scholarly journals Implications of Efficient Hepatic Delivery by Tenofovir Alafenamide (GS-7340) for Hepatitis B Virus Therapy

2015 ◽  
Vol 59 (6) ◽  
pp. 3563-3569 ◽  
Author(s):  
Eisuke Murakami ◽  
Ting Wang ◽  
Yeojin Park ◽  
Jia Hao ◽  
Eve-Irene Lepist ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Human Hepatocytes ◽  
Target Tissue ◽  
Small Contribution ◽  
Primary Human Hepatocytes ◽  
B Virus ◽  
Tenofovir Alafenamide ◽  
Pharmacologically Active

ABSTRACTTenofovir alafenamide (TAF) is a prodrug of tenofovir (TFV) currently in clinical evaluation for treatment for HIV and hepatitis B virus (HBV) infections. Since the target tissue for HBV is the liver, the hepatic delivery and metabolism of TAF in primary human hepatocytesin vitroand in dogsin vivowere evaluated here. Incubation of primary human hepatocytes with TAF resulted in high levels of the pharmacologically active metabolite tenofovir diphosphate (TFV-DP), which persisted in the cell with a half-life of >24 h. In addition to passive permeability, studies of transfected cell lines suggest that the hepatic uptake of TAF is also facilitated by the organic anion-transporting polypeptides 1B1 and 1B3 (OATP1B1 and OATP1B3, respectively). In order to inhibit HBV reverse transcriptase, TAF must be converted to the pharmacologically active form, TFV-DP. While cathepsin A is known to be the major enzyme hydrolyzing TAF in cells targeted by HIV, including lymphocytes and macrophages, TAF was primarily hydrolyzed by carboxylesterase 1 (CES1) in primary human hepatocytes, with cathepsin A making a small contribution. Following oral administration of TAF to dogs for 7 days, TAF was rapidly absorbed. The appearance of the major metabolite TFV in plasma was accompanied by a rapid decline in circulating TAF. Consistent with thein vitrodata, high and persistent levels of TFV-DP were observed in dog livers. Notably, higher liver TFV-DP levels were observed after administration of TAF than those given TDF. These results support the clinical testing of once-daily low-dose TAF for the treatment of HBV infection.

scholarly journals Pharmacology of β-l-Thymidine and β-l-2′-Deoxycytidine in HepG2 Cells and Primary Human Hepatocytes: Relevance to Chemotherapeutic Efficacy against Hepatitis B Virus

2002 ◽  
Vol 46 (6) ◽  
pp. 1728-1733 ◽  
Author(s):  
B. Hernandez-Santiago ◽  
L. Placidi ◽  
E. Cretton-Scott ◽  
A. Faraj ◽  
E. G. Bridges ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Hepg2 Cells ◽  
Hepatitis Virus ◽  
Parent Compound ◽  
Human Hepatocytes ◽  
Primary Human Hepatocytes ◽  
B Virus ◽  
Intracellular Concentrations

ABSTRACT β-l-Thymidine (l-dT) and β-l-2′-deoxycytidine (l-dC) are potent and highly specific inhibitors of hepatitis B virus (HBV) replication both in vivo and in vitro (50% effective concentrations, 0.19 to 0.24 μM in 2.2.15 cells). The intracellular metabolisms of l-dT and l-dC were investigated in HepG2 cells and primary cultured human hepatocytes. l-dT and l-dC were extensively phosphorylated in both cell types, with the 5′-triphosphate derivative being the predominant metabolite. In HepG2 cells, the 5′-triphosphate levels were 27.7 ± 12.1 and 72.4 ± 1.8 pmol/106 cells for l-dT and l-dC, respectively. In primary human hepatocytes, the 5′-triphosphate levels were 16.5 ± 9.8 and 90.1 ± 36.4 pmol/106 cells for l-dT and l-dC, respectively. Furthermore, a choline derivative of l-dCDP was detected at concentrations of 15.8 ± 1.8 and 25.6 ± 0.1 pmol/106 cells in human hepatocytes and HepG2 cells, respectively. In HepG2 cells exposed to l-dC, the 5′-monophosphate and 5′-triphosphate derivatives of β-l-2′-deoxyuridine (l-dUMP and l-dUTP, respectively) were also observed, reaching intracellular concentrations of 6.7 ± 0.4 and 18.2 ± 1.0 pmol/106 cells, respectively. In human hepatocytes, l-dUMP and l-dUTP were detected at concentrations of 5.7 ± 2.4 and 43.5 ± 26.8 pmol/106 cells, respectively. It is likely that deamination of l-dCMP by deoxycytidylate deaminase leads to the formation of l-dUMP, as the parent compound, l-dC, was not a substrate for deoxycytidine deaminase. The intracellular half-lives of l-dTTP, l-dCTP, and l-dUTP were at least 15 h, with intracellular concentrations of each metabolite remaining above their respective 50% inhibitory concentrations for the woodchuck hepatitis virus DNA polymerase for as long as 24 h after removal of the drug from cell cultures. Exposure of HepG2 cells to l-dT in combination with l-dC led to concentrations of the activated metabolites similar to those achieved with either agent alone. These results suggest that the potent anti-HBV activities of l-dT and l-dC are associated with their extensive phosphorylation.

In vitro experimental infection of primary human hepatocytes with hepatitis B virus

1994 ◽  
Vol 106 (3) ◽  
pp. 664-673 ◽  
Author(s):  
Peter R. Galle ◽  
Jens Hagelstein ◽  
Burkhard Kommerell ◽  
Martin Volkmann ◽  
Peter Schranz ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Experimental Infection ◽  
Human Hepatocytes ◽  
Primary Human Hepatocytes ◽  
B Virus

Regulatory Function of Interferon‐Inducible 44 Like for Hepatitis B Virus cccDNA in Primary Human Hepatocytes

2021 ◽  
Author(s):  
Takuto Nosaka ◽  
Tatsushi Naito ◽  
Yosuke Murata ◽  
Hidetaka Matsuda ◽  
Masahiro Ohtani ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Human Hepatocytes ◽  
Regulatory Function ◽  
Primary Human Hepatocytes ◽  
B Virus

scholarly journals Smc5/6-antagonism by HBx is an evolutionary-conserved function of hepatitis B virus infection in mammals

2017 ◽  
Author(s):  
Fabien Filleton ◽  
Fabien Abdul ◽  
Laetitia Gerossier ◽  
Alexia Paturel ◽  
Janet Hall ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Antiviral Activity ◽  
Positive Selection ◽  
Host Species ◽  
Human Hepatocytes ◽  
Arms Race ◽  
Restriction Factor ◽  
Primary Human Hepatocytes ◽  
B Virus

AbstractInfection with Hepatitis B virus (HBV) is a major cause of liver disease and cancer in humans. HBVs (family Hepadnaviridae) have been associated with mammals for millions of years. Recently, the Smc5/6 complex, known for its essential housekeeping functions in genome maintenance, was identified as an antiviral restriction factor of human HBV. The virus has however developed a counteraction mechanism by degrading the complex via its regulatory HBx protein. Whether the antiviral activity of the Smc5/6 complex against hepadnaviruses is an important and evolutionary-conserved function is unknown. Here, we used a combined evolutionary and functional approach to address this question. We first performed phylogenetic and positive selection analyses of the six Smc5/6 complex subunits and found that they have been highly conserved in primates and mammals. Yet, the Smc6 subunit showed marks of adaptive evolution, potentially reminiscent of virus-host “arms-race” We then functionally tested the HBx from six very divergent hepadnaviruses now naturally infecting primates, rodents, and bats. Despite little sequence homology, we demonstrate that these HBx efficiently degraded mammalian Smc5/6 complexes, independently of the host species and of the sites under positive selection. Importantly, all also rescued the replication of an HBx-deficient HBV in primary human hepatocytes. These findings point to an evolutionary-conserved requirement for Smc5/6 inactivation by HBx, showing that the Smc5/6 antiviral activity has been an important defense mechanism against hepadnaviruses in mammals. Interestingly, Smc5/6 may further be a restriction factor of other yet unidentified viruses that have driven some of its adaptation.ImportanceInfection with hepatitis B virus (HBV) led to 887000 human deaths in 2015. HBV has been co-evolving with mammals for millions of years. Recently, the Smc5/6 complex, known for its essential housekeeping functions, was identified as a restriction factor of human HBV antagonized by the regulatory HBx protein. Here, we address whether the antiviral activity of Smc5/6 is an important evolutionary-conserved function. We found that all six subunits of Smc5/6 have been conserved in primates with only Smc6 showing signatures of “evolutionary arms-race” Using evolutionary-guided functional assays that include infections of primary human hepatocytes, we demonstrate that HBx from very divergent mammalian HBVs could all efficiently antagonize Smc5/6, independently of the host species and sites under positive selection. These findings show that the Smc5/6 antiviral activity against HBV is an important function in mammals. It also raises the intriguing possibility that Smc5/6 restricts other, yet unidentified viruses.

SAT-165-Identification of chromatin-accessible domains on the host genome and hepatitis B virus mini-chromosome in infected primary human hepatocytes

2019 ◽  
Vol 70 (1) ◽  
pp. e702
Author(s):  
Oceane Floriot ◽  
Giuseppe Rubens Pascucci ◽  
Matteo Pallocca ◽  
Francesca de Nicola ◽  
Michel Rivoire ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Human Hepatocytes ◽  
Host Genome ◽  
Primary Human Hepatocytes ◽  
B Virus

Molecular signature of hepatitis B virus regulation by interferon‐γ in primary human hepatocytes

2019 ◽  
Vol 50 (3) ◽  
pp. 292-302
Author(s):  
Takuto Nosaka ◽  
Tatsushi Naito ◽  
Hidetaka Matsuda ◽  
Masahiro Ohtani ◽  
Katsushi Hiramatsu ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Interferon Γ ◽  
Human Hepatocytes ◽  
Molecular Signature ◽  
Primary Human Hepatocytes ◽  
B Virus

(-)-cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine (524W91) inhibits hepatitis B virus replication in primary human hepatocytes.

1996 ◽  
Vol 40 (2) ◽  
pp. 520-523 ◽  
Author(s):  
L D Condreay ◽  
J P Condreay ◽  
R W Jansen ◽  
M T Paff ◽  
D R Averett
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Virus Replication ◽  
Human Hepatocytes ◽  
Hbv Replication ◽  
Primary Human Hepatocytes ◽  
B Virus ◽  
20 Nm

The anti-hepatitis B virus (HBV) activity of (-)-cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine (524W91) in cultures of primary human hepatocytes was examined. 524W91 was anabolized to the active 5'-triphosphate in these cells. HBV replication was equally inhibited in cultures incubated with 524W91 when the drug was added 24 h preinfection, at infection, or 24 h postinfection. 524W91 inhibited HBV replication by 50% at less than 20 nM in human hepatocytes.

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