ABSTRACT
Type III phosphatidylinositol-4-kinase beta (PI4KIIIß) was previously implicated in hepatitis C virus (HCV) replication by small interfering RNA (siRNA) depletion and was therefore proposed as a novel cellular target for the treatment of hepatitis C. Medicinal chemistry efforts identified highly selective PI4KIIIß inhibitors that potently inhibited the replication of genotype 1a and 1b HCV replicons and genotype 2a virus in vitro. Replicon cells required more than 5 weeks to reach low levels of 3- to 5-fold resistance, suggesting a high resistance barrier to these cellular targets. Extensive in vitro profiling of the compounds revealed a role of PI4KIIIß in lymphocyte proliferation. Previously proposed functions of PI4KIIIß in insulin secretion and the regulation of several ion channels were not perturbed with these inhibitors. Moreover, PI4KIIIß inhibitors were not generally cytotoxic as demonstrated across hundreds of cell lines and primary cells. However, an unexpected antiproliferative effect in lymphocytes precluded their further development for the treatment of hepatitis C.
Subject(s)
1-Phosphatidylinositol 4-Kinase/antagonists & inhibitors , Antiviral Agents/pharmacology , Hepacivirus/drug effects , Animals , Antiviral Agents/adverse effects , Bone Marrow Cells/cytology , Bone Marrow Cells/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Cells, Cultured , Humans , Male , Mice , Rats , Rats, Sprague-DawleyABSTRACT
This work details the development of a model for the rapid evaluation of drug metabolism in an integrated fashion using in situ architecture of the liver. A Krumdieck tissue slicer was used to generate slices from 10-mm cores of rat liver (approximately 250-microm thick). Initial unsuccessful efforts with 6-well plate-based incubation were overcome with the use of a dynamic (rotating) incubation in 23-ml liquid scintillation vials containing titanium mesh supports for the slice. Incubation of 1 slice/5 ml of a Krebs-Henseleit solution buffered with HEPES showed a <2% increase over the initial 25% release of lactate dehydrogenase over 2 h of incubation at 37 degrees C under ambient oxygen conditions. Coupled O-dealkylase and conjugative metabolism of alkoxycoumarin derivatives was shown to be linear for both 7-methoxy- and 7-ethoxycoumarin (100 microM) with a low amount of nonconjugated 7-hydroxycoumarin (7-HC) at all time points. Metabolic profiles for 7-methoxy- and 7-ethoxycoumarin were compared between slice and microsomal incubations generated from the same tissue. The use of 7-HC as a primary substrate not only provided an assessment of the capacity-based differences in oxidative versus conjugative metabolism but also capacity-based differences in glucuronidation and sulfation. These studies underscore the physiological fact that phase I metabolism has a lower capacity for substrate metabolism than phase II metabolism. Additionally, this technique provides a model for examination of pharmacodynamic and pharmacokinetic influences in the context of maintenance of the in situ architecture of the liver.
Subject(s)
Coumarins/metabolism , Liver/metabolism , Models, Biological , Umbelliferones/metabolism , Animals , In Vitro Techniques , Male , Methods , Rats , Rats, Sprague-DawleyABSTRACT
The effects of waterborne nitrite (3 mg/l NO2) on channel catfish were studied to evaluate changes in hematological parameters and phase I-II biotransformation in liver slices. Nitrite-exposed fish had significantly higher methemoglobin, blood and liver nitrite, and significantly lower pO2 than control fish. Total phase I-mediated metabolism of 7-ethoxycoumarin (EC) was not altered in nitrite-exposed fish compared with control fish (291 +/- 43 and 312 +/- 20 pmol/mg/h, respectively). However, phase II glucuronosyltransferase-mediated metabolism of 7-hydroxycoumarin (HC), both as a phase I metabolite of EC and as a parent substrate, was elevated in nitrite-exposed fish (204 +/- 17 and 1007 +/- 103 pmol/mg/h, respectively) as compared to control fish (149 +/- 14 and 735 +/- 87 pmol/mg/h) (P < 0.05). Sulfotransferase-mediated metabolism of HC (as a metabolite of EC and as a parent substrate) was not notably altered in nitrite-exposed fish (95 +/- 16 and 617 +/- 33 pmol/mg protein/h, respectively) as compared with control fish (118 +/- 24 and 575 +/- 55 pmol/mg/h, respectively). These studies indicate that in vivo nitrite exposure and associated changes in hematological parameters do not appear to affect hepatic phase I EC biotransformation in channel catfish. However, subtle but significant changes in phase II glucuronidation, but not sulfation activity, were observed. The mechanism of these alterations is unclear. However, the data suggest that environmentally realistic concentrations of nitrite may affect the dynamics of conjugative metabolism in exposed fish.
