Cellular Pharmacology of the Anti-Hepatitis B Virus Agent β- l -2′,3′-Didehydro-2′,3′-Dideoxy-N ⁴ -Hydroxycytidine: Relevance for Activation in HepG2 Cells

Abstract

A large number of nucleoside analogues have been described as inhibitors of hepatitis B virus (HBV) and HIV replication. Recently L-nucleoside analogues in particular have gained increasing interest. They are characterized by an opposite configuration from that of the natural D-nucleoside analogues and represent one of the most attractive groups of antiretroviral compounds, including ß-L-2Ј,3Ј-dideoxy-3-thiacytidine (3TC) and its, and ß-L-2Ј,3Ј-didehydro-2Ј,3Ј-dideoxy-2Ј-fluoro-cytidine (L-2ЈFd4C) (3,5,22).Some of them not only have been found to be more potent than their corresponding D-nucleosides but seem to exhibit lower cytotoxicity and have been proved to be effective and selective agents for the treatment of chronic hepatitis B virus infections (4). However, only long-term therapy with a single nucleoside for several years was shown to be able to completely suppress HBV DNA in serum of patients and to reverse the progression of the disease. The disadvantage connected with such therapy regimens is the development of drug-resistant HBV strains (22). Therefore, the challenge will be to develop more-efficient drugs for shorter treatment regimens and to combine them to reach synergistic or at least additive drug action. This approach has been described not only as being highly efficient for the treatment of HIV infections but also as preventing the development of resistant mutants. Therefore, AIDS therapy is considered a model for future therapy of chronic HBV infections (17).Recently we described a series of new ß-L-N 4 -hydroxydeoxycytidine and ß-L-5-methyl-deoxycytidine derivatives as inhibitors of HBV replication. Between them, ß-L-2Ј,3Ј-didehydro-2Ј,3Ј-dideoxy-N 4 -hydroxycytidine (L-Hyd4C) (Fig. 1) emerged as the most effective in suppression of virus production in HepG2.2.15 cells (50% effective dose [ED 50 ] ϭ 0.03 M), displaying an extremely low cytotoxicity (50% cytotoxic dose [CD 50 ] for HepG2 cells ϭ 2,500 M) (12).These encouraging features have prompted us to investigate the cellular pharmacology of L-Hyd4C in a hepatic cell line. This included the activation of this unnatural L-deoxycytidine nucleoside to its 5Ј-mono-, 5Ј-di-, and 5Ј-triphosphate, the search for other metabolites, and the estimation of the intracellular halflives (t 1/2 ) of the 5Ј-di-and 5Ј-triphosphate of (This work was presented in part at BIT's 5th Anniversary Congress of International Drug Discovery Science and Technology, 7 to 13 November 2007, Xi'an and Beijing, China.) MATERIALS AND METHODSCompounds. The synthesis and characterization of L-Hyd4C and its 5Ј-triphosphate were described elsewhere (E. Matthes, M. von Janta-Lipinski, H. Will, H. Sirma, and A. High-performance liquid chromatography (HPLC) analysis by methods described below showed 12% of radioactive contamination of the tritiated products which were separated by HPLC.Acetonitrile (gradient grade), tetrabutylammonium phosphate (monobasic, purissimum), and 3-deazauridine (deazaUR) were obtained from Sigma-Aldrich (Taufkirchen, Germany). Ammonium dihydrogen ph...

Therapeutic Inhibitory Activities of N‐Hydroxy Derived Cytidines: A Patent Overview

“…In 2010, Matthes et al , have investigated and reported β‐L‐2′,3′‐didehydro‐2′,3′‐dideoxy‐N 4 ‐Hydroxycytidine 45 as potent inhibitor of HBV virus in HepG2 cells (Figure 35). [72] The biological evaluation results showed that compound 45 had moderately potent antiviral activity and lowers cytotoxicity of HBV replication against HepG2 cells to 5’‐triphosphate in cells with 50 % Effective dose (ED 50 ) of 0.03 μM.…”

Status of antiviral therapeutics against rabies virus and related emerging lyssaviruses

“…Beyond ribavirin, ribonucleoside analogs present an exciting option for the discovery of broad-spectrum lyssavirus inhibitors. For instance, the ribonucleoside analogy N 4hydroxycytidine reportedly blocks both seasonal and pathogenic influenza virus strains, respiratory syncytial virus, Ebola virus, chikungunya virus, and hepatitis B and C viruses [182][183][184][185][186][187][188] with excellent pharmacological properties [182]. If antiviral activity equally extends to RABV and related lyssaviruses, compounds like N 4 -hydroxycytidine may present an example for a viable next-generation therapeutic option to address the rabies challenge.…”

Strong Antiviral Activity of the New L-Hydroxycytidine Derivative, L-HYD4FC, In HBV-Infected Human Chimeric UPA/SCID Mice

“…Recently we described the hydroxylation of the amino group of some of these l-deoxycytidine analogue-produced compounds, which maintained their strong anti-HBV activity but exhibited markedly reduced cytotoxicity [6]. The most effective compound was b-l-2′,3′didehydro-2′,3′-dideoxy-N 4 -hydroxcytidine (l-Hyd4C; 50% effective dose in HepG2.2.15 cells 0.03 mM and 50% cytotoxic dose in HepG2 cells 2,500 mM) [6,7]. For the first in vivo study of this new group of compounds, a 5-fluoromodified derivative of the l-Hyd4C, the b-l-2′,3′-didehydro-2′,3′-dideoxy-N 4 -5fluorocytidine (l-Hyd4FC) predicting the most favourable pharmacokinetic properties was selected.…”

“…To establish HBV infection in mice, human chimeric mice received a single intraperitoneal injection of mouse-derived HBV-positive serum (2×10 7 HBV DNA copies, genotype D, hepatitis B e antigen [HBeAg]-positive). Only mice that had been infected with HBV for ≥10 weeks and hence displayed stable viraemia levels of >10 7 HBV DNA copies/ml were used for the study.…”

“…To investigate the efficacy of the new compound l-Hyd4FC (Figure 1A) to reduce HBV viraemia in vivo, human chimeric uPA mice with stable viraemia (median 4×10 7 HBV DNA copies/ml; n=7) received daily injections of 50 mg/kg l-Hyd4FC. As control, chimeric mice displaying similar viraemia levels were either injected with saline (n=4) or received 4 weeks of oral administration of 30 mg/kg/day lamivudine (n=4).…”