The results of this study demonstrate the potent activity of PMEA on serum and liver DHBV DNA levels, confirming the original in vitro findings of Heijtink et al. (15
). PMEA produced a rapid antiviral response which was maintained during treatment. However, a relapse was seen on the cessation of therapy. These results indicate an antiviral efficacy equivalent to those of penciclovir and famciclovir (21
) and greater than that of ganciclovir (23
) when comparable dosages (10 mg/kg/day) were used.
PMEA may differ from other experimental nucleoside analogues evaluated for activity against HBV since it appears to reduce the viral protein and DNA loads within BDEC, a response which has not previously been documented with other nucleoside analogues (21
). This effect was detected when individual cell types were compared by using immunohistochemical stains for the detection of DHBcAg and DHBpreS1Ag, even though there was only a modest antiviral effect on total liver DHBcAg and DHBpreS1Ag, as measured by immunoblot hybridization. The likely explanation for this discrepancy is that the immunoblotting technique is extremely sensitive and is unable to detect changes in the very high levels of intrahepatic viral protein expression that occur in this model. Interestingly, there was a suggestion that this agent may also affect the intrahepatic levels of viral CCC DNA and RNA; however, the differences between the groups were not statistically significant (Fig. ).
The antiviral activity in BDEC is of importance because other studies have demonstrated that the levels of hepadnaviral proteins and nucleic acids in BDEC (4
) were unchanged during famciclovir (21
) and ganciclovir (23
) treatment. The reasons for this may include failure to deliver the antiviral agent to the site in adequate concentrations or the inability of this particular cell type to take up the agent or convert it to its active form. The demonstration of activity against hepadnaviruses within BDEC may indicate an important difference in the liver’s handling of this compound (21
). PMEA is an acyclic phosphonate, and so it requires only two phosphorylation steps within the cell to attain the active form. Phosphorylation by cellular enzymes to the triphosphate form is required by most nucleoside analogues, the initial phosphorylation step being the most critical (9
). This initial requirement may explain the poor activities of most nucleoside analogues in infected nonhepatocytes. The rate-limiting and initial phosphorylation step by adenosine kinase is therefore not required for PMEA. The rate-limiting step becomes adenylate kinase, a nucleotide kinase, to convert it to the active diphosphate form.
In contrast to the liver, there was no demonstrable antiviral effect in pancreatic cells during PMEA treatment. Pancreatic islet cells are derived from the neural crest and may express a different complement of salvage enzymes including kinases and phosphorylases or may not receive the antiviral agent in sufficient concentrations for the agent to have any significant effect. Further evaluation is required to identify the reasons for the lack of effect of PMEA in the pancreas.
PMEA is active only as a parenterally administered compound (28
), and an orally available form of PMEA has been tested in clinical trials with humans for the treatment of human immunodeficiency virus (7
), herpes simplex virus, and cytomegalovirus (28
) infections, with no adverse effects being reported to date. The bis(pivaloyloxymethyl) derivatives of PMEA have increased oral bioavailabilities and improved anti-herpes simplex virus and anti-human immunodeficiency virus potencies in cell culture, with greater uptake into cells compared to the amount of parent compound taken up (35
). A phase I study with patients with chronic hepatitis B has been completed (13
), and it showed substantial inhibition of viremia over the 4-week treatment period. Hepatitic flares were documented in 50% of the treated patients, requiring withdrawal from the study by two patients, but patients with increases in hepatic transaminase levels had more sustained HBV suppression posttherapy. Phase II studies are in progress.
PMEA has also been shown to possess immunomodulatory effects, in addition to direct antiviral activity, including dose-dependent increases in NK cell activity and interferon alpha production (6
). It is difficult to assess the usefulness of this immunostimulatory effect of PMEA in the congenital model of DHBV infection since the animal is essentially immunotolerant of the hepadnaviral infection. Immunostimulation plays an important role in viral clearance during the immunoelimination phase of chronic HBV infection in humans (27
), as demonstrated during successful interferon alpha therapy (26
). In the phase I study of Gilson et al. (13
), one patient experienced two hepatitic flares, the first during the treatment phase and the second during the follow-up period. It is interesting to speculate whether these multiple hepatitic flares might represent an initial NK cell-induced flare followed by a further interferon alpha-enhanced flare during the follow-up period. Further studies of the immunomodulating effects of nucleoside and nucleotide analogues to help provide further insight into the mechanisms of action of such agents, including PMEA, are clearly indicated.
The development of new nucleoside analogues for the treatment of chronic hepatitis B is of considerable clinical importance. However, major issues must be resolved. Relapse following the cessation of therapy is almost universal, certainly as a result of the fact that persistent CCC DNA species remain functional within hepatocytes (8
) but also probably as a result of persistent viral infection in nonhepatocytes, and more recently, antiviral resistance has been documented (1
). In the current study, PMEA was shown to reduce viral replication in both hepatocytes and BDEC and thus may offer the important advantage of reducing the viral burden at sites that other nucleoside analogues do not affect. In addition, it may prove to be useful in the treatment of patients who have a relapse of HBV replication following the development of antiviral resistance since it does not appear to be affected by the same mutations as lamivudine (1
). However, cell-based assays will need to be performed in order to confirm this apparent lack of cross-resistance. Further clinical studies of PMEA against chronic hepatitis B certainly appear to be warranted.