Discovery of ß-d-2'-deoxy-2'-dichlorouridine nucleotide prodrugs as potent inhibitors of hepatitis C virus replication.

Discovery of sofosbuvir has radically changed hepatitis C treatment and nucleoside/tide NS5B inhibitors are now viewed as one of the key components in combination therapies with other direct-acting antiviral agents. As part of our program to identify new nucleoside inhibitors of HCV replication, we now wish to report on the discovery of ß-d-2'-deoxy-2'-dichlorouridine nucleotide prodrugs as potent inhibitors of HCV replication. Although, cytidine analogues have long been recognized to be metabolized to both cytidine and uridine triphosphates through the action of cytidine deaminase, uridine analogues are generally believed to produce exclusively uridine triphosphate. Detailed investigation of the intracellular metabolism of our newly discovered uridine prodrugs, as well as of sofosbuvir, has now revealed the formation of both uridine and cytidine triphosphates. This occurs, not only in vitro in cell lines, but also in vivo upon oral dosing to dogs.

Don't forget to look down - collaborative approaches to predator conservation.

Finding effective ways of conserving large carnivores is widely recognised as a priority in conservation. However, there is disagreement about the most effective way to do this, with some favouring top-down 'command and control' approaches and others favouring collaboration. Arguments for coercive top-down approaches have been presented elsewhere; here we present arguments for collaboration. In many parts of the developed world, flexibility of approach is built into the legislation, so that conservation objectives are balanced with other legitimate goals. In the developing world, limited resources, poverty and weak governance mean that collaborative approaches are likely to play a particularly important part in carnivore conservation. In general, coercive policies may lead to the deterioration of political legitimacy and potentially to non-compliance issues such as illegal killing, whereas collaborative approaches may lead to psychological ownership, enhanced trust, learning, and better social outcomes. Sustainable hunting/trapping plays a crucial part in the conservation and management of many large carnivores. There are many different models for how to conserve carnivores effectively across the world, research is now required to reduce uncertainty and examine the effectiveness of these approaches in different contexts.

Generation of mice transgenic for human CYP2C18 and CYP2C19: characterization of the sexually dimorphic gene and enzyme expression.

CYP2C19 is an important enzyme for human drug metabolism, and it also participates in the metabolism of endogenous substrates, whereas the CYP2C18 enzyme is not expressed in human liver despite high mRNA expression. Mice transgenic for the human CYP2C18 and CYP2C19 genes were generated. Quantitative mRNA analysis showed CYP2C18 and CYP2C19 transcripts in liver, kidneys, and heart to be expressed in a sexually dimorphic manner, with male mice having 2- to 100-fold higher levels. Transcript levels in the small intestine were somewhat higher than liver but were similar in both sexes. Transgene mRNA expression was much lower in lung and brain and least in the heart. Immunoblotting using an antipeptide antiserum, reactive with human CYP2Cs and mouse CYP2C70, revealed increased immunoreactive protein in liver microsomes from heterozygous transgenic male mice and a concomitant increase in 5'-hydroxylation of R-omeprazole and S-mephenytoin intrinsic clearance, consistent with CYP2C19 overexpression. A CYP2C18-specific antiserum showed that this enzyme was not expressed in livers or kidneys from heterozygous transgenic mice, but the antiserum had high affinity for recombinant CYP2C18 expressed in COS-7 cells. It is concluded that 1) both the CYP2C18 and CYP2C19 genes are subject to sexually dimorphic regulation in murine liver, kidney, and heart; 2) the CYP2C18 protein is not expressed in murine liver or kidney despite high levels of the corresponding mRNA; and 3) this transgenic model may be suitable for studying sex-dependent regulation of the human CYP2C genes and possibly serve as an in vivo model for CYP2C19-dependent drug metabolism.

Generic three-column parallel LC-MS/MS system for high-throughput in vitro screens.

A parallel three-column LC-MS/MS system for quantitative high-throughput in vitro screens is described. The robust novel system is composed of three LC pumps, an autosampler and a triple quadrupole mass spectrometer used in combination with three valves and three analytical LC columns in parallel configuration. Two of the three valves work in unison to select which column receives the injection, and the third valve selects which column is to be in line with the mass spectrometer. Improved sample throughput is achieved without sacrificing chromatographic separation quality or sensitivity. To demonstrate the applicability of the system, pools of five compounds (phenacetin, S-mephenytoin, bufuralol, midazolam and clomethiazole) were analyzed, together with an internal standard. The results show that the sample throughput can be increased significantly by reducing analysis time to 3 min per sample as compared to 8 min with a general gradient single-column system. Analysis of the five compounds shows an accuracy of 81-108% and a precision (given as relative standard deviation) of 1.5-14%. The system was further applied to samples from a metabolic stability assay in liver microsomes.