10.1128/AAC.44.3.551-560.2000.

2000, 44(3):551. DOI:Antimicrob. Agents Chemother. ShawDanni Colledge, Gilda Civitico, Stephen Locarnini and Tim and AdefovirCombinations of Penciclovir, Lamivudine, In Vitro Antihepadnaviral Activities of

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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY,0066-4804/00/$04.0010

Mar. 2000, p. 551–560 Vol. 44, No. 3

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

In Vitro Antihepadnaviral Activities of Combinations ofPenciclovir, Lamivudine, and Adefovir

DANNI COLLEDGE, GILDA CIVITICO, STEPHEN LOCARNINI, AND TIM SHAW*

Victorian Infectious Diseases Reference Laboratory, North Melbourne, Victoria, 3051, Australia

Received 7 June 1999/Returned for modification 27 August 1999/Accepted 7 December 1999

Penciclovir {9-[2-hydroxy-1-(hydroxymethyl)-ethoxymethyl]guanine [PCV]}, lamivudine ([2]-b-L-2*,3*-dideoxy-3*-thiacytidine [3TC]), and adefovir (9-[2-phosphonylmethoxyethyl]-adenine [PMEA]) are potentinhibitors of hepatitis B virus (HBV) replication. Lamivudine has recently received approval for clinical useagainst chronic human HBV infection, and both PCV and PMEA have undergone clinical trials against HBVin their respective prodrug forms {famciclovir and adefovir dipivoxil [bis-(POM)-PMEA]}. Since multidrugcombinations are likely to be used to control HBV infection, investigation of potential interactions betweenPCV, 3TC, and PMEA is important. Primary duck hepatocyte cultures which were either acutely or congenitallyinfected with the duck hepatitis B virus (DHBV) were used to investigate in vitro interactions between PCV,3TC, and PMEA. Here we show that the anti-DHBV effects of all the combinations containing PCV, 3TC, andPMEA are greater than that of each of the individual components and that their combined activities areapproximately additive or synergistic. These results may underestimate the potential in vivo usefulness ofPMEA-containing combinations, since there is evidence that PMEA has immunomodulatory activity and, atleast in the duck model of chronic HBV infection, is capable of inhibiting DHBV replication in cells other thanhepatocytes, the latter being unaffected by treatment with either PCV or 3TC. Further investigation of theantiviral activities of these drug combinations is therefore required, particularly since each of the componentdrugs is already in clinical use.

It has been estimated that approximately 5% of the world’shuman population is chronically infected with the hepatitis Bvirus (HBV) (23) and as a direct consequence are at dramat-ically increased risk of developing cirrhosis, hepatocellular car-cinoma, or decompensated liver disease (23, 36). Antiviralchemotherapy remains the only option for controlling HBVinfection in these individuals, for whom the HBV vaccineswhich are now available provide no benefit (23). Unfortu-nately, the development of effective chemotherapy for treat-ment of chronic HBV infection has proven difficult due to avariety of factors which are both virus and host dependent (32).At present, the only licensed treatment for chronic hepatitis Binfection in most countries is alpha interferon, use of which isonly partially effective and frequently limited by the occurrenceof adverse side effects (23). Although the use of nucleoside andnucleoside analogs as antihepadnaviral agents has been simi-larly disappointing (9, 30), prospects for their future use haveimproved dramatically, as several of the more recently devel-oped analogs have been found to be potent and selective in-hibitors of HBV replication (10, 32). These analogs fall intotwo broad categories: (i) those which have modified cyclic oracyclic sugar configurations and (ii) those which have the “un-natural” L configuration. Of the recently developed analogswhich have already been used clinically or are about to enterpreliminary clinical trials against HBV, all representatives ofthe first category are purine derivatives, whereas all represen-tatives of the second category are pyrimidine derivatives. Pen-ciclovir {9-[2-hydroxy-1-(hydroxymethyl)-ethoxymethyl]guanine[PCV]}, a deoxyguanosine analog, and lamivudine ([2]-b-L-29,39-dideoxy-39-thiacytidine [3TC]), a deoxycytidine analog,

are representative of the first and second categories, respec-tively.

PCV was originally developed as an antiherpesvirus agent(33). Its antihepadnaviral activity was first demonstrated in theduck model of HBV infection (26, 31, 33), and it has alreadyundergone preliminary clinical trials against chronic HBV in-fection in its orally available form, famciclovir (27). 3TC wasoriginally developed as an inhibitor of retroviral reverse tran-scriptase and was later shown to possess potent antihepadna-viral activity (33); following successful clinical trials againstchronic HBV infection (16, 24), it was approved for clinical useagainst chronic HBV in several countries, including the UnitedStates, Canada, and members of the European Union. BecausePCV and 3TC have been in clinical use for some time asantiherpesvirus and antiretrovirus agents, respectively, there isalready a considerable body of literature attesting to theirsafety and efficacy (33).

Treatment with either PCV or 3TC alone causes a rapid andsubstantial decrease in viremia in most chronically HBV-in-fected patients, and it has recently been shown that the de-crease is sufficient to restore the antiviral T-cell responsewithin 2 weeks of the initiation of treatment in a subset of3TC-treated patients (6). We have previously shown that the invitro anti-duck HBV (anti-DHBV) activities of PCV and 3TCare synergistic (11), and preliminary reports of the successfulclinical use of this combination both simultaneously (16) andsequentially (37) have already appeared.

Adefovir (9-[2-phosphonylmethoxyethyl]adenine [PMEA]),an acyclic dAMP analog, is a third compound which, in its oralprodrug form, adefovir dipivoxil [the bis(pivaloyloxy-methyl)-ester of PMEA (2)], has shown promise in preliminary clinicaltrials against chronic HBV infection (18); it has also under-gone clinical trials against human immunodeficiency virus(HIV) (4, 13). PMEA has broad-spectrum antiviral activity,being active against herpesviruses, retroviruses, and hepadna-viruses (28). The antihepadnaviral activity of PMEA was first

* Corresponding author. Mailing address: Victorian Infectious Dis-eases Reference Laboratory, Locked Bag 815, Carlton South, Victoria,3053, Australia. Phone: 61 3 9342 2615. Fax: 61 3 9342 2666. E-mail:Tim.Shaw@nwhcn.org.au.

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observed in human hepatocellular carcinoma cells stably trans-fected with HBV and in primary duck hepatocytes infectedwith DHBV (21, 22).

The encouraging results from preliminary clinical trials ofPCV, 3TC, and PMEA are tempered by a large body of expe-rience suggesting that long-term remissions of chronic HBVinfection following chemotherapy are uncommon, with mostpatients experiencing a relapse or a rebound in viremia afterdrug administration is stopped (10, 23). This phenomenon isattributable to two important characteristics of chronic hepad-naviral infection: (i) the potential for reinitiation of infectionby viral particles released from cell and tissue sites which mayact as sanctuaries for potentially infectious virus, even duringaggressive antiviral therapy (26, 29, 30), and (ii) the persistenceof intranuclear viral supercoiled, covalently closed circular(CCC) DNA, the hepadnaviral genomic species which servesas the template for transcription of viral mRNA (mediated byhost cell RNA polymerase II) but which is only indirectlyinvolved in DNA replication and is minimally affected by de-oxynucleoside triphosphate (dNTP) analogs (8). These char-acteristics ensure that chemotherapeutic control of chronicHBV infection is most likely to require long-term treatment.Such long-term treatment may be compromised by cumulativedrug toxicity and also carries increased risks for developmentof viral resistance. Although to date no consistent dose-relatedtoxic effects have been observed during clinical use of PCV,3TC, or PMEA, strains of HBV resistant to PCV and 3TC havealready been reported (1, 3, 40). In both cases resistant virusmay become predominant after less than 12 months’ antiviraltherapy in as many as 25% of treated patients, depending onthe clinical setting (41). Consequently, it is becoming increas-ingly apparent that no single antiviral agent will be able to

suppress chronic hepadnaviral infection in the long term (9–11). Preliminary indications of the efficacy of drug combina-tions can be obtained from in vitro experiments; accordingly,we have investigated the effects of combinations containingPCV, 3TC, and PMEA on DHBV replication in primary duckhepatocytes (PDH). Here we report that all possible combina-tions of these drugs behave additively or synergistically in thissystem. Although these observations are not necessarily pre-dictive of in vivo efficacy, use of such combinations may also bebeneficial in vivo, and their use may inhibit the rate of devel-opment of viral resistance. Indeed, since 3TC, PCV, andPMEA are already in clinical use, further investigation of pos-sible interactions in terms of pharmacology, toxicology, andpossible viral cross-resistance would be appropriate.

MATERIALS AND METHODS

Chemicals and drugs. All chemicals and reagents were of analytical grade orof the highest grade obtainable commercially. Lamivudine and penciclovir wereprovided by SmithKline Beecham Pharmaceuticals, King of Prussia, Pa., andPMEA was supplied by Gilead Sciences, Foster City, Calif. Stock solutions (50mM) of 3TC and PCV were prepared in distilled water or dimethyl sulfoxide,respectively, and stored at room temperature in light-proof containers. Stocksolutions of PMEA were prepared when required and were discarded after 1 day.Serial working dilutions of drugs and drug combinations at 100 times the final

FIG. 1. Drug combination assay protocol. (A) Diagram illustrating how com-bination assays were set up in 12-well cell culture plates. PDH were plated on day1, and triplicate cultures exposed to either A, B, A plus B, or no drug (control),where A and B are the concentrations of drugs A and B, respectively. After 9days’ incubation, intracellular accumulation of DHBV RI was assayed by dot blothybridization with a 32P-labeled full-length DHBV cDNA probe. (B) Autora-diograph showing an example of results obtained after processing cell lysatesderived from six 12-well plates. In rows A to F, columns 1 through 3, 4 through6, 7 through 9, and 10 through 12 correspond to triplicate cultures from a singleplate. The total drug concentration (A 1 B) which is present in columns 7through 9 is shown (as a micromolar concentration) on the right, at the end ofeach row. There were no samples in row G. Row H contained DHBV DNAstandards. In this example, drug A was PMEA and drug B was PCV. They werepresent at equimolar concentrations. Note that dot blot data presented in tablesand graphs were derived directly from scintillation counting, (which gives linearresponses over a much wider range), not from densitometry of autoradiographs.(C) Intracellular DHBV levels during culture. PDH were plated on day 0 andincubated overnight in drug-free medium. They were harvested immediatelyafter plating and at 2-day intervals thereafter until day 12, and lysates wereassayed for DHBV DNA. Exponential curves were fitted to the data from days4 onward and were used to estimate the doubling time (approximately 1.5 days)for intracellular DHBV in the absence of inhibitors of replication. In the pres-ence of 1.0 mM penciclovir, a concentration which was not cytotoxic but wassufficient to completely block DHBV replication, intracellular DHBV DNAdecreased with a half-life of 3.3 days, close to the estimated half-life of DHBVCCC DNA under similar conditions (see reference 8). (Data were pooled fromthree separate experiments; DHBV signals were standardized as fractions of theday 0 signal; error bars represent standard deviations.)

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concentration were prepared in sterile isotonic saline and added to cell culturemedium immediately before each medium change. The final concentration ofdimethyl sulfoxide in cell culture medium never exceeded 0.2% (vol/vol). Drugconcentrations, purity, and stability were checked by UV spectrophotometry andhigh-performance liquid chromatography (HPLC).

PDH isolation and culture. One-day-old Pekin-Aylesbury cross ducks congen-itally infected with an Australian strain of DHBV were obtained from a com-mercial supplier (7, 31). DHBV-free ducklings were obtained from a differentsupplier, and DHBV-infected and uninfected ducklings were housed separately.Viremia was monitored by serum dot blot hybridization as previously described(7). Primary hepatocytes were obtained from livers of 7- to 14-day-old congen-itally infected or virus-free ducklings and seeded into 12-well plastic cultureplates (ICN Biomedicals, Aurora, Ohio) at a density of approximately 0.75 3 106

to 1.0 3 106 PDH per well (7, 29). PDH were allowed to attach overnight beforethe first medium change (on day 1 postplating) and were maintained at 37°C ina humidified incubator under 5% CO2 with medium changes every second day(7). Under these conditions, DHBV replicative intermediates (RI), includingCCC DNA, accumulate intracellularly from day 3 or 4 onwards, paralleled bysecretion of virions into the culture medium (7, 8, 11, 17, 38, 39). The slightdecrease in intracellular DHBV during the first 2 or 3 days in culture is due toloss of cells (see Fig. 1). Like primary hepatocytes of other species, PDH inculture show negligible mitotic activity. Intracellular CCC DNA, the least abun-dant intracellular DHBV RI, becomes easily detectable after about day 6 post-

plating (7, 8, 38, 39). Antiviral assays were performed on two separate occasions,using PDH from different ducklings for each assay. Antiviral effects were as-sessed by monitoring viral DNA replication and virus-specific protein synthesis atthe end of treatment.

Cytotoxic and cytostatic effects of treatment. In experiments conducted inparallel with antiviral assays, PDH congenitally infected with DHBV were ex-posed continuously from day 1 postplating to PMEA, PCV, and 3TC alone or incombination at total concentrations of 0, 0.25, 0.5, 1.0, 2.0, or 4.0 mM for 9 days.On day 10, cell viability was assessed by microscopic examination immediatelybefore each medium change and by neutral red uptake (31). Since PDH arequiescent, they cannot be used to assess potential cytostatic (antiproliferative)effects, for which a well-characterized human hepatocyte-derived cell line,Huh-7, was used. For these assays, Huh-7 cells were seeded into 96-well flat-bottom microtiter plates at a density of approximately 104 cells per well inDulbecco’s modified Eagle medium supplemented with 5% (vol/vol) fetal calfserum. They were allowed to attach for 4 h, after which the medium was replacedby fresh medium with or without PMEA, PCV, or 3TC alone or in combinationat total concentrations of 0, 0.25, 0.5, 1.0, 2.0, or 4.0 mM (three sets of triplicatesfor each treatment). After 2, 4, or 6 days, cell viability was assessed by neutral reduptake. Huh-7 monolayers in drug-free control wells reached confluence on day4. Neutral red uptake data were fitted to exponential decay equations of the formy 5 a 3 102bx, where y is the percentage of dye uptake relative to the averagevalue (defined as 100%) for untreated controls and x is the drug concentration.

FIG. 2. Cytotoxic or cytostatic effects of PCV, 3TC, and PMEA alone and in combination. (A) PDH cultures were exposed continuously to PCV, 3TC, and PMEAalone or in an equimolar combination for 9 days from day 1 postplating. Viability was estimated by extrapolation of exponential plots of neutral red uptake, expressedas a percentage relative to the mean uptake in drug-free controls (y axis, log scale) versus drug concentration (x axis, linear scale). (B) Sets of growing Huh-7 cell cultureswere exposed continuously to PCV, 3TC, and PMEA alone or in an equimolar combination for 2, 4, or 6 days, after which when cell numbers and viability were assessedby neutral red uptake. Cytotoxicity increased with exposure time, as shown by this plot of CC50s (estimated as for PDH) (y axis, log scale) versus day of assay (x axis,linear scale).

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The a and b parameters define the curves’ maxima and slopes, respectively.CC50s (the drug concentrations which reduced uptake to 50% of the controlvalue) were estimated by solving each equation for the x (drug concentration)value corresponding to a y (uptake percentage) value of 50.

Antiviral activities of PMEA, PCV, and 3TC alone or in combination incongenitally infected PDH. Replicate sets of PDH monolayers congenitally in-fected with DHBV were continuously exposed to PCV, 3TC, and PMEA aloneor in combination at various concentrations and concentration ratios for 9 daysbeginning on day 1 postplating. Total drug concentrations (in micromolar con-centrations) were 10, 5, 0, and 10 halving dilutions from 2 to 0.0078125. Eachconcentration and concentration ratio was assayed in triplicate (Fig. 1). Cellswere harvested on day 10 postplating, and antiviral effects were assessed bymonitoring viral DNA replication and virus-specific protein synthesis. In exper-iments using PMEA in combination with PCV or 3TC, the drugs were present atfixed molar ratios (PMEA to PCV or PMEA to 3TC) of either 0:1, 1:9, 1:4, 1:1,4:1, or 1:0. A wide range of concentrations and concentration ratios was used toincrease the chances of detection of possible anomalies in combination behavior.In initial experiments in which all three drugs were present in combination, theconcentration ratio was 1:1:1. The ratio was modified for subsequent experiments(see below).

Antiviral activities of PMEA, PCV, and 3TC alone or in combination inacutely infected PDH. One set of studies was carried out using acutely infectedPDH, in which the viral load is lower and the effects of drugs on early replicationevents can be assessed more easily compared to those for chronically infectedPDH. Uninfected PDH cultures were prepared as described above and allowedto attach overnight before infection with DHBV. Infection was achieved byremoving the medium and then incubating the cell monolayers for 1 h withDHBV-positive serum diluted in culture medium to a final multiplicity of infec-tion of approximately 5 to 10 viral genome equivalents per cell (300 ml/well).Pooled sera from 4- to 5-week-old ducklings with high DHBV titers were used asthe inoculum. Mock infection of control PDH was performed using DHBV-freeduck serum. After an hour, inocula were removed and replaced by fresh mediumwith or without test compounds at various concentrations as above. Cultureswere maintained for as long as 9 days after infection.

Inhibition of DHBV-specific protein synthesis and CCC DNA production byPMEA, PCV, and 3TC alone or in combination. Two separate sets of sampleswere subjected to further analysis. They were derived from cells infected eitheracutely or chronically with DHBV. Drug concentrations were chosen so that eachinhibitor was present at a concentration expected to cause 25 to 50% inhibitionof DHBV DNA replication based on previous experience. Samples were pre-pared for assay of DHBV-specific protein synthesis and CCC DNA productionby immunoblotting or Southern blotting, respectively. Further details are pro-vided in the legend to Fig. 5.

Preparation of radioactive probe, detection of DHBV DNA replication, andanalysis of viral replicative species. Southern hybridization was performed, andDNA dot blots were probed with a full-length DHBV DNA clone labeled with[a-32P]dCTP as described previously (7, 8, 31) by using the Random Primer Plusextension kit (Dupont-NEN, Boston, Mass.). Total cellular DNA was extractedfrom cell lysates and probed for DHBV DNA by dot blot hybridization asdescribed previously (7, 31). Intracellular DHBV replicative species were ana-lyzed by Southern blot hybridization after electrophoresis through 1.5% agarosegels and capillary transfer to positively charged nylon membranes (7, 31). Intra-cellular DHBV CCC DNA was extracted from lysates by a specific enrichmentprocedure (39) and analyzed by Southern blotting as previously described (7, 31).Hybridization conditions and autoradiographic procedures have been describedin detail previously (7, 31).

Detection of DHBV-specific protein synthesis. Immunoblotting was performedas described elsewhere (31). Polyclonal rabbit antibodies to the carboxy-terminalpart of the DHBV core protein or monoclonal antibodies to the DHBV pre-Santigen were used to stain immunoblots. Bound antibody was detected using anenhanced chemiluminescence (ECL) kit (Amersham Australia, North Ryde,New South Wales, Australia), according to the manufacturer’s instructions. De-tailed procedures have been reported previously (7, 31).

Quantitation of antiviral effects and data analysis. DNA dot blots were au-toradiographed to visualize bound probe. The amount of bound probe on eachblot was then quantitated directly by liquid scintillation counting using a Micro-plate Liquid Scintillation Counter (Top Count; Packard Instruments, Meriden,Conn.). Image densities in autoradiographs following Southern blot analysis orECL (immunoblot) exposures following immunoblotting were quantitated bydensitometry. Viral replication levels in drug-treated PDH were expressed aspercentages relative to the mean values for drug-free controls. Two-dimensionaldose-response plots for individual drugs and drug combinations were generatedwith the aid of TableCurve2D, a graphics-statistics software package from JandelScientific (San Rafael, Calif.).

Definitions and modelling of drug interactions. Various methods have beenemployed for analysis of drug interactions, generating a large body of literatureon the subject (for comprehensive reviews, see Berenbaum [5] and Greco et al.[19]), in which definition of the outcome expected from the use of drug combi-nations is crucial, because opposing phenomena are defined as significantlygreater (synergy) or significantly less (antagonism) than the “expected” outcome.Two main alternative approaches, termed “Bliss independence” and “Loweadditivity,” have been generally accepted and widely used as the basis for pre-

FIG. 3. (A) Diagram illustrating parameters of the logistic dose-responsecurve. The curve is described by the equation y 5 k 1 a/(11[x/b]c), where xrepresents the dose (drug concentration) and y represents the response (virusreplication expressed as a percentage of that in untreated control cultures). k isa constant corresponding to the minimum value of y (i.e., the lower plateau of thecurve); a defines the curves’ amplitude, being the difference between the maxi-mum and minimum values of y. The b value defines the transition center, whichis the x value at half the amplitude. The value of c controls the transition width(distances on the x axis on either side of b corresponding to the middle 2/3 of a).In this form of the equation, the slope of the curve becomes steeper as cincreases. Note that the x axis is logarithmic and the y axis is linear. In the assaysdescribed here, k 5 0 and the amplitude a, representing the amount of viralreplication in uninfected controls, is by definition 100%. b therefore correspondsto the EC50 (the drug concentration which reduces viral replication to 50% of thecontrol value). (B) Application of dose-response curve analysis to a set of data.After 9 days’ continuous exposure of chronically DHBV-infected PDH to eitherPMEA, 3TC, PCV, or equimolar mixtures, intracellular DNA was extracted anddot blots were prepared and probed as described in the text. The mean probesignal from drug-treated PDH was expressed as a percentage of the mean fromuntreated controls and plotted (on the y axis) versus the total drug concentration(x axis). Each data point represents the mean of triplicates. This example showsdata from experiment 2 (see Table 1), in which the total concentrations requiredto inhibit intracellular DHBV replication by 50% (EC50s) for PMEA, PCV, 3TC,PCV plus 3TC, and PMEA plus PCV plus 3TC) were 0.17, 0.43, 0.64, 0.28, and0.2 mM, respectively. The last two values correspond to 0.14 and 0.1 mM for eachcomponent.

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dicting the effects of drug combinations. Although each has particular advantagesand disadvantages, only the latter is free from implicit assumptions about mech-anisms of action (5, 19).

In the Bliss independence model, the combined effect (Exyz) of drugs incombination is the product of the individual effects (Exy, Ey, and Ez, respectively),and can be calculated from the equation Exyz 5 (Ex)(Ey)Ez), in which the effectE represents fractional viral replication rather than inhibition of replication.

An alternative model, known as Lowe additivity, is based on a definition ofzero interaction which can be expressed as (dx/Dx 1 dy/Dy 1 dz/Dz) 5 1, whereDx, Dy, and Dz are the doses of individual drugs required to produce the sameeffect as the effect produced by doses dx, dy, and dz in combination. Antagonism(.1) or synergy (,1) is indicated if the sum of the expression (the “combinationindex”) (dx/Dx 1 dy/Dy 1 dz/Dz) is significantly different from 1. Experimentaldata were analyzed using both Bliss independence and Lowe additivity models.In addition, three-dimensional dose-response surfaces (19) which described theactivity of each drug combination were generated by using the TableCurve3Dprogram (Jandel Scientific), which fitted a surface to all data points for each setof experiments without reference to preconceptions of the nature of the druginteraction or shapes of individual dose-response curves. Coordinates of pointson each dose-response surface were compared with predictions from the twomain theoretical models. Further details are provided in the legends to Fig. 3 and4 and in the footnote to Table 1.

RESULTS

Cytotoxic and cytostatic effects of drug treatment. PDHmonolayers remained intact for the duration of all experi-ments, and on microscopic examination, there were no observ-

able differences between treated and untreated PDH in anti-viral assays, nor were there measurable differences in neutralred uptake. Significant toxicity occurred only when PDH wereexposed continuously to drug concentrations in the millimolarrange. After 9 days’ continuous exposure, the concentrationsrequired to cause a 50% reduction in cell viability as measuredby neutral red uptake were estimated (by extrapolation) to beapproximately 3, 5, and 7 mM for PMEA, PCV, and 3TC,respectively, and 4.5 mM (total concentration) for an equimo-lar combination of the three drugs (Fig. 2A). The estimatedCC50s for the three drugs in combination were consistent withadditive cytotoxic effects according to both the Bliss and Lowemodels. Additive cytotoxic effects were also observed for two-drug combinations (data not shown). The cytostatic effects ofPMEA, PCV, 3TC, and equimolar mixtures of each in combi-nation were estimated using Huh-7 cells. The effects were bothdose and time dependent. After 2 days’ exposure, the concen-trations required to cause 50% inhibition of cell proliferation(CC50s) were estimated by extrapolation to be .7 mM forPMEA and .10 mM for PCV, 3TC, and an equimolar mixtureof all three drugs; by day 4, when drug-free controls reachedconfluence, estimated CC50s were estimated as approximately3, 5, and 6 mM for PMEA, PCV, and 3TC, respectively, and

TABLE 1. Anti-DHBV efficacies of PCV, 3TC, and PMEA alone and in combination in congenitally infected PDHa

Drug or combination and molar ratio

Equation parameter (mean 6 SD) Correlationcoefficient

r2C.I. (Lowe)

%Inhibitionexpectedat EC50(Bliss)

Interaction

a b c

Expt 1: PMEA in combination with PCV or 3TC

PMEA 100 6 1.4 0.12 6 0.01 1.5 6 0.08 1.0PCV 100 6 5.1 0.13 6 0.03 0.94 6 0.15 0.953TC 100 6 4.5 0.20 6 0.02 2.0 6 0.38 0.97

PMEA-PCV (4:1) 100 6 5.6 0.05 6 0.01 0.87 6 0.12 0.96 0.41 6 0.13 19 6 5.9 SYNPMEA-PCV (1:1) 100 6 5.0 0.04 6 0.01 0.77 6 0.09 0.97 0.32 6 0.10 14 6 4.3 SYNPMEA-PCV (1:4) 99.8 6 5.5 0.05 6 0.01 0.90 6 0.12 0.93 0.39 6 0.12 20 6 6.2 SYNPMEA-PCV (1:9) 100 6 6.5 0.14 6 0.04 0.87 6 0.17 0.99 1.09 6 0.33 51 6 16 ADD

PMEA-3TC (4:1) 96.9 6 4.8 0.06 6 0.01 0.89 6 0.12 0.97 0.46 6 0.15 21 6 6.9 SYNPMEA-3TC (1:1) 100 6 5.0 0.13 6 0.01 2.2 6 0.40 0.98 0.87 6 0.29 32 6 10.5 ADDPMEA-3TC (1:4) 98.4 6 3.6 0.12 6 0.01 1.5 6 0.16 0.99 0.68 6 0.22 17 6 5.6 SYNPMEA-3TC (1:9) 102 6 4.5 0.13 6 0.01 2.0 6 0.28 0.98 0.69 6 0.23 20 6 6.6 SYN

Expt 2: PMEA in combination with both PCV and 3TC

PMEA 104 6 4.2 0.17 6 0.01 2.47 6 0.30 0.99PCV 99.8 6 4.7 0.43 6 0.04 1.8 6 0.3 0.993TC 102.7 6 6.8 0.64 6 0.08 1.7 6 0.4 0.96PCV 1 3TC (1:1) 100 6 2.31 0.28 6 0.02 2.35 6 0.23 0.99 0.54 6 0.12 31 6 6.7 SYNPMEA 1 PCV 1 3TC (1:1:1) 99.9 6 3.5 0.2 6 0.03 0.7 6 0.17 0.98 0.71 6 0.29 36 6 14 ADD

a Congenitally DHBV-infected PDH were harvested after 10 days’ in vitro culture (i.e., after 9 days of continuous exposure to drugs. Calculations are based on relativesignal intensities of probe signals from dot blots of total intracellular DNA probed with a full-length 32P-labeled DHBV cDNA probe. Parameters describing eachdose-response curve were derived from analysis of 12 data points, each of which represented the mean of triplicate assays. Logistic dose-response curves of the formy 5 k 1 a/(1 1 [x/b]c) (see Fig. 3) were fitted to each data set with the aid of TableCurve2D, which also calculated the equation parameters and correlation coefficients.Listed b values correspond to EC50s in micromolar concentrations (see Fig. 3). For drug combinations, the b values listed correspond to the total drug concentration,not the concentration of any single component. EC50s for individual components of combinations can be calculated by dividing the b value by the fractionalconcentration of that component. Combination indicies (C.I.) were estimated at the EC50 using the Lowe additivity formula (see the text). The percent inhibitionexpected at the observed EC50 for each drug combination was calculated using the Bliss independence formula (see the text). Errors in calculations were assumed tobe additive, and interactions were defined as synergistic (SYN) if the C.I. was significantly less than 1 and/or the expected percent inhibition at the EC50 was significantlyless than 50. The upper 95% confidence interval can be obtained by adding twice the standard deviation to each estimate. Synergy was significant at the P , 0.05 levelfor all but two combinations rated as synergistic. Synergy between PMEA and 3TC at ratios of 1:4 and 1:9 was significant at a P value of ,0.1 based on the C.I. andat a P value of ,0.05 based on the Bliss calculation. In the remaining three cases, estimates of C.I. and expected percent inhibition were not significantly different from1 or 50, respectively, so the interaction was defined as additive (ADD). Most importantly, antagonism between drugs in combination was not observed (but see Fig.3B).

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approximately 5.8 mM (total concentration) for an equimolarcombination of the three drugs. The corresponding concentra-tions were reduced by day 6 to approximately 1, 2.5, 3, and 2.2mM (Fig. 2B). The estimated CC50s at each time point for thethree drugs in combination were consistent with approximatelyadditive cytostatic effects according to either the Lowe or theBliss model. Approximately additive cytostatic effects were alsoobserved for two-drug combinations (data not shown).

Anti-DHBV activity of PMEA, PCV, and 3TC alone or incombination in congenitally infected PDH. PMEA, PCV, and3TC alone and in all combinations caused dose-dependentinhibition of DHBV replication in congenitally infected PDH.Concentrations which produced 50% inhibition of replication

(EC50s) were estimated from logistic dose-response equations(Fig. 3) fitted to each data set. The concentrations required toproduce a particular inhibition end point for each drug andcombination varied between different sets of congenitally in-fected PDH cultures. Results, which are summarized in Table1, show that the effects of all drug combinations were eithersynergistic or not significantly different from additive, regard-less of which model of drug interaction was used. Analysis ofthe three-drug combination suggested synergy at low total drugconcentrations and slight antagonism at higher drug concen-trations; however, results were not significantly different fromadditivity except at extremes of the concentration range (Fig.3B, Table 1).

Three-dimensional graphical analysis of data. The resultsfor the two- and three-drug combinations were plotted in threedimensions, and a dose-response surface was fitted to all datain each set by unweighted nonlinear regression analysis withthe aid of TableCurve3D (Fig. 4). The best-fit dose-responsesurfaces generated by fitting all data in each set were consistentwith the Bliss independence model, in which the combinedeffect of two drugs in combination is the product of the indi-vidual effects (see above). As shown in Fig. 4, the majority ofindividual experimental data points lay within one standarddeviation of a Bliss independence dose-response surface.

Further experiments were undertaken to assess the effects ofPMEA, PCV, and 3TC and combinations on viral protein andCCC DNA synthesis in congenitally infected PDH. As outlinedearlier, for these experiments individual inhibitor concentra-tions were chosen which, when used alone, could be expectedto cause reductions in viral DNA replication of 25 to 50%. Anextraction procedure which enriches for CCC DNA (39) fol-lowed by Southern blotting was used for the analysis of DHBVCCC DNA levels, while immunoblotting was used to assessviral protein synthesis. The results of these studies are shown inFig. 5. Overall, these experiments showed viral protein synthe-sis to be less sensitive to drug treatments than viral DNAsynthesis. Concentrations of individual drugs which reducedtotal intracellular DNA by approximately 25 to 50% had com-parable effects on CCC DNA synthesis, with inhibition by drugcombinations consistent with predictions from the Bliss inde-pendence model. By contrast, treatment with individual inhib-itors had no significant effect on core antigen expression andbarely significant effects (,20% reduction, slightly greaterthan 1 standard deviation) on pre-S antigen expression, al-though all combinations caused inhibition (approaching 50%)of both core and pre-S antigen synthesis (Fig. 5A).

Anti-DHBV activities of PMEA, PCV, and 3TC alone or incombination in acutely infected PDH. Acute infections wereperformed to determine the effects of drug treatments on earlyDHBV replication, namely, the events leading to the accumu-lation of DHBV CCC DNA by the direct conversion of DNAwithin infecting virus particles and the early intracellular recy-cling of immature progeny virions to the nucleus. Drug con-centrations used in these experiments were identical to thoseused in corresponding experiments with chronically infectedPDH. Because drugs are added at around the time of infection,the viral load is significantly lower in acutely infected cells thanin chronically infected cells incubated ex vivo for equivalenttimes. As a consequence, drug responses are enhanced due tothe absence of a pre-existing intracellular pool of viral DNA orprotein (Fig. 5B).

DISCUSSION

Here we confirm and extend our earlier observations (11, 34;D. Colledge, S. Locarnini, and T. Shaw, Abstr. 38th Intersci.

FIG. 4. Three-dimensional dose-response plot illustrating inhibition ofDHBV DNA replication in chronically infected PDH by PMEA in combinationwith PCV (A) or 3TC (B) at molar ratios (PMEA to PCV or PMEA to 3TC) of4:1, 1:1, 1:4, or 1:9. The amount of DHBV replication was expressed as apercentage relative to replication in untreated controls (set at 100%) and plottedon the z (vertical) axis against the concentration of each drug using the x axis forPCV or 3TC and the y axis for PMEA. Although they do not show individualdrug dose responses, logarithmic axes have been used because they show theresponse to drug combinations much more clearly than do linear axes. A best-fitdose-response surface (shown as a mesh) has been fitted to each set of points byunweighted nonlinear regression analysis with the TableCurve3D program,which was given the option of fitting dose-response surfaces “expected” on thebasis of different drug interaction models. The best-fit surfaces in each case werefound to be those predicted by the Bliss independence model of drug interaction.Each data point shown represents the average of three determinations. Pointslying above or below the surface suggest antagonism or synergy, respectively.Those which lie within 1 standard deviation of the fitted surface are indicated byopen circles, and those which lie within 2 standard deviations of the fitted surfaceare indicated by filled circles. Most experimental data points lie within 1 standarddeviation of the fitted dose-response surfaces, and no data points lie beyond 2standard deviations.

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Conf. Antimicrob. Agents Chemother., abstr. 159, p. 360,1998) by showing that at clinically achievable concentrations,the antiviral effects of all two-drug combinations containingPCV, 3TC, and PMEA are additive or synergistic. Further-more, the anti-DHBV effect of combinations containing allthree drugs is also approximately additive as measured byinhibition of intracellular DHBV replication, although this isnot consistently reflected by comparable inhibition of virus-specific protein synthesis. Conclusions about the effects of drugcombinations on viral DNA synthesis were similar regardlessof the method used to analyze results, demonstrating thatdespite controversy over which drug interaction model(s) isappropriate for application in particular cases (5, 19), the dif-

ferent approaches lead to similar conclusions when applied tobiological data, which is typically too noisy to provide theaccuracy required to support subtle distinctions required bytheoretical arguments.

Combination chemotherapy has a number of recognized ad-vantages over monotherapy and in the future will probablybecome the most effective approach to control chronic humanHBV infection (9–11). Rationally chosen drug combinationshave the potential to minimize the risk of drug toxicity and toreduce the probability of development of viral resistance, bothimportant considerations during long-term therapy (5, 9, 11,19). Theoretically it should be possible to design therapeuticregimens using two or more drugs having complementary ac-

FIG. 5. Inhibition of viral CCC DNA (indicated by open circles) and protein synthesis (bar graphs) by PMEA, PCV, and 3TC alone and in combination.Concentrations of PMEA, PCV, and 3TC in these experiments were 0.1, 0.125, and 0.125 mM, respectively. Southern blots and immunoblots were prepared, stained,and analyzed as described previously (9, 26), and results are expressed as percentages of the average probe signal density from untreated controls. Values for eachparameter are the means of duplicate or triplicate determinations, with standard deviations represented by error bars. Arrows above and below each bar forcombinations indicate expected values for pre-S1 and core antigens, respectively, applying the Bliss independence formula to results for each drug alone. Results arefrom representative experiments using chronically (A) and acutely (B) infected PDH.

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tivities (5, 9, 19). Hepadnaviruses depend completely on thehost cells’ enzymatic machinery for their supply of deoxynucle-otides, since their genomes do not encode enzymes for de-oxynucleoside salvage or dNTP synthesis (32, 33). Pathways fordeoxynucleoside salvage and de novo dNTP synthesis are rel-atively inactive in hepatocytes and presumably in other cells(such as pancreatic islet cells and bile duct epithelial cells)which are known to be susceptible to HBV infection, sincethese cell types turn over slowly and most are normally quies-cent (32, 33). Ideally, drugs used in combination should useindependent processes for uptake and activation and have dif-ferent mechanisms or sites of action. To minimize administra-tion frequency and make patient compliance with dosageschedules easier, the active metabolites should have long, andpreferably similar, intracellular half-lives, and it is also desir-able that the agents be orally available and able to be admin-istered simultaneously without adverse or unpredictable phar-macological and pharmacokinetic interactions.

PCV and 3TC appear to be transported into the cell andactivated enzymatically by different sets of cellular enzymes(33) (Fig. 6). Both specifically inhibit the hepadnaviral DNApolymerase/reverse transcriptase by competing with the corre-

sponding dNTPs for incorporation into nascent DNA and act-ing as chain terminators after incorporation (11, 33, 35). Spe-cific interference with the unique dGTP-dependent step whichprimes hepadnaviral reverse transcription (12) is an additionalmechanism by which PCV exerts antihepadnaviral activity andwhich is not shared by 3TC. Both PCV (as famciclovir) and3TC are orally available, and each has been used for HBVmonotherapy with varying success (15, 24, 25, 27). Regardlessof more-consistent responses obtained with 3TC (23), emer-gence of resistance appears almost inevitable during long-termmonotherapy (3, 33, 40), necessitating the development of newdrugs and therapeutic regimes.

We have previously shown that the in vitro antihepadnaviralactivities of PCV and 3TC are synergistic (11) and that eachacted additively or synergistically in combination with PMEA(35; Colledge et al., 38th ICAAC) in the test system describedhere. PMEA has low oral bioavailability, a problem which hasbeen overcome by the development of prodrugs including thebis(pivaloyloxymethyl) derivative (bis-[POM]-PMEA; adefovirdipivoxil) (2), which has reached phase II and III trials againstHIV (4, 13) and phase I and II trials against HBV (18, 20).

A recent report from this laboratory (30) has described an in

FIG. 6. The main enzymatic pathways for nucleotide salvage, synthesis, and interconversion. PCV probably enters the cell via purine base transporters (a), resistsphosphorylysis by purine nucleoside phosphorylase (b), and is probably initially phosphorylated by deoxyguanosine (GdR) kinase (c). 3TC follows the deoxycytidine(CdR) salvage pathway, entering the cell via pyrimidine nucleoside transporters (d); it resists deamination by deoxycytidine deaminase (e) and is initially phosphorylatedby deoxycytidine kinase (f). PMEA enters the cell by endocytosis (g), bypasses the initial phosphorylation, and is phosphorylated by AMP kinase or PRPP synthetase(h) to produce the monophosphate. The terminal anabolites PCV-TP, 3TC-TP, and PMEA-DP compete with dGTP, dCTP, and dATP, respectively, for incorporationby HBV polymerase/reverse transcriptase (j). In addition, PCV-TP blocks the priming of reverse transcription. Pathways for uridine (UR) and thymidine (TdR)metabolism are also shown: the main pathway to thymidine triphosphate (TPP) in hepatocytes is via UMP rather than salvage of thymidine.

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vivo evaluation of PMEA in the duck model of chronic HBVinfection. Using immunohistochemical and in situ DNA hy-bridization techniques, it was found that treatment with PMEAreduced viral protein and DNA loads in bile duct epithelialcells (BDEC) as well as hepatocytes (30). This observation issignificant because the reservoir of DHBV in BDEC is refrac-tory to treatment with nucleoside analogs such as PCV and3TC, presumably because BDEC lack the enzymatic machineryrequired for their uptake or phosphorylation (29, 30). Since itis a dAMP analog, PMEA is able to bypass the initial phos-phorylation site, which is critical and often limiting for theactivity of most nucleoside analogs (32, 33) (Fig. 6). Cellularenzymes convert PMEA to its diphosphate, a dATP analogwhich acts as an obligatory chain terminator after incorpora-tion into nascent DNA (2, 28). Its selectivity depends on rel-atively high affinity for HBV DNA polymerase/reverse tran-scriptase compared to host cell DNA polymerases (28).Significantly, mutations in the HBV polymerase gene whichconfer resistance to 3TC do not confer cross-resistance toPMEA in vitro (40). In addition, PMEA has immunomodula-tory activity and has been shown to stimulate interferon alphaproduction and natural killer cell activity (14, 28). Whether theresults presented here are predictive of efficacy against humanHBV infection remains to be established, since they may beinfluenced by species-dependent differences in the behavior ofkey cellular and/or viral enzymes (e.g., transmembrane trans-porters, cellular deoxynucleoside kinases, and viral DNA poly-merase/reverse transcriptases).

Our results probably underestimate the potential efficacy oftriple-drug combination for at least two main reasons. First,the immunostimulatory potential of PMEA cannot be reliablyassessed in congenital DHBV infection because (i) immuneresponses are typically species-specific and (ii) ducks are, inany case, essentially immunotolerant of hepadnaviral infection.Secondly, the assay system described here uses an isolatedpopulation of primary hepatocytes, relatively free of other livercell types (7). Consequently, the putative advantages ofPMEA—the ability to stimulate immunity and the ability toinhibit viral replication in hepatocytes as in other cells harbor-ing virus—cannot be adequately assessed using this system.

Available evidence, although limited (2, 28, 32, 33), indicatesthat PCV, 3TC, and PMEA each use a different pathway forcellular uptake and activation (Fig. 6), and although the activeanabolites of each must compete for recognition by the HBVDNA polymerase/reverse transcriptase, each pairs with a dif-ferent template site, since each is an analog of a differentdNTP. Neither PCV nor 3TC affects hepadnaviral replicationin BDEC, a site where PMEA is active (30); nor does PCV or3TC share the immune-stimulating capacity of PMEA (14). Inaddition, 3TC-resistant HBV mutants do not display significantcross-resistance to PMEA (40), and while 3TC resistance alsoconfers resistance to PCV, the converse has not been observed(41). Together, these observations provide a sound rationalefor the use of PCV, 3TC, and PMEA in combination, andfurther investigation of their combined anti-HBV activity isjustified, particularly since each is already being used clinically.In particular, the issues of tissue-specific activity, possiblecross-resistance, and potential for cumulative in vivo toxicityrequire further investigation.

ACKNOWLEDGMENTS

This work was supported by grants from the National Health andMedical Research Council of Australia and an educational grant fromGilead Sciences. T.S. was partly supported by the Australian govern-ment under a syndicated research and development scheme.

Specific antibodies to DHBV proteins were a gift from Allison

Jilbert of the Institute for Medical and Veterinary Science, Adelaide,South Australia. Klaus Esser of SmithKline Beecham Pharmaceuticalskindly arranged supply of penciclovir and lamivudine. We thank ScottBowden and two anonymous reviewers who reviewed the manuscriptand suggested improvements.

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