Inhibition of the replication of the DNA polymerase M550Vmutation variant of human hepatitis B virus by adefovir,tenofovir, L-FMAU, DAPD, penciclovir and lobucavirC. Ying,1 E. De Clercq,1 W. Nicholson,2 P. Furman2 and J. Neyts1 1Laboratory of Virology and Chemotherapy,

Department of Microbiology and Immunology, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Leuven, Belgium, 2Triangle

Pharmaceuticals, Durham, NC, USA

Received 13 August 1999; accepted for publication 22 October 1999

INTRODUCTION

There are more than 350 million carriers of hepatitis B virus

(HBV) worldwide. Infection with this virus may ultimately

lead to severe liver disease, including liver failure, cirrhosis

and hepatocellular carcinoma [1]. Currently, only lamivu-

dine and interferon-a (IFN-a) are approved for the treatment

of chronic HBV infection. However, treatment with IFN-aresults in variable and often unsatisfactory response rates

and is associated with serious side-effects [2]. Lamivudine is

effective in reducing HBV titres and is well tolerated [3].

However, following long-term treatment, resistance against

lamivudine may develop [4]. Similarly to the human

immunode®ciency virus (HIV) reverse transcriptase, ®ve

conserved regions (A to E) have been proposed in the HBV

DNA polymerase. The YMDD polymerase motif is located in

the C domain of both the HBV and HIV polymerases.

A methionine to isoleucine or valine mutation in this motif is

associated with resistance to lamivudine. The M550I

mutation (amino acid numbering system according to [5])

occurs independently of other mutations, whereas the

M550V mutation is also associated with the L524M

mutation [5±7].

Other compounds that are currently under clinical or pre-

clinical study for the treatment of chronic HBV infections

include famciclovir (the diacetyl ester of 6¢-deoxypenciclovir),

adefovir dipivoxil [the bis(pivaloyloxymethyl) ester of adefovir

(PMEA, or 9-(2-phosphonylmethoxyethyl)adenine]), tenofo-

vir [(R)-9-(2-phosphonylmethoxypropyl)adenine (PMPA)], L-

FMAU [1-(2¢-deoxy-2¢-¯uoro-b-L-arabinosyl)-5-methylura-

cil] and lobucavir [R(1a,2b,3a)]-9-[2,3-bis(hydroxymeth-

yl)cyclobutyl]guanine. Famciclovir is currently under

investigation in phase III clinical trials. This compound, alone

or in combination with other agents, results in a decrease

of HBV DNA levels in patients with chronic HBV infection

and/or in patients with HBV reinfection following liver

Abbreviations: DAPD, 1-b-2,6-diaminopurine dioxalane; DXG,

dioxalane guanine; HBV, hepatitis B virus.

Correspondence: J. Neyts, Rega Institute for Medical Research,

Minderbroedersstraat 10, B-3000 Leuven, Belgium.

Journal of Viral Hepatitis, 2000, 7, 161±165

Ó 2000 Blackwell Science Ltd

SUMMARY. Several nucleoside analogues (penciclovir, lob-

ucavir, dioxalane guanine [DXG], 1-b-2,6-diaminopurine

dioxalane [DAPD], L-FMAU, lamivudine) and acyclic nucleo-

side phosphonate analogues (adefovir, tenofovir) that are in

clinical use, in clinical trials or under preclinical development

for the treatment of hepatitis B virus (HBV) infections, were

evaluated for their inhibitory effect on the replication of a la-

mivudine-resistant HBV variant containing the methionine

® valine substitution (M550V) in the polymerase nucleo-

side-binding domain. The antiviral activity was determined

in the tetracycline-responsive HepAD38 and HepAD79 cells,

which are stably transfected with either a cDNA copy of the

wild-type pregenomic RNA or with cDNA containing the

M550V mutation. As expected, lamivudine was much less

(» 200-fold) effective at inhibiting replication of the M550V

mutant virus than the wild-type virus. In contrast, adefovir,

tenofovir, lobucavir, L-FMAU, DXG and DAPD proved almost

equally effective against both viruses. A second objective of

this study was to directly compare the antiviral potency of the

anti-HBV agents in HepG2 2.2.15 cells (which are routinely

used for anti-HBV drug-screening purposes) with that in

HepAD38 cells. HepAD38 cells produce much larger quan-

tities of HBV than HepG2 2.2.15 cells, and thus allow drug

screening in a multiwell plate format. All compounds were

found to be almost equally effective at inhibiting HBV repli-

cation in HepAD38 cells (as in HepG2 2.2.15 cells), except for

penciclovir, which was clearly less effective in HepAD38 cells.

Keywords: adefovir, DAPD, HBV, lamivudine, L-FMAU, pen-

ciclovir, tenofovir.

transplantation. Famciclovir is well tolerated upon long-term

treatment, but resistant viruses may develop [8]. Lobucavir

is, akin to penciclovir, a purine nucleoside analogue and it

inhibits, besides the replication of herpesviruses, HIV and HBV

[9]. This compound has entered phase II/III clinical trials for

the treatment of HBV infections. However, these studies have

been recently stopped. In a placebo-controlled study, lobu-

cavir, given orally (at 200 mg four times daily or 200 mg

twice daily for 28 days2 ), reduced HBV DNA levels by 2.8 logs,

and the viral DNA levels became undetectable

(< 2.5 pg ml)1) in four out of 17 subjects within 4 weeks.

Oral doses of lobucavir, as high as 400 mg kg)1 four times

daily,3 were well tolerated [10].

Dioxalane guanine (DXG) and its derivative, 1-b-2,6-di-

aminopurine dioxalane (DAPD) are purine nucleoside ana-

logues that inhibit HIV and HBV replication in vitro [11,12].

Pharmacokinetic studies indicate that DAPD is rapidly con-

verted to DXG, which is in fact the active metabolite.

In woodchucks experimentally infected with woodchuck

hepatitis virus (WHV), DAPD proved as effective as lamivu-

dine in reducing serum levels of circulating viral DNA when

administered for 12 weeks. L-FMAU is an L-nucleoside ana-

logue that has potent anti-HBV activity [13,14]. It is phos-

phorylated intracellularly by cytosolic thymidine kinase,

deoxycytidine kinase and mitochondrial deoxypyrimidine

kinase [15]. L-FMAU, even at concentrations up to 200 lM,

did not adversely affect mitochondrial function in hepatoma

cells [14]. Short-term administration (5 days) of this

compound (40 mg kg)1) to ducks experimentally infected

with duck hepatitis virus resulted in a signi®cant decrease in

viraemia. Histologically, no evidence of liver toxicity was

observed [16]. L-FMAU proved effective in woodchucks

chronically infected with WHV. When given at a dose of

10 mg kg)1 for 12 weeks, viral recrudescence did not occur

in the majority of the animals after cessation of therapy, and

molecular evidence of viral infection in the liver remained

undetectable during a 36-week post-treatment period

[17,18].

Adefovir and tenofovir belong to the class of the acyclic

nucleoside phosphonate analogues: these compounds were

initially developed to bypass the ®rst phosphorylation step by

viral kinases that is needed to activate various, antivirally

active, nucleoside analogues [19,20]. Adefovir is active

against herpesviruses, HIV and HBV [21]. Adefovir dipivoxil

has entered multicentre clinical phase III trials for the

treatment of HBV infection. Phase II studies for the treat-

ment of HBV infection have shown a 4 log10 (99.99%)

median reduction in HBV DNA levels following once daily

(oral) administration of the compound (at 10- or 30-mg

daily doses). Additional studies have shown that a 12-week

course of therapy can lead to loss of the HBV `e' antigen and

seroconversion [22,23]. The diphosphorylated form of ade-

fovir (PMEApp) has proved to be similarly effective in in-

hibiting recombinant wild-type HBV DNA polymerase as

recombinant HBV DNA polymerase carrying the lamivudine

resistance mutations M550I, M550V and L524M/M550V

[24; amino acid numbering used in the original manuscript

was M552I, M552V and L526M)]. Three liver transplant

recipients who were unresponsive to lamivudine responded

favourably to treatment with adefovir dipivoxil [25]. Also

tenofovir, a close congener of adefovir, is not only a potent

inhibitor of HIV replication, but also a potent anti-HBV agent

(C. Ying et al., unpublished4 ). The potential of tenofovir as an

antiviral drug has already been shown in animal models for

retrovirus infections [26,27]. Tenofovir proved very effective

in chronic and perinatal simian immunode®ciency virus

(SIV) infection in macaques and was well tolerated. The

compound has entered phase II clinical trials for the treat-

ment of HIV infection [26±28]. In animals, tenofovir appears

to be associated with few side-effects, which makes it an

attractive candidate for the treatment of chronic HBV in-

fections. From in vitro studies, as well as from animal and

clinical studies, it has become evident that resistance of

herpesviruses, HIV or HBV to acyclic nucleoside phospho-

nates, such as adefovir and tenofovir, does not readily de-

velop [reviewed in reference 28]. This characteristic

obviously offers additional bene®t for the treatment of

chronic viral infections such as those caused by HBV.

Although the diphosphorylated form of adefovir

(PMEApp) has equipotent inhibitory activity against wild-

type lamivudine-resistant (M550I, M550V, M550Y,

L526M) HBV DNA polymerase [24; amino acid numbering

M552I, M552V and L528M in the original manuscript]

and adefovir resulted in a virological response in three

HBV-infected transplant recipients who were unresponsive

to lamivudine [25], the effect of adefovir, and of its con-

gener tenofovir, on the replication of lamivudine-resistant

HBV has so far not been evaluated. We have now deter-

mined the effects of adefovir and tenofovir on the replica-

tion of the DNA polymerase M550V HBV mutant. We also

assessed whether this mutant virus had altered sensitivity

to penciclovir, lobucavir, L-FMAU, DXG and DAPD. To

achieve this, we employed the tetracycline-responsive

HepAD38 and HepAD79 cell lines [29,30]. These are

hepatoma cells that have been stably transfected with a

cDNA copy of the pregenomic RNA of wild-type virus or

with cDNA containing an A ® G mutation in the ®rst

position of the DNA polymerase gene codon 550 [315 , no.

539 in the original manuscript]. Withdrawal of tetracycline

from the culture medium of HepAD38 or HepAD79 cells

resulted in the initiation of viral DNA synthesis. Because

HepAD38 cells produce much larger quantities of HBV

DNA than HepG2 2.2.15 cells [29; and C. Ying et al.,

unpublished6 ], HepAD38 cells, unlike HepG2 2.2.15 cells,

allow anti-HBV drug screening in multiwell plate format.

We also studied whether HBV replication in HepAD38 cells

and HepG2 2.2.15 cells would be equally susceptible to

anti-HBV agents and, thus, whether HepG2 2.2.15 cells

could be replaced by HepAD38 cells for anti-HBV drug-

screening purposes.

Ó 2000 Blackwell Science Ltd, Journal of Viral Hepatitis, 7, 161±165

162 C. Ying et al.

MATERIALS AND METHODS

Compounds

Adefovir and tenofovir were obtained from Gilead Sciences

(Foster City, CA),7 lamivudine was from Glaxo Smithkline

(Stevenage, UK), penciclovir from Roche8;9 (Welwyn Garden

City, UK), lobucavir from Bristol-Myers Squibb10 (Wallingford,

CT) and DAPD, DXG and L-FMAU from Triangle Pharma-

ceuticals (Durham, NC).

Cell and culture conditions

HepG2 2.2.15 cells were grown in minimal essential

medium (MEM)11 medium supplemented with 10% fetal calf

serum (FCS), 2 mM L-glutamine, 0.02 U ml)1 insulin,

50 lg ml)1 gentamycin, 2.5 lg ml)1 fungizone,

100 lg ml)1 vancomycin and 50 nM dexamethasone. Cells

were seeded in 25-cm2 culture ¯asks at a density of 3 ´ 106.

Two days after reaching con¯uency, different dilutions of the

drugs in MEM containing 2% FCS were added. Medium was

changed at days 3 and 6, and fresh medium, either with or

without drug, was added. Total cellular DNA was isolated

(Qiagen Blood Kit; Qiagen12 , Hieten, Germany) at day 9 of the

experiment. HepAD38 and HepAD79 cells were maintained

in Dulbecco's MEM (DMEM)/F12 (50 : 50) medium supple-

mented with 10% FCS, 50 lg ml)1 penicillin, 50 lg ml)1

streptomycin, 100 lg ml)1 kanamycin (P/S/K),

400 lg ml)1 G418 and 0.3 lg ml)1 tetracycline. Cells were

seeded in six-well plates at a density of 0.1 ´ 106 cells cm)2.

After 3 days, cell cultures were washed ®ve times with pre-

warmed phosphate-buffered saline (PBS) after which they

were further incubated with tetracycline-free DMEM/F12

medium supplemented with 10% FCS, antibiotics and serial

dilutions of the different drugs. On day 3, medium was re-

moved and fresh medium added. Total cellular DNA was

extracted 6 days after the start of the experiment.

Quanti®cation of HBV DNA

DNA was blotted onto a nylon membrane (Hybond-N;

Amersham, Little Chalfont, Bucks, UK13 ) and UV cross-linked

after which prehybridization was carried out for 1 h at 42 °C

followed by overnight hybridization at 42 °C with

25 ng ml)1 of a digoxigenin-labelled HBV-speci®c probe.

The latter, spanning a 523-bp fragment in the core gene

of the HBV genome, was generated by polymerase chain

reaction (PCR) ampli®cation using the primer pair:

5¢-CTGTGGAGTTACTCTCGTTTTTGC-3¢ and 5¢-CTAACA-

TTGAGATTCCCGAGATTG-3¢. The PCR reaction contained

5 ll 10´ PCR buffer (Gibco, Paisley, Strathclyde, UK),

100 lM each of dATP, dGTP and dCTP, 67 lM dTTP,

33 lM dig-dUTP (Boehringer Mannheim, Mannheim, Ger-

many), 1 lM each primer and 300 ng template DNA, as

well as 1 ll Amplitaq DNA polymerase (5 U ml)1). The fol-

lowing PCR programme was run: 1 cycle of 5 min at 94 °C,

30 cycles of 30 s at 94 °C, 60 s at 57 °C and 30 s at 72 °C,

and 1 cycle of 7 min at 72 °C. The fragment generated was

gel-puri®ed and stored at )20 °C. Following hybridization,

the membranes were washed twice with 2´ sodium saline

citrate (SSC)14 , 0.1% sodium dodecyl sulphate (SDS) for

10 min at room temperature followed by two washes of

15 min each in 0.1 ´ SSC, 0.1% SDS at 65 °C. After incu-

bation with 1% blocking buffer (Boehringer Mannheim) for

2 ´ 15 min, the membranes were incubated with an anti-

digoxigenin antibody conjugated to alkaline phosphatase

(antidigoxigenin-AP, Fab fragments) (Boehringer Mann-

heim) for 1 h followed by detection of chemiluminescence by

using standard methods. The signal was quanti®ed densito-

metrically, as described previously [30].

RESULTS AND DISCUSSION

The 50% effective concentration (EC50) values for inhibition

of HBV DNA synthesis in HepG2 2.2.15, HepAD38 and

HepAD79 cells are presented in Table 1. As expected, the

virus produced in the HepAD79 cells proved markedly less

(» 200-fold) susceptible to the antiviral activity of lamivu-

dine than the virus produced in the HepAD38 cells. Ladner

et al. [31] reported a 26-fold reduced activity of lamivudine

in HepAD79 cells as compared to HepAD38 cells. In tran-

sient transfection assays, HepG2 cells that had been trans-

fected with a plasmid containing the cDNA of the HBV

pregenomic RNA with the M550V mutation proved 330-fold

less sensitive to lamivudine [32]. Also ()) FTC (L(-)-2¢,3¢-dideoxy-S-¯uoro-3¢-thiacytidine)15;16 and ddC (2¢,3¢-dide-

oxycytidine)15;16 were less effective in HepAD79 cells than in

HepAD38 cells [32]. As demonstrated here, the acyclic

nucleoside phosphonate analogues adefovir and tenofovir,

were equally effective (tenofovir) or almost equally effective

(adefovir) in HepAD38 and HepAD79 cells. Furthermore,

lobucavir, L-FMAU, DAPD and DXG proved equipotent at

inhibiting HBV replication in HepAD79 and HepAD38 cells.

A second objective of the present study was to directly

compare the ef®cacy of the anti-HBV agents in HepAD38

and HepG2 2.2.15 cells. While penciclovir inhibited viral

DNA synthesis in HepG2 2.2.15 cells with an EC50 value of

3.5 lg ml)1, the compound was > 10-fold less active in

HepAD38 or HepAD79 cells as compared to HepG2 2.2.15

cells (Table 1). The reduced ef®cacy of penciclovir in

HepAD38 cells could be attributed to a reduced phosphory-

lation of the compound in HepAD38 cells as compared to

HepG2 2.2.15 cells. In contrast to penciclovir, the other

compounds studied were almost equally effective (adefovir,

tenofovir) or slightly less effective (LBV, DXG, L-FMAU) at

inhibiting HBV replication in HepAD38 as compared to

HepG2 2.2.15 cells. Thus, the HepAD38 cell system can be

used for drug-screening purposes, although some com-

pounds (such as penciclovir) may be quantitatively less

effective in this system.

Ó 2000 Blackwell Science Ltd, Journal of Viral Hepatitis, 7, 161±165

Inhibition of the DNA polymerase M550V mutation variant of HBV 1631

In conclusion, we have demonstrated that adefovir, ten-

ofovir, lobucavir, L-FMAU, DAPD and DXG are equally

effective at inhibiting the replication of wild-type HBV DNA

as well as the replication of a lamivudine-resistant variant

carrying the M550V mutation in the DNA polymerase.

Together with the ®ndings of Xiong et al. [24], who dem-

onstrated that adefovir diphosphate (PMEApp) is equally

effective at inhibiting the enzyme activity of wild-type and

HBV DNA polymerase variants containing the lamivudine-

resistance mutations M550I or M550V or L524M/M550V,

our observations lend further support to the use of adefovir

and tenofovir in the treatment of infections with lamivudine-

resistant HBV variants. Moreover, DAPD and L-FMAU are

drugs that have potential for the treatment of HBV infections

with lamivudine-resistant M550V variants.

ACKNOWLEDGEMENTS

J. Neyts is a postdoctoral research assistant from the `Fonds

voor Wetenschappelijk Onderzoek (FWO) ± Vlaanderen'.

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Lamivudine (3TC) 0.0016 � 0.0005 0.0037 � 0.0009 0.82 � 0.1 221

Adefovir (PMEA) 0.03 � 0.01 0.05 � 0.01 0.14 � 0.1 2.8

Tenofovir (PMPA) 0.04 � 0.02 0.05 � 0.02 0.05 � 0.01 1

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Inhibition of the DNA polymerase M550V mutation variant of HBV 1651