. The mixed BA and non-BA model was the most accurate and provided higher area under the receiver operating characteristic (ROC) and smaller akaike information criterion (AIC) values compared to both non-BA and MELD (models for end stage liver disease) models. Therefore, the mixed BA and non-BA model could be used to predict the development of ascites in patients diagnosed with liver disease at early stages of intervention. This will help physicians to make a better decision when treating hepatobiliary disease-related ascites., Competing Interests: The authors state that this study has no conflicts of interest., (Copyright © 2022 Wenkuan Li et al.)

There are several challenges associated with LC-MS/MS bioanalytical method development and validation. Low and variable recovery of some analytes, especially the more hydrophobic ones, is often challenging. Analytes can be lost to various extents throughout the process of sample collection, storage, before, during, and/or after sample preparation and analysis. The calculation of overall extraction recovery can detect problems of low recovery during sample preparation but does not identify the source(s) of analyte losses. Low overall analyte recovery is the net result of losses that can happen for multiple reasons at all steps of sample preparation and analysis. Therefore, identifying the source(s) of analyte loss during sample preparation can help guide the optimization the bioanalysis conditions to minimize these losses. In this article we propose a practical protocol to systematically identify and quantify the sources of low analyte recovery. This allows the proper choice of strategies to optimize the relevant bioanalytical conditions to minimize analyte losses and improve overall recovery., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

RNA interference (RNAi) molecules have tremendous potential for cancer therapy but are limited by insufficient potency after intravenous (IV) administration. We previously found that polymer complexes (polyplexes) formed between 3'-cholesterol-modified siRNA (Chol-siRNA) or DsiRNA (Chol-DsiRNA) and the cationic diblock copolymer PLL[30]-PEG[5K] greatly increase RNAi potency against stably expressed LUC mRNA in primary syngeneic murine breast tumors after daily IV dosing. Chol-DsiRNA polyplexes, however, maintain LUC mRNA suppression for ~48 h longer after the final dose than Chol-siRNA polyplexes, which suggests that they are the better candidate formulation. Here, we directly compared the activities of Chol-siRNA polyplexes and Chol-DsiRNA polyplexes in primary murine 4T1 breast tumors against STAT3, a therapeutically relevant target gene that is overexpressed in many solid tumors, including breast cancer. We found that Chol-siSTAT3 polyplexes suppressed STAT3 mRNA in 4T1 tumors with similar potency (half-maximal ED 50 0.3 mg/kg) and kinetics (over 96 h) as Chol-DsiSTAT3 polyplexes, but with slightly lower activity against total Stat3 protein (29% vs. 42% suppression) and tumor growth (11.5% vs. 8.6% rate-based T/C ratio) after repeated IV administration of equimolar, tumor-saturating doses every other day. Thus, both Chol-siRNA polyplexes and Chol-DsiRNA polyplexes may be suitable clinical candidates for the RNAi therapy of breast cancer and other solid tumors.

Treatment and prevention of human immunodeficiency virus type one (HIV-1) infection was transformed through widespread use of antiretroviral therapy (ART). However, ART has limitations in requiring life-long daily adherence. Such limitations have led to the creation of long-acting (LA) ART. While nucleoside reverse transcriptase inhibitors (NRTI) remain the ART backbone, to the best of our knowledge, none have been converted into LA agents. To these ends, we transformed tenofovir (TFV) into LA surfactant stabilized aqueous prodrug nanocrystals (referred to as NM1TFV and NM2TFV), enhancing intracellular drug uptake and retention. A single intramuscular injection of NM1TFV, NM2TFV, or a nanoformulated tenofovir alafenamide (NTAF) at 75 mg/kg TFV equivalents to Sprague Dawley rats sustains active TFV-diphosphate (TFV-DP) levels ≥ four times the 90% effective dose for two months. NM1TFV, NM2TFV and NTAF elicit TFV-DP levels of 11,276, 1,651, and 397 fmol/g in rectal tissue, respectively. These results are a significant step towards a LA TFV ProTide., (© 2021. The Author(s).)

Background: Multiple tissue reservoirs are established soon after HIV infection, and some tissues may also be pharmacological sanctuaries. Parenteral administration of antiretroviral (ARV) drugs for treatment and prevention of HIV infection is an active area of drug development. The influence of route of administration on ARV tissue pharmacokinetics is not known., Objectives: To investigate ARV pharmacokinetics in lymphatic and select non-lymphatic tissues (e.g. brain and testes) after intramuscular and subcutaneous administration compared with oral in BALB/c mice., Methods: Tissue concentrations of cobicistat, efavirenz, elvitegravir, maraviroc, rilpivirine, tenofovir alafenamide and tenofovir disoproxil fumarate were determined. The tissue penetration ratio (TPR) was the primary measure for comparison; a change in TPR arises from factors affecting tissue distribution controlling for changes in systemic bioavailability., Results: Intramuscular and subcutaneous delivery increased TPRs in the lymph node and spleen for 27 of 28 (96%) drug administration events. Decreased TPRs, however, were found in some tissues such as the brain and testes., Conclusions: These results demonstrate a change in route of drug administration from oral to intramuscular or subcutaneous can change tissue uptake. This has implications for HIV pharmacotherapy. For example, HIV persists in lymphoid tissues despite long-term oral ARV therapy, and low ARV concentrations have been found in lymphoid tissues. The improved ARV lymphatic tissue bioavailability with intramuscular and subcutaneous administration allows future studies to investigate these routes of drug administration as a therapeutic manoeuvre to limit viral persistence and eliminate viral sanctuaries in the lymphatic tissues, which is a prerequisite for eradication of HIV., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The present study describes the discovery and characterization of a series of N -(1-(1,1-dioxidotetrahydrothiophen-3-yl)-3-methyl-1 H -pyrazol-5-yl)acetamide ethers as G protein-gated inwardly-rectifying potassium (GIRK) channel activators. From our previous lead optimization efforts, we have identified a new ether-based scaffold and paired this with a novel sulfone-based head group to identify a potent and selective GIRK1/2 activator. In addition, we evaluated the compounds in tier 1 DMPK assays and have identified compounds that display nanomolar potency as GIRK1/2 activators with improved metabolic stability over the prototypical urea-based compounds., Competing Interests: C. D. W. is an owner of WaveFront Biosciences and ION Biosciences – sellers of the Panoptic kinetic imaging plate reader and the thallium-sensitive fluorescent indicators used in these studies., (This journal is © The Royal Society of Chemistry.)

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.

Background: Cholestatic liver diseases are characterized by an accumulation of toxic bile acids (BA) in the liver, blood and other tissues which lead to progressive liver injury and poor prognosis in patients., Aim: To discover and validate prognostic biomarkers of cholestatic liver diseases based on the urinary BA profile., Methods: We analyzed urine samples by liquid chromatography-tandem mass spectrometry and investigated the use of the urinary BA profile to develop survival models that can predict the prognosis of hepatobiliary diseases. The urinary BA profile, a set of non-BA parameters, and the adverse events of liver transplant and/or death were monitored in 257 patients with cholestatic liver diseases for up to 7 years. The BA profile was characterized by calculating BA indices, which quantify the composition, metabolism, hydrophilicity, formation of secondary BA, and toxicity of the BA profile. We have developed and validated the bile-acid score (BAS) model (a survival model based on BA indices) to predict the prognosis of cholestatic liver diseases., Results: We have developed and validated a survival model based on BA (the BAS model) indices to predict the prognosis of cholestatic liver diseases. Our results demonstrate that the BAS model is more accurate and results in higher true-positive and true-negative prediction of death compared to both non-BAS and model for end-stage liver disease (MELD) models. Both 5- and 3-year survival probabilities markedly decreased as a function of BAS. Moreover, patients with high BAS had a 4-fold higher rate of death and lived for an average of 11 mo shorter than subjects with low BAS. The increased risk of death with high vs low BAS was also 2-4-fold higher and the shortening of lifespan was 6-7-mo lower compared to MELD or non-BAS. Similarly, we have shown the use of BAS to predict the survival of patients with and without liver transplant (LT). Therefore, BAS could be used to define the most seriously ill patients, who need earlier intervention such as LT. This will help provide guidance for timely care for liver patients., Conclusion: The BAS model is more accurate than MELD and non-BAS models in predicting the prognosis of cholestatic liver diseases., Competing Interests: Conflict-of-interest statement: The authors declare that there is no conflict of interests in this study., (©The Author(s) 2021. Published by Baishideng Publishing Group Inc. All rights reserved.)

Background: Hepatobiliary diseases result in the accumulation of toxic bile acids (BA) in the liver, blood, and other tissues which may contribute to an unfavorable prognosis., Aim: To discover and validate diagnostic biomarkers of cholestatic liver diseases based on the urinary BA profile., Methods: We analyzed urine samples by liquid chromatography-tandem mass spectrometry and compared the urinary BA profile between 300 patients with hepatobiliary diseases vs 103 healthy controls by statistical analysis. The BA profile was characterized using BA indices, which quantifies the composition, metabolism, hydrophilicity, and toxicity of the BA profile. BA indices have much lower inter- and intra-individual variability compared to absolute concentrations of BA. In addition, BA indices demonstrate high area under the receiver operating characteristic curves, and changes of BA indices are associated with the risk of having a liver disease, which demonstrates their use as diagnostic biomarkers for cholestatic liver diseases., Results: Total and individual BA concentrations were higher in all patients. The percentage of secondary BA (lithocholic acid and deoxycholic acid) was significantly lower, while the percentage of primary BA (chenodeoxycholic acid, cholic acid, and hyocholic acid) was markedly higher in patients compared to controls. In addition, the percentage of taurine-amidation was higher in patients than controls. The increase in the non-12α-OH BA was more profound than 12α-OH BA (cholic acid and deoxycholic acid) causing a decrease in the 12α-OH/ non-12α-OH ratio in patients. This trend was stronger in patients with more advanced liver diseases as reflected by the model for end-stage liver disease score and the presence of hepatic decompensation. The percentage of sulfation was also higher in patients with more severe forms of liver diseases., Conclusion: BA indices have much lower inter- and intra-individual variability compared to absolute BA concentrations and changes of BA indices are associated with the risk of developing liver diseases., Competing Interests: Conflict-of-interest statement: The authors declare that there is no conflict of interests in this study., (©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.)

RNA interference molecules have tremendous potential for cancer therapy but are limited by insufficient potency after i.v. administration. We previously found that Chol-DsiRNA polyplexes formed between cholesterol-modified dicer-substrate siRNA (Chol-DsiRNA) and the cationic diblock copolymer PLL[30]-PEG[5K] greatly increase the activity of Chol-DsiRNA against a stably expressed reporter mRNA in primary murine syngeneic breast tumors after daily i.v. dosing. Here, we provide a more thorough preliminary preclinical study of Chol-DsiRNA polyplexes against the therapeutically relevant target protein, STAT3. We found that Chol-DsiSTAT3 polyplexes greatly increase plasma exposure, distribution, potency, and therapeutic activity of Chol-DsiSTAT3 in primary murine syngeneic 4T1 breast tumors after i.v. administration. Furthermore, inactive Chol-DsiCTRL polyplexes are well tolerated by healthy female BALB/c mice after chronic i.v. administration at 50 mg Chol-DsiCTRL/kg over 28 days. Thus, Chol-DsiRNA polyplexes may be a good candidate for Phase I clinical trials to improve the treatment of breast cancer and other solid tumors., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Alcohol-associated liver disease is accompanied by dysregulation of bile acid metabolism and gut barrier dysfunction. Peroxisome proliferator-activated receptor-delta (PPARδ) agonists are key metabolic regulators and have anti-inflammatory properties. Here, we evaluated the effect of the selective PPAR-delta agonist seladelpar (MBX-8025) on gut barrier function and bile acid metabolism in a mouse model of ethanol-induced liver disease. Wild type C57BL/6 mice were fed LieberDeCarli diet containing 0%-36% ethanol (caloric) for 8 weeks followed by a single binge of ethanol (5 g/kg). Pair fed mice received an isocaloric liquid diet as control. MBX-8025 (10 mg/kg/d) or vehicle were added to the liquid diet during the entire feeding period (prevention), or during the last 4 weeks of Lieber DeCarli diet feeding (intervention). In both prevention and intervention trials, MBX-8025 protected mice from ethanol-induced liver disease, characterized by lower serum alanine aminotransferase (ALT) levels, hepatic triglycerides, and inflammation. Chronic ethanol intake disrupted bile acid metabolism by increasing the total bile acid pool and serum bile acids. MBX-8025 reduced serum total and secondary bile acids, and the total bile acid pool as compared with vehicle treatment in both prevention and intervention trials. MBX-8025 restored ethanol-induced gut dysbiosis and gut barrier dysfunction. Data from this study demonstrates that seladelpar prevents and treats ethanol-induced liver damage in mice by direct PPARδ agonism in both the liver and the intestine., (Copyright © 2020 Elsevier Inc. All rights reserved.)

HPMA copolymer-based dexamethasone prodrug (P-Dex) and PEG-based dexamethasone prodrug (PEG-Dex, ZSJ-0228) were previously found to passively target the inflamed kidney and provide potent and sustained resolution of nephritis in NZB/WF1 lupus-prone mice. While both prodrug nanomedicines effectively ameliorate lupus nephritis, they have demonstrated distinctively different safety profiles. To explore the underlining mechanisms of these differences, we conducted a head-to-head comparative PK/BD study of P-Dex and PEG-Dex on NZB/WF1 mice. Overall, the systemic organ/tissue exposures to P-Dex and Dex released from P-Dex were found to be significantly higher than those of PEG-Dex. The high prodrug concentrations were sustained in kidney for only 24 h, which cannot explain their lasting therapeutic efficacy (>1 month). P-Dex showed sustained presence in liver, spleen and adrenal gland, while the presence of PEG-Dex in these organs was transient. This difference in PK/BD profiles may explain PEG-Dex' superior safety than P-Dex., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: The mechanistic target of rapamycin complex 1 (mTORC1) is a nutrient-sensing pathway and a key regulator of amino acid and glucose metabolism. Dysregulation of the mTOR pathways is implicated in the pathogenesis of metabolic syndrome, obesity, type 2 diabetes, and pancreatic cancer., Objectives: We investigated the impact of inhibition of mTORC1/mTORC2 and synergism with metformin on pancreatic tumor growth and metabolomics., Methods: Cell lines derived from pancreatic tumors of the KPC (Kras G12D/+ ; p53 R172H/+ ; Pdx1-Cre) transgenic mice model were implanted into the pancreas of C57BL/6 albino mice ( n  = 10/group). Two weeks later, the mice were injected intraperitoneally with daily doses of 1 ) Torin 2 (mTORC1/mTORC2 inhibitor) at a high concentration (TH), 2 ) Torin 2 at a low concentration (TL), 3 ) metformin at a low concentration (ML), 4 ) a combination of Torin 2 and metformin at low concentrations (TLML), or 5 ) DMSO vehicle (control) for 12 d. Tissues and blood samples were collected for targeted xenometabolomics analysis, drug concentration, and cell signaling., Results: Metabolomic analysis of the control and treated plasma samples showed differential metabolite profiles. Phenylalanine was significantly elevated in the TLML group compared with the control (+426%, P  = 0.0004), whereas uracil was significantly lower (-38%, P  = 0.009). The combination treatment reduced tumor growth in the orthotopic mouse model. TLML significantly decreased pancreatic tumor volume (498 ± 104 mm 3 ; 37%; P  < 0.0004) compared with control (1326 ± 134 mm 3 ; 100%), ML (853 ± 67 mm 3 ; 64%), TL (745 ± 167 mm 3 ; 54%), and TH (665 ± 182 mm 3 ; 50%) (ANOVA and post hoc tests). TLML significantly decreased tumor weights (0.66 ± 0.08 g; 52%) compared with the control (1.28 ± 0.19 g; 100%) ( P  < 0.002)., Conclusions: The combination of mTOR dual inhibition by Torin 2 and metformin is associated with an altered metabolomic profile and a significant reduction in pancreatic tumor burden compared with single-agent therapy, and it is better tolerated., (Copyright © The Author(s) on behalf of the American Society for Nutrition 2020.)

Cocaine addiction remains a major public concern throughout the world especially in developed countries. In the last three decades, significant achievements have led to a greater understanding of the signaling pathways involved in the development of cocaine addiction; however, there are no FDA-approved treatments available to reverse or block this brain disease due to either the unsatisfactory therapeutic efficacy or severe side effects. Previous studies have demonstrated that chronic exposure to cocaine elevates levels of cyclic AMP (cAMP) as a neuroadaptative response in reward-related brain regions. Phosphodiesterase 4 (PDE4) inhibitors, which elevate cAMP levels, have been shown to block cocaine-mediated behavioral changes related to psychoactive and reinforcing properties. Unfortunately, previously studied PDE4 inhibitors induce severe side-effects, which limit their clinical usage. In this study, we identified a novel PDE4B inhibitor, KVA-D-88, with an improved selectivity profile compared to previous compounds (e.g., rolipram). Pharmacokinetic studies have shown that this compound is brain penetrant and preferably acts on PDE4B compared to PDE4D in vitro , alluding to less unwanted side effects with KVA-D-88 in vivo . Interestingly, pretreatment with KVA-D-88 significantly inhibited cocaine-induced hyperlocomotor activity. In cocaine self-administering mice with differential schedules, KVA-D-88 strikingly decreased the number of active nose-pokes and cocaine infusions and reduced the break point. Taken together, our findings demonstrate that this novel PDE4 inhibitor, KVA-D-88, could inhibit cocaine-mediated rewarding effects implying its potential clinical usage for cocaine addiction.

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.

We previously reported that fish oil in combination with cyclooxygenase (COX) inhibitors exerts enhanced hypolipidemic and anti-inflammatory effects in mice. Here, we sought to determine the effects of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) in combination with naproxen (NX), a COX inhibitor, on dyslipidemia and gene expression in adipose tissue (AT) in humans. Obese dyslipidemic patients were randomly assigned to one of these interventions for 12 wk: 1) Standard nutrition counseling (control), 2) ω-3 PUFAs (2 g twice daily), 3) NX (220 mg twice daily), and 4) ω-3 PUFAs (2 g twice daily) + NX (220 mg twice daily). The serum triglycerides showed a trend towards a reduction and a significant reduction (P<0.05) in ω-3 and ω3 + NX-treated subjects, respectively, compared to control. The mRNA expression of vascular cell adhesion molecule-1 (Vcam1), an inflammatory marker, increased significantly in AT of ω-3 PUFA-treated subjects but not in ω-3 PUFAs+NX-treated group. The plasma level of glycine-conjugated hyodeoxycholic acid, a secondary bile acid with hypolipidemic property, increased significantly in ω-3 PUFAs + NX-treated group. Our data suggest that combining NX with ω-3 PUFAs increases their effectiveness in reducing serum TG and favorably altering AT gene expression and plasma bile acid profile., (Published by Elsevier Inc.)

Nucleoside reverse transcriptase inhibitors (NRTIs) are prodrugs that require intracellular phosphorylation to active triphosphate nucleotide metabolites (NMs) for their pharmacological activity. However, monitoring these pharmacologically active NMs is challenging due to their instability, high hydrophilicity, and their low concentrations in blood and tissues. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is the gold standard technique for the quantification of NRTIs and their phosphorylated NMs. In this review, an overview of the publications describing the quantitative analysis of intracellular and total tissue concentration of NMs is presented. The focus of this review is the comparison of the different approaches and challenges associated with sample collection, tissue homogenization, cell lysis, cell counting, analyte extraction, sample storage conditions, and LC-MS analysis. Quantification methods of NMs via LC-MS can be categorized into direct and indirect methods. In the direct LC-MS methods, chromatographic retention of the NMs is accomplished by ion-exchange (IEX), ion-pairing (IP), hydrophilic interaction (HILIC), porous graphitic carbon (PGC) chromatography, or capillary electrophoresis (CE). In indirect methods, parent nucleosides are 1st generated from the dephosphorylation of NMs during sample preparation and are then quantified by reverse phase LC-MS as surrogates for their corresponding NMs. Both approaches have advantages and disadvantages associated with them, which are discussed in this review., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Herein we report the synthesis, SAR, and biological evaluation of a series of 1 H -pyrrolo[2,3- b ]pyridine-2-carboxamide derivatives as selective and potent PDE4B inhibitors. Compound 11h is a PDE4B preferring inhibitor and exhibited acceptable in vitro ADME and significantly inhibited TNF-α release from macrophages exposed to pro-inflammatory stimuli (i.e., lipopolysaccharide and the synthetic bacterial lipopeptide Pam3Cys). In addition, 11h was selective against a panel of CNS receptors and represents an excellent lead for further optimization and preclinical testing in the setting of CNS diseases., Competing Interests: The authors declare no competing financial interest.

Antiretroviral therapy (ART) has improved the quality of life in patients infected with HIV-1. However, complete viral suppression within anatomical compartments remains unattainable. This is complicated by adverse side effects and poor adherence to lifelong therapy leading to the emergence of viral drug resistance. Thus, there is an immediate need for cellular and tissue-targeted long-acting (LA) ART formulations. Herein, we describe two LA prodrug formulations of darunavir (DRV), a potent antiretroviral protease inhibitor. Two classes of DRV prodrugs, M1DRV and M2DRV, were synthesized as lipophilic and hydrophobic prodrugs and stabilized into aqueous suspensions designated NM1DRV and NM2DRV. The formulations demonstrated enhanced intracellular prodrug levels with sustained drug retention and antiretroviral activities for 15 and 30 days compared to native DRV formulation in human monocyte-derived macrophages. Pharmacokinetics tests of NM1DRV and NM2DRV administered to mice demonstrated sustained drug levels in blood and tissues for 30 days. These data, taken together, support the idea that LA DRV with sustained antiretroviral responses through prodrug nanoformulations is achievable.

A long acting (LA) hydrophobic and lipophilic lamivudine (3TC) was created as a phosphoramidate pronucleotide (designated M23TC). M23TC improved intracellular delivery of active triphosphate metabolites and enhanced antiretroviral and pharmacokinetic (PK) profiles over the native drug. A single treatment of human monocyte derived macrophages (MDM) with nanoformulated M23TC (NM23TC) improved drug uptake, retention, intracellular 3TC triphosphates and antiretroviral activities in MDM and CD4 + T cells. PK tests of NM23TC administered to Sprague Dawley rats demonstrated sustained prodrug and drug triphosphate levels in blood and tissues for 30 days. The development of NM23TC remains a substantive step forward in producing LA slow effective release antiretrovirals for future clinical translation., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

While antiretroviral therapy (ART) has revolutionized treatment and prevention of human immunodeficiency virus type one (HIV-1) infection, regimen adherence, viral mutations, drug toxicities and access stigma and fatigue are treatment limitations. These have led to new opportunities for the development of long acting (LA) ART including implantable devices and chemical drug modifications. Herein, medicinal and formulation chemistry were used to develop LA prodrug nanoformulations of emtricitabine (FTC). A potent lipophilic FTC phosphoramidate prodrug (M2FTC) was synthesized then encapsulated into a poloxamer surfactant (NM2FTC). These modifications extended the biology, apparent drug half-life and antiretroviral activities of the formulations. NM2FTC demonstrated a >30-fold increase in macrophage and CD4+ T cell drug uptake with efficient conversion to triphosphates (FTC-TP). Intracellular FTC-TP protected macrophages against an HIV-1 challenge for 30 days. A single intramuscular injection of NM2FTC, at 45 mg/kg native drug equivalents, into Sprague Dawley rats resulted in sustained prodrug levels in blood, liver, spleen and lymph nodes and FTC-TP in lymph node and spleen cells at one month. In contrast, native FTC-TPs was present for one day. These results are an advance in the transformation of FTC into a LA agent., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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 half-life and improve tissue biodistribution. These results warrant further development for human use., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: The secondary lymphoid tissues (LTs), lymph nodes (LNs) and gut-associated lymphoid tissue (GALT) are considered reservoirs for HIV. Antiretrovirals (ARVs) have lower penetration into LT. In vitro models predictive of ARV LT penetration have not been established., Objectives: To develop an in vitro model of LT bioavailability using human lymphoid endothelial cells (HLECs) and investigate its predictability with in vivo pharmacokinetic (PK) studies in mice., Methods: ARV bioavailability in HLECs was evaluated at the maximum plasma concentration (Cmax) observed in HIV-infected patients. ARVs were: abacavir, atazanavir, darunavir, dolutegravir, efavirenz, elvitegravir, emtricitabine, maraviroc, raltegravir, rilpivirine, ritonavir, tenofovir disoproxil fumarate and the PK booster cobicistat. The LT PK of representative drugs showing high (efavirenz), intermediate (dolutegravir) and low (emtricitabine) HLEC bioavailability was investigated in BALB/c mice given 50/10/30 mg/kg efavirenz/dolutegravir/emtricitabine orally, daily for 3 days. The concordance of in vitro and in vivo ARV bioavailability was examined., Results: ARVs showed high (>67th percentile; rilpivirine, efavirenz, elvitegravir and cobicistat), intermediate (67th-33rd percentile; ritonavir, tenofovir disoproxil fumarate, dolutegravir and maraviroc) and low (<33rd percentile; atazanavir, darunavir, raltegravir, emtricitabine and abacavir) HLEC bioavailability. The hierarchy of efavirenz, dolutegravir and emtricitabine bioavailability in LN, gut and brain tissues of mice was: efavirenz>dolutegravir>emtricitabine., Conclusions: ARVs displayed distinct HLEC penetration patterns. PK studies of representative ARVs in LT of mice were concordant with HLEC bioavailability. These findings support further development of this approach and its translational predictability in humans., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The present study describes the discovery and characterization of a series of 5-aryl-2H-tetrazol-3-ylacetamides as G protein-gated inwardly-rectifying potassium (GIRK) channels activators. Working from an initial hit discovered during a high-throughput screening campaign, we identified a tetrazole scaffold that shifts away from the previously reported urea-based scaffolds while remaining effective GIRK1/2 channel activators. In addition, we evaluated the compounds in Tier 1 DMPK assays and have identified a (3-methyl-1H-pyrazol-1-yl)tetrahydrothiophene-1,1-dioxide head group that imparts interesting and unexpected microsomal stability compared to previously-reported pyrazole head groups., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

: 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.

One of the mechanisms of drug-induced liver injury (DILI) involves alterations in bile acid (BA) homeostasis and elimination, which encompass several metabolic pathways including hydroxylation, amidation, sulfation, glucuronidation and glutathione conjugation. Species differences in BA metabolism may play a major role in the failure of currently used in vitro and in vivo models to predict reliably the DILI during the early stages of drug discovery and development. We developed an in vitro cofactor-fortified liver S9 fraction model to compare the metabolic profiles of the four major BAs (cholic acid, chenodeoxycholic acid, lithocholic acid and ursodeoxycholic acid) between humans and several animal species. High- and low-resolution liquid chromatography-tandem mass spectrometry and nuclear magnetic resonance imaging were used for the qualitative and quantitative analysis of BAs and their metabolites. Major species differences were found in the metabolism of BAs. Sulfation into 3-O-sulfates was a major pathway in human and chimpanzee (4.8%-52%) and it was a minor pathway in all other species (0.02%-14%). Amidation was primarily with glycine (62%-95%) in minipig and rabbit and it was primarily with taurine (43%-81%) in human, chimpanzee, dog, hamster, rat and mice. Hydroxylation was highest (13%-80%) in rat and mice followed by hamster, while it was lowest (1.6%-22%) in human, chimpanzee and minipig. C6-β hydroxylation was predominant (65%-95%) in rat and mice, while it was at C6-α position in minipig (36%-97%). Glucuronidation was highest in dog (10%-56%), while it was a minor pathway in all other species (<12%). The relative contribution of the various pathways involved in BA metabolism in vitro were in agreement with the observed plasma and urinary BA profiles in vivo and were able to predict and quantify the species differences in BA metabolism. In general, overall, BA metabolism in chimpanzee is most similar to human, while BA metabolism in rats and mice is most dissimilar from human., (Copyright © 2018 John Wiley & Sons, Ltd.)

Maintenance of bile acid (BA) homeostasis is essential to achieve their physiologic functions and avoid their toxic effects. The marked differences in BA composition between preclinical safety models and humans may play a major role in the poor prediction of drug-induced liver injury using preclinical models. We compared the composition of plasma and urinary BAs and their metabolites between humans and several animal species. Total BA pools and their composition varied widely among different species. Highest sulfation of BAs was observed in human and chimpanzee. Glycine amidation was predominant in human, minipig, hamster and rabbit, while taurine amidation was predominant in mice, rat and dogs. BA profiles consisted primarily of tri-OH BAs in hamster, rat, dog and mice, di-OH BAs in human, rabbit and minipig, and mono-OH BA in chimpanzee. BA profiles comprised primarily hydrophilic and less toxic BAs in mice, rat, pig and hamster, while it primarily comprised hydrophobic and more toxic BAs in human, rabbit and chimpanzee. Therefore, the hydrophobicity index was lowest in minipig and mice, while it was highest in rabbit, monkey and human. Glucuronidation and glutathione conjugation were low in all species across all BAs. Total concentration of BAs in urine was up to 10× higher and more hydrophilic than plasma in most species. This was due to the presence of more tri-OH, amidated, sulfated and primary BAs, in urine compared to plasma. In general, BA profiles of chimpanzee and monkeys were most similar to human, while minipig, rat and mice were most dissimilar to human., (Copyright © 2018 John Wiley & Sons, Ltd.)

Abacavir pronucleotide nanoformulations (NM3ABC) were prepared as a novel long acting slow effective release antiretroviral therapy. Single NM3ABC treatment of human monocyte-derived macrophages produced sustained intracellular carbovir-triphosphate and antiretroviral activities for up to 30 days.

Alcoholic liver disease (ALD) is associated with changes in the intestinal microbiota. Functional consequences of alcohol-associated dysbiosis are largely unknown. The aim of this study was to identify a mechanism of how changes in the intestinal microbiota contribute to ALD. Metagenomic sequencing of intestinal contents demonstrated that chronic ethanol feeding in mice is associated with an over-representation of bacterial genomic DNA encoding choloylglycine hydrolase, which deconjugates bile acids in the intestine. Bile acid analysis confirmed an increased amount of unconjugated bile acids in the small intestine after ethanol administration. Mediated by a lower farnesoid X receptor (FXR) activity in enterocytes, lower fibroblast growth factor (FGF)-15 protein secretion was associated with increased hepatic cytochrome P450 enzyme (Cyp)-7a1 protein expression and circulating bile acid levels. Depletion of the commensal microbiota with nonabsorbable antibiotics attenuated hepatic Cyp7a1 expression and reduced ALD in mice, suggesting that increased bile acid synthesis is dependent on gut bacteria. To restore intestinal FXR activity, we used a pharmacological intervention with the intestine-restricted FXR agonist fexaramine, which protected mice from ethanol-induced liver injury. Whereas bile acid metabolism was only minimally altered, fexaramine treatment stabilized the gut barrier and significantly modulated hepatic genes involved in lipid metabolism. To link the beneficial metabolic effect to FGF15, a nontumorigenic FGF19 variant-a human FGF15 ortholog-was overexpressed in mice using adeno-associated viruses. FGF19 treatment showed similarly beneficial metabolic effects and ameliorated alcoholic steatohepatitis., Conclusion: Taken together, alcohol-associated metagenomic changes result in alterations of bile acid profiles. Targeted interventions improve bile acid-FXR-FGF15 signaling by modulation of hepatic Cyp7a1 and lipid metabolism, and reduce ethanol-induced liver disease in mice. (Hepatology 2018;67:2150-2166)., (© 2017 by the American Association for the Study of Liver Diseases.)

Nucleoside reverse transcriptase inhibitors (NRTIs) require intracellular phosphorylation to active triphosphate (TP) nucleotide metabolites before they can inhibit the HIV reverse transcriptase. However, monitoring these pharmacologically active TP metabolites is challenging due to their instability and their low concentrations at the pg/ml levels in blood and tissues. The combination of lamivudine (3TC) and abacavir (ABC) is one of the first lines for HIV therapy. Therefore, a sensitive, selective, accurate, and precise LC-MS/MS method was developed and validated for the simultaneous quantification of 3TC- and ABC-TP metabolites in mouse blood and tissues. Calibration curves were linear over the range of 10-100,000 pg/ml for 3TC-TP and 4-40,000 pg/ml for carbovir-TP (CBV-TP; phosphorylated metabolite of ABC). This corresponds to 2.1-21,322 fmol/10 6 cells for 3TC-TP and 0.8-8000 fmol/10 6 cells for CBV-TP. Accuracy and precision were less than 15% for all quality control sample (QCs), and absolute extraction recovery of were >65% for 3TC-TP and >90% for CBV-TP. The method was optimized to ensure stability of TP samples and standards during sample collection, preparation, analysis, and storage conditions. This method has enhanced sensitivity and requires smaller amounts of blood and tissue samples compared to previous LC-MS/MS methods for 3TC- and CBV-TP quantification. The developed method was successfully applied to characterize the pharmacokinetic profile of TP metabolites in mouse peripheral blood mononuclear cells (PBMCs), spleen, lymph nodes, and liver cells. In addition, another direct, simple, and high-throughput method for the quantification of TP standards was developed and used for the analysis of stability samples., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Aim: While the  therapeutic potential for current long-acting (LA) antiretroviral therapy (ART) is undeniable, ligand-decorated nanoformulated LA-ART could optimize drug delivery to viral reservoirs. The development of decorated ART hinges, however, on formulation processes and manufacture efficiencies. To this end, we compared manufacture and purification techniques for ligand-decorated antiretroviral drug nanocrystals., Materials & Methods: Ligand-decorated nanoparticle manufacturing was tested using folic acid (FA) nanoformulated cabotegravir., Results: Direct manufacturing of FA-cabotegravir resulted in stable particles with high drug loading and monocyte-macrophage targeting. A one step 'direct' scheme proved superior over differential centrifugation or tangential flow filtration facilitating particle stability and preparation simplicity and efficiency., Conclusion: Direct manufacturing of FA nanoparticles provides a path toward large-scale clinical grade manufacturing of cell-targeted LA-ART.

The differences among individual eicosanoids in eliciting different physiological and pathological responses are largely unknown because of the lack of valid and simple analytical methods for the quantification of individual eicosanoids and their metabolites in serum, sputum and bronchial alveolar lavage fluid (BALF). Therefore, a simple and sensitive LC-MS/MS method for the simultaneous quantification of 34 eicosanoids in human serum, sputum and BALF was developed and validated. This method is valid and sensitive with a limit of quantification ranging from 0.2 to 3 ng/mL for the various analytes, and has a large dynamic range (500 ng/mL) and a short run time (25 min). The intra- and inter-day accuracy and precision values met the acceptance criteria according to US Food and Drug Administration guidelines. Using this method, detailed eicosanoid profiles were quantified in serum, sputum and BALF from a pilot human study. In summary, a reliable and simple LC-MS/MS method to quantify major eicosanoids and their metabolites was developed and applied to quantify eicosanoids in human various fluids, demonstrating its suitability to assess eicosanoid biomarkers in human clinical trials., (Copyright © 2017 John Wiley & Sons, Ltd.)