Intracellular Metabolism of β-L-ddAMP-bis(tbutylSATE), a Potent Inhibitor of Hepatitis B Virus Replication

Abstract

β-L-ddAMP-bis(tbutylSATE) is a potent inhibitor of HBV replication with an EC₅₀ = 0.1 μM. Following a 0-to72-hrs exposure of human hepatocytes to a 10 μM [2′,3′^?3H] β-L-ddAMP-bis(tbutylSATE), the pharmacologically active β-L-ddATP was the predominant metabolite attaining a concentration of 268.53 ± 107.97 pmoles/10⁶ cells at 2 hrs. In Hep-G2 cell, β-L-ddATP accounted for 146.8 ± 29.8 pmoles/10⁶ cells at 2 hrs with an half life of approximately 5.4 hrs. This study reveals that extensive intracellular concentrations of β-L-ddATP after incubation of cells to the parent drug is accounting for its potent antiviral activity.     

Deranged Tyrosine Metabolism in Cirrhosis

In normal individuals, the main route for tyrosine degradation is the hepatic pathway tyrosine→4-hydroxyphenylpyruvic acid→homogentisic acid→CO₂. Quantitatively minor pathways, in large part extrahepatic, are: tyrosine→tyramine→octopamine and tyrosine→dopa→catecholamines.In cirrhosis, the main hepatic pathway is blocked to varying degrees at the first three stages. This appears to be due to lack of activity of the enzymes tyrosine transaminase, PHPA oxidase, and HGA oxidase, the first step being rate limiting. Hypertyrosinemia and tyrosine intolerance result.With the main hepatic pathway partially blocked, an abnormally large amount of tyrosine passes into the normally minor extrahepatic pathway leading to the false neurotransmitters tyramine and octopamine. Overproduction of these amines ensues and they accumulate in the body fluid.The false neurotransmitters can displace catecholamines from their storage sites in the peripheral and central nervous system, and thereby disrupt adrenergic processes in arterioles, kidneys, and brain. Their accumulation in cirrhotic patients may play a role in the pathogenesis of hepatic encephalopathy, hepatorenal syndrome, and hyperdynamic circulation.     

Induction of antiproliferative effect by diosgenin through activation of p53,release of apoptosis-inducing factor （AIF） and modulation of caspase-3 activity in different human cancer cells

Previously, we demonstrated that a plant steroid, diosgenin, altered cell cycle distribution and induced apoptosis in the human osteosarcoma 1547 cell line. The objective of this study was to investigate if the antiproliferative effect of diosgenin was similar for different human cancer cell lines such as laryngocarcinoma HEp-2 and melanoma M4Beu cells. Moreover, this work essentially focused on the mitochondrial pathway. We found that diosgenin had an important and similar antiproliferative effect on different types of cancer cells. In addition, our new results show that diosgenin-induced apoptosis is caspase-3 dependent with a fall of mitochondrial membrane potential, nuclear localization of AIF and poly (ADP-ribose) polymerase cleavage. Diosgenin treatment also induces p53 activation and cell cycle arrest in the different cell lines studied.     

A model-dependent approach to correlate accelerated with real-time release from biodegradable microspheres

The purpose of this study was to determine the feasibility of applying accelerated in vitro release testing to correlate or predict long-term in vitro release of leuprolide poly(lactideco-glycolide) microspheres. Peptide release was studied using a dialysis technique at 37°C and at elevated temperatures (50°C–60°C) in 0.1 M phosphate buffered saline (PBS) pH 7.4 and 0.1 M acetate buffer pH 4.0. The data were analyzed using a modification, of the Weibull equation. Peptide release was temperature dependent and complete within 30 days at 37°C and 3 to 5 days at the elevated temperatures. In vitro release profiles at the elevated temperatures correlated well with release at 37°C. The shapes of the release profiles at all temperatures were similar. Using the modified Weibull equation, an increase in temperature was characterized by an increase in the model parameter, α, a scaling factor for the apparent rate constant. Complete release at 37°C was shortened from ∼30 days to 5 days at 50°C, 3.5 days at 55°C, 2.25 days at 60°C in PBS pH 7.4, and 3 days at 50°C in acetate buffer pH 4.0. Values for the model parameter β indicated that the shape of the release profiles at 55°C in PBS pH 7.4 (2.740) and 50°C in 0.1 M acetate buffer pH 4.0 (2.711) were similar to that at 37°C (2.577). The E_a for hydration and erosion were determined to be 42.3 and 19.4 kcal/mol, respectively. Polymer degradation was also temperature dependent and had an E_a of 31.6 kcal/mol. Short-term in vitro release studies offer the possibility of correlation with long-term release, thereby reducing the time and expense associated with longterm studies. Accelerated release methodology could be useful in the prediction of long-term release from extended release microsphere dosage forms and may serve as a quality control tool for the release of clinical or commercial batches. Selected for the 2005 AAPS Outstanding Graduate Student Research Award in Pharmaceutical Technologies Sponsored by Solvay Pharmaceuticals.     

Studies of the specificity of an antibody directed against probenecid. Investigations with probenecid analogs

An antibody against probenecid was obtained in rabbits immunized with probenecid conjugated to bovine serum albumin. The antibody exhibited a high degree of specificity (competitive assay) as shown by studies with 25 analogs which included isomers, homologs and metabolites. Interestingly, three analogs (2′-hydroxy, glycine and methyl ester) had a higher affinity than probenecid. The side-chain metabolites all had much lower affinity than the parent drug. Radioimmunoassay was carried out with dextran-coated charcoal and was sensitive to about 1 nanogram. This raises the possibility of radioimmunoassay of probenecid in plasma and tissue.     

Herbivory by the insect diaphorina citri?induces greater change in citrus plant volatile profile than does infection by the bacterium,Candidatus Liberibacter asiaticus

The volatile organic compound (VOC) profile in plant leaves often changes after biotic and abiotic stresses. Monitoring changes in VOCs in plant leaves could provide valuable information about multitrophic interactions. In the current study, we investigated the effect of Asian citrus psyllid (ACP) infestation, citrus greening pathogen (Candidatus Liberibacter asiaticus [CLas]) infection, and simultaneous attack by ACP and CLas on the VOC content of citrus leaves. Leaf volatiles were extracted using hexane and analyzed with gas chromatography-mass spectrometry (GC-MS). Although ACP is a phloem-sucking insect that causes minimal damage to plant tissues, the relative amount of 21 out of the 27 VOCs increased 2- to 10-fold in ACP-infested plants. The relative amount of d-limonene, β-phelandrene, citronellal, and undecanal were increased 4- to 20- fold in CLas-infected plants. A principle component analysis (PCA) and cluster analysis (CA) showed that VOC patterns of ACP-infested and CLas-infected plants were different from each other and were also different from the controls, while the VOC pattern of double-attacked plants was more like that of the controls than that of ACP-infested or CLas-infected plants. VOC amounts from leaves were compromised when plants were attacked by ACP and CLas. The results of this study showed that a simple direct extraction of citrus leaf volatiles could be successfully used to discriminate between healthy and CLas-infected plants. Information about the effects of insect and pathogen attack on the VOC content profile of plants might contribute to a better understanding of biotic stress.     

In Vitro and In Vivo Metabolism and Pharmacokinetics of bis [(T-Butyl)-S-acyl-2-thioethyl]-β-L-2′,3′-dideoxy-5-fluorocytidine Monophosphate

Abstract

Exposure to 10 & M L-FddCMP-bisSATE led to formation of intracellular L-FddCTP levels of 410.1^± 46.2 and 242.1 ± 13.2 pmol/10⁶ cells in unstimulated and PHAstimulated PBM cells, respectively; whereas, exposure of cells to the parent nucleoside, L-FddC, generated 5-10-fold less L-FddCTP. In Hep-G2 cells and EGF/HGF stimulated and unstimulated primary cultured hepatocytes, the active metabolite reached 113 ± 29, 23.9 ± 15.6, and 20.6 ± 10.5 pmol/10⁶ cells. Three other metabolites, L-FddCMP-monoSATE, L-FddCMP-SH, and M I, were detected intracellularly and extracellularly in all cell types examined. Intravenous administered dose of 3 mg/kg L-FddCMP-bisSATE to rhesus monkeys resulted in plasma concentration levels of 2.06 ± 1.00 and 0.39 ± 0.15 & M of L-FddCMP-monoSATE and L-FddC, respectively, while the prodrug was completely cleared metabolically within 15 min. Following oral administration of an equivalent dose, the absolute oral bioavailability of L-FddC derived from L-FddCMP-bisSATE administration was 65%.     

Steering vaccinomics innovations with anticipatory governance and participatory foresight

Vaccinomics is the convergence of vaccinology and population-based omics sciences. The success of knowledge-based innovations such as vaccinomics is not only contingent on access to new biotechnologies. It also requires new ways of governance of science, knowledge production, and management. This article presents a conceptual analysis of the anticipatory and adaptive approaches that are crucial for the responsible design and sustainable transition of vaccinomics to public health practice. Anticipatory governance is a new approach to manage the uncertainties embedded on an innovation trajectory with participatory foresight, in order to devise governance instruments for collective "steering" of science and technology. As a contrast to hitherto narrowly framed "downstream impact assessments" for emerging technologies, anticipatory governance adopts a broader and interventionist approach that recognizes the social construction of technology design and innovation. It includes in its process explicit mechanisms to understand the factors upstream to the innovation trajectory such as deliberation and cocultivation of the aims, motives, funding, design, and direction of science and technology, both by experts and publics. This upstream shift from a consumer "product uptake" focus to "participatory technology design" on the innovation trajectory is an appropriately radical and necessary departure in the field of technology assessment, especially given that considerable public funds are dedicated to innovations. Recent examples of demands by research funding agencies to anticipate the broad impacts of proposed research--at a very upstream stage at the time of research funding application--suggest that anticipatory governance with foresight may be one way how postgenomics scientific practice might transform in the future toward responsible innovation. Moreover, the present context of knowledge production in vaccinomics is such that policy making for vaccines of the 21st century is occurring in the face of uncertainties where the "facts are uncertain, values in dispute, stakes high and decisions urgent and where no single one of these dimensions can be managed in isolation from the rest." This article concludes, however, that uncertainty is not an accident of the scientific method, but its very substance. Anticipatory governance with participatory foresight offers a mechanism to respond to such inherent sociotechnical uncertainties in the emerging field of vaccinomics by making the coproduction of scientific knowledge by technology and the social systems explicit. Ultimately, this serves to integrate scientific and social knowledge thereby steering innovations to coproduce results and outputs that are socially robust and context sensitive.     

Study of p53 expression and post‐transcriptional modifications after GSM‐900 radiofrequency exposure of human amniotic cells

The potential effects of radiofrequency (RF) exposure on the genetic material of cells are very important to determine since genome instability of somatic cells may be linked to cancer development. In response to genetic damage, the p53 protein is activated and can induce cell cycle arrest allowing more time for DNA repair or elimination of damaged cells through apoptosis. The objective of this study was to investigate whether the exposure to RF electromagnetic fields, similar to those emitted by mobile phones of the second generation standard, Global System for Mobile Communications (GSM), may induce expression of the p53 protein and its activation by post‐translational modifications in cultured human cells. The potential induction of p53 expression and activation by GSM‐900 was investigated after in vitro exposure of human amniotic cells for 24 h to average specific absorption rates (SARs) of 0.25, 1, 2, and 4 W/kg in the temperature range of 36.3–39.7 °C. The exposures were carried out using a wire‐patch cell (WPC) under strictly controlled conditions of temperature. Expression and activation of p53 by phosphorylation at serine 15 and 37 were studied using Western blot assay immediately after three independent exposures of cell cultures provided from three different donors. Bleomycin‐exposed cells were used as a positive control. According to our results, no significant changes in the expression and activation of the p53 protein by phosphorylation at serine 15 and 37 were found following exposure to GSM‐900 for 24 h at average SARs up to 4 W/kg in human embryonic cells. Bioelectromagnetics 34:52–60, 2013. © 2012 Wiley Periodicals, Inc.