In vitro activity and resistance profile of samatasvir, a novel NS5A replication inhibitor of hepatitis C virus.

The hepatitis C virus (HCV) nonstructural 5A (NS5A) protein is a clinically validated target for drugs designed to treat chronic HCV infection. This study evaluated the in vitro activity, selectivity, and resistance profile of a novel anti-HCV compound, samatasvir (IDX719), alone and in combination with other antiviral agents. Samatasvir was effective and selective against infectious HCV and replicons, with 50% effective concentrations (EC50s) falling within a tight range of 2 to 24 pM in genotype 1 through 5 replicons and with a 10-fold EC50 shift in the presence of 40% human serum in the genotype 1b replicon. The EC90/EC50 ratio was low (2.6). A 50% cytotoxic concentration (CC50) of >100 µM provided a selectivity index of >5 × 10(7). Resistance selection experiments (with genotype 1a replicons) and testing against replicons bearing site-directed mutations (with genotype 1a and 1b replicons) identified NS5A amino acids 28, 30, 31, 32, and 93 as potential resistance loci, suggesting that samatasvir affects NS5A function. Samatasvir demonstrated an overall additive effect when combined with interferon alfa (IFN-α), ribavirin, representative HCV protease, and nonnucleoside polymerase inhibitors or the nucleotide prodrug IDX184. Samatasvir retained full activity in the presence of HIV and hepatitis B virus (HBV) antivirals and was not cross-resistant with HCV protease, nucleotide, and nonnucleoside polymerase inhibitor classes. Thus, samatasvir is a selective low-picomolar inhibitor of HCV replication in vitro and is a promising candidate for future combination therapies with other direct-acting antiviral drugs in HCV-infected patients.

Role of paracellular junction complexes in baculovirus-mediated gene transfer to nondividing rat hepatocytes.

Gene delivery to differentiated hepatocytes is notoriously difficult. Hepatocytes plated on collagen-coated dishes and maintained in dimethyl sulfoxide (DMSO)-supplemented medium acquire paracellular junctions, arrange themselves in multicellular islands and are an excellent in vitro model for studying liver function. Baculovirus-mediated gene delivery to hepatocytes in this culture system is restricted to peripheral cells of the islands. However, this limitation can be overcome by transient calcium depletion of the cells prior to and during baculovirus infection. Examination of the mechanism underlying this process revealed that calcium depletion was accompanied by a transient loss of intercellular contacts and paracellular junction complex integrity, increased distance between adjoining cells, and internalization of the tight junction protein, zona occludens ZO-1. Internalization of ZO-1 was accompanied by baculovirus infection of internal cells of hepatocyte islands. When calcium levels were restored, paracellular junction complex integrity returned to normal by 12 h. No permanent alterations in hepatocyte ultrastructure and albumin mRNA, and protein expression were caused by this gene transfer method. Loss in paracellular junction complex integrity exposes the basolateral (sinusoidal) surface of hepatocytes resulting in homogeneous baculovirus-mediated gene delivery to approximately 75% of the cells in long-term DMSO culture. We conclude that the use of recombinant baculovirus as a vector in combination with transient calcium depletion is a highly efficient method for delivering exogenous genes to hepatocytes without loss of hepatic differentiation.

Transient disruption of intercellular junctions enables baculovirus entry into nondividing hepatocytes.

Baculovirus infection has extended the capabilities for transfection of exogenous genes into a variety of mammalian cell types. Because rat hepatocytes plated on collagen-coated dishes and maintained in dimethyl sulfoxide (DMSO)-supplemented chemically defined medium are an excellent model system for studying liver function in vitro, we investigated the ability of baculoviruses to infect and deliver exogenous genes to cells in this culture system. Efficient delivery to hepatocytes in short-term culture becomes restricted to peripheral cells, or "edge" cells, as the hepatocytes acquire intercellular junctions and form islands with time in culture. This barrier to baculovirus entry can be overcome, and the percentage of internal cells within the hepatocyte islands that are infected with the baculovirus can be increased more than 100-fold, when cells are subjected to transient calcium depletion before and during infection. These findings suggest that at least in some cell types, such as hepatocytes, baculovirus entry may require contact with the basolateral surface. We conclude from this study that recombinant baculovirus infection following transient depletion of extracellular calcium results in delivery of exogenous genes to at least 75% of hepatocytes in long-term DMSO culture, thereby making it possible for the first time to carry out gain-of-function and loss-of-function studies in this cell system.