Structure-Guided In Vitro to In Vivo Pharmacokinetic Optimization of Propargyl-Linked Antifolates.

Pharmacokinetic/pharmacodynamic properties are strongly correlated with the in vivo efficacy of antibiotics. Propargyl-linked antifolates, a novel class of antibiotics, demonstrate potent antibacterial activity against both Gram-positive and Gram-negative pathogenic bacteria, including multidrug-resistant Staphylococcus aureus Here, we report our efforts to optimize the pharmacokinetic profile of this class to best match the established pharmacodynamic properties. High-resolution crystal structures were used in combination with in vitro pharmacokinetic models to design compounds that not only are metabolically stable in vivo but also retain potent antibacterial activity. The initial lead compound was prone to both N-oxidation and demethylation, which resulted in an abbreviated in vivo half-life (∼20 minutes) in mice. Stability of leads toward mouse liver microsomes was primarily used to guide medicinal chemistry efforts so robust efficacy could be demonstrated in a mouse disease model. Structure-based drug design guided mitigation of N-oxide formation through substitutions of sterically demanding groups adjacent to the pyridyl nitrogen. Additionally, deuterium and fluorine substitutions were evaluated for their effect on the rate of oxidative demethylation. The resulting compound was characterized and demonstrated to have a low projected clearance in humans with limited potential for drug-drug interactions as predicted by cytochrome P450 inhibition as well as an in vivo exposure profile that optimizes the potential for bactericidal activity, highlighting how structural data, merged with substitutions to introduce metabolic stability, are a powerful approach to drug design.

Performance of the ImmunoCard STAT! E. coli O157:H7 test for detection of Escherichia coli O157:H7 in stools.

ImmunoCard STAT! E. coli O157:H7 (Meridian Diagnostics, Inc., Cincinnati, Ohio) is a novel rapid (10-min) test for the presence of Escherichia coli O157:H7 in stools. The test may be performed either directly on stool specimens or on an overnight broth culture of stool. In a multicenter prospective study, 14 of 14 specimens positive by culture for E. coli O157:H7 were positive by the ImmunoCard STAT! O157:H7 test, and there were no false positives from 263 culture-negative specimens. In a retrospective study, the test was positive in 339 (81%) of 417 stored culture-positive specimens and the specificity was 95% (98 of 103 specimens). No false positives were associated with alternate stool pathogens. The ImmunoCard STAT! O157:H7 test has high sensitivity and specificity.

Variable expression of leukocyte cytosolic broad-specificity beta-glucosidase activity.

The cytosolic beta-glucosidase activity that is found in a variety of mammalian tissues has no clearly defined function. In vitro assay conditions under which the broad-specificity beta-glucosidase hydrolyzes glucocerebroside at a significant rate have not been described. Nonetheless, it has been suggested that this enzyme might play an accessory role with lysosomal glucocerebrosidase in catalyzing the hydrolysis of glucosylceramide. However, this hypothesis would require that activity of both enzymes be low in severe cases of Gaucher disease in which there are pathological accumulations of glucosylceramide in one or more of the affected organs, i.e. spleen, liver and bone marrow. Information is lacking regarding the normal range of cytosolic beta-glucosidase activity in humans. p-Nitrophenyl-beta-D-mannoside was found to be a potent inhibitor (Ki = 0.068 mM) of cytosolic beta-glucosidase. In parallel studies, the activity of glucocerebrosidase was found to be minimally affected by p-nitrophenyl-beta-D-mannoside at concentrations as high as 2.5 mM. This information was used to design an assay system that would allow us to estimate glucocerebrosidase and cytosolic beta-glucosidase activities in extracts of human leukocytes. Average cytosolic beta-glucosidase activity with 4-heptyl-umbelliferyl-beta-D-glucoside as a substrate was 8.8 nmol/h/mg protein in leukocytes from 356 subjects (range, 0.2-28). Average leukocyte glucocerebrosidase specific activity was 16 nmol/h/mg protein (range 5.3-45.7). No correlation was observed between cytosolic beta-glucosidase and glucocerebrosidase activity for control and Gaucher heterozygote populations (r = 0.19 and 0.25, respectively). The wide range of leukocyte cytosolic beta-glucosidase activity in individuals tested in this study indicates that a substantial proportion of the population may lack sufficient cytosolic beta-glucosidase activity to assist a defective lysosomal glucocerebrosidase in hydrolyzing glucosylceramide.