ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, JUlY 1993, p. 1539-15420066-4804/93/071539-04$02.00/0Copyright X 1993, American Society for Microbiology

Suramin Prevents Duck Hepatitis B Virus Infection In VivoWOLF-BERNHARD OFFENSPERGER,l SILKE OFFENSPERGER,l EIKE WALTER,2

HUBERT E. BLUM,2* AND WOLFGANG GEROK'

Department ofMedicine, University of Freiburg, DW-7800 Freiburg, Germany, 1 and Department of Medicine,Medical Clinic B, University Hospital Zurich, CH-8091 Zurich, Switzerland2

Received 4 December 1992/Accepted 13 April 1993

The effect of suramin on duck hepatitis B virus (DHBV) infection was investigated in vivo. Suraminpretreatment of Pekin ducklings completely prevented DHBV infection. In contrast, suramin given at the timeof or after inoculation with DHBV did not inhibit viral infection, replication, or gene expression. These dataindicate that suramin effectively blocks the early stages of DHBV infection in vivo.

Suramin, a polysulfonated naphthylurea, was the firstclinically useful antiparasitic drug and is still one of the mostcommonly used antitrypanosomal agents. Apart from itsantiparasitic effects, suramin has been demonstrated to havea number of biochemical activities (7). Most notably,suramin has been shown to inhibit a number of enzymes,such as hexokinase, lysozyme (11), thrombin, plasma kal-likrein (5), and protein kinase C (6). Since suramin wasshown to bind to a number of growth factors (8) and proteinkinase C is involved in tumor promotion and differentiation,the antitumor activity of suramin has been studied in patientswith metastatic adrenocortical carcinoma (18) and metastaticprostate cancer (9). In addition to its antiparasitic andantitumor properties, suramin has been shown to stronglyinhibit duck hepatitis B virus (DHBV) DNA polymerase (19,20) and the reverse transcriptase activities of a number ofretroviruses (3), including the human immunodeficiencyvirus (13). A clinical trial in 10 patients with AIDS demon-strated that suramin indeed inhibits human immunodefi-ciency virus replication; immunological parameters and clin-ical outcomes were not affected, however (1).

After the discovery of DHBV (12), Pekin ducks havebecome a widely used animal model to study hepadnaviralinfection. By using this model, antiviral agents can bestudied in vitro in replicative complexes isolated fromDHBV-infected livers (16) or in primary duck hepatocytecultures (18) as well as in vivo in DHBV-infected Pekinducks (15). In primary duck hepatocyte cultures, suraminblocks infection when it is present before and during incu-bation of the cells with DHBV (17). Since suramin inhibitsDHBV infection in vitro, we tested the effect of suramin onDHBV infection in vivo.

Pekin ducks were used as an in vivo model of DHBVinfection. One-day-old ducklings were purchased from a

German duck farm. Blood was taken from the foot vein and

was tested for the presence of DHBV DNA by dot blothybridization (14). DHBV DNA-negative ducklings were

infected at the age of 2 or 3 days by intravenous injection of100 ,ul of DHBV-positive serum (about 109 virions per ml).Treatment of the animals with suramin (Germanin; Bayer,Leverkusen, Germany) was started before, during, or afterDHBV infection of the animals. After 14 days, the ducklingswere sacrificed. Livers and sera were stored at -80°C untilanalysis. The number of animals per experimental groupcould be kept very low (three ducklings treated with

* Corresponding author.

suramin, one nontreated duckling as a negative control)because in our hands DHBV infection of more than 500ducklings between days 1 and 5 after hatching resulted in a

100% infection rate. The study protocol was approved by thegovernmental committee on the treatment of laboratoryanimals.For Southern blot analysis, DNA was extracted from liver

tissues as described previously (15). DNA samples were

electrophoresed on a horizontal 1.25% agarose slab gel,denatured, and transferred to Hybond N membranes (Am-ersham Buchler, Braunschweig, Germany), as recom-

mended by the manufacturer. After prehybridization, theblots were hybridized with a full-length DHBV DNA probeand labeled by nick translation with [32P]dCTP (AmershamBuchler) to a specific activity of about 109 dpm/,ug (22).For Western blot (immunoblot) analyses, serum samples

were denatured, subjected to sodium dodecyl sulfate-12%polyacrylamide gel electrophoresis, and transferred to nitro-cellulose membranes (Schleicher & Schuell, Dassel, Germa-ny). The membrane was incubated overnight with a poly-clonal antibody against DHBV presurface (pre-S) andsurface (S) antigens (DHBpre-S/S) raised in rabbits. Thefollowing incubations with swine anti-rabbit immunoglobu-lin, peroxidase-antiperoxidase complex, and substrate solu-tion were carried out according to the instructions of themanufacturer (Dakopatts, Hamburg, Germany). In DHBV-positive control sera, this antibody detected a major proteinwith a molecular mass of about 36 kDa (pre-S/S protein),some minor species with different molecular masses becauseof the different degree of glycosylation, and a minor proteinwith a molecular mass of 18 kDa (S protein).

In a first experiment, three Pekin ducks were treated withsuramin prior to infection with DHBV-positive serum; one

Pekin duck served as a negative control. Ducklings, weigh-ing about 50 g at hatching and about 150 g after 2 weeks,received 40 mg of suramin per kg of body weight byintravenous injection on days 2, 3, 4, 7, and 10. On day 3, theducklings were infected by the intravenous injection of 100,ul of DHBV-positive serum. The ducks were sacrificed on

day 14. DNA was extracted from the livers and analyzed bySouthern blot hybridization as described above. There was

no difference in the amount ofDNA extracted from the liversof suramin-treated and nontreated animals. As shown in Fig.1, the untreated control animal (Fig. 1, lane 1) was success-

fully infected with DHBV, resulting in the presence ofvarious replicative forms of DHBV DNA in the liver,ranging from short single-stranded DNA species of minus-strand polarity to complete double-stranded DNA molecules

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FIG. 1. Effect of suramin on DHBV infection in vivo determined by Southern blot analysis of DNAs isolated from duck livers. Lanes 1,5, and 9, DNAs from the livers of control animals without suramin treatment; lanes 2 to 4, DNAs from the livers of three animals infectedwith DHBV on day 3 and treated with suramin beginning on day 2; lanes 6 to 8, DNAs from the livers of three animals infected with DHBVon day 3 and treated with suramin beginning on day 3; lanes 10 to 12, DNAs from the livers of three animals infected with DHBV on day 3and treated with suramin beginning on day 4. Size markers are HindIII-digested bacteriophage lambda DNA and 10 pg of DHBV DNA. Theautoradiographic exposure time was 1 day at -80°C.

migrating in the 3.0-kbp position. In contrast, in the threeanimals pretreated with suramin (Fig. 1, lanes 2 to 4), noviral DNA could be detected.Western blot analyses of serum from the untreated control

animal showed the expected pre-S/S and S proteins, demon-strating successful infection of the animal (Fig. 2, lane 1). Incontrast, animals pretreated with suramin were negative forDHBpre-S/S antigens in serum (Fig. 2, lanes 2 to 4).

In order to distinguish between a block ofDHBV infectionand inhibition of DHBV replication after infection, suramintherapy was started at the same time as DHBV infection or1 day after DHBV infection. Ducklings were infected withDHBV-positive serum on day 3 as described above. Suramin(40 mg/kg) was given on days 3, 4, 5, 8, and 11. The duckswere sacrificed on day 14. The untreated control duck (Fig.1, lane 5) was again successfully infected. Different from thedata obtained after pretreatment of the ducklings withsuramin (Fig. 1, lanes 2 to 4), suramin given at the time ofinoculation with DHBV-positive serum did not preventinfection but resulted in a productive infection with DHBVDNA replication in the liver (Fig. 1, lanes 6 to 8). Also at theprotein level, suramin given at the time of DHBV infectiondid not block viral infection and resulted in DHBpre-S/Santigen synthesis and export (Fig. 2, lanes 6 to 8) that wereindistinguishable from those for the untreated control animal(Fig. 2, lane 5).

In a further experiment, three ducklings were treated withsuramin starting 1 day after DHBV infection on day 4, andtreatment was continued on days 5, 6, 9, and 12. Oneduckling served as a negative control. On day 14 the animalswere sacrificed and analyzed for DHBV DNA and DHBpre-

S/S antigens as described above. As demonstrated in Fig. 1and 2, respectively, treated (lanes 10 to 12) and untreated(lanes 9) animals showed identical amounts of viral replica-tive intermediates in liver and viral antigens in serum.Suramin therefore does not affect viral replication and geneexpression in DHBV-infected hepatocytes in vivo.Our data demonstrate that the infection of Pekin ducklings

with DHBV can be effectively blocked by pretreatment ofthe animals with suramin. The dose of suramin used in our invivo study is about two times higher than that used in in vivostudies in humans (1) and ducks (19). Even at this dose, thetoxic effects of suramin were not observed during theshort-term administration of the drug over 10 to 12 days.Studies to define the minimal dose required for the preven-tion of DHBV infection and to specifically address thequestion of toxic side effects are in progress.

Since suramin given at the time of inoculation with DHBVor later does not prevent viral infection, we assume thatsuramin acts at a very early step of the infection process,such as at the viral attachment or uptake stage. Thesefindings extend the observations of Petcu et al. (17) andourselves, demonstrating the inhibition of DHBV infectionof primary duck hepatocytes in vitro by suramin (Fig. 3). Sofar, the molecular mode of action of suramin that leads to ablock of DHBV infection is not known. It is conceivable thatsuramin affects endocytosis of DHBV, viral uncoating, orcycling of receptors. Since known inhibitors of viral attach-ment to target cells, e.g., sulfated polysaccharides such asdextran sulfate or heparin, do not affect DHBV infection invitro (Fig. 3) or in vivo (14), it is unlikely that suramin actsthrough this mechanism.

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FIG. 2. Effect of suramin on viral pre-S/S protein expression determined by Western blot analysis of sera obtained from DHBV-infectedducks. Lanes 1, 5, and 9, sera from control animals without suramin treatment; lanes 2 to 4, sera from three animals infected with DHBV onday 3 and treated with suramin beginning on day 2; lanes 6 to 8, sera from three animals infected with DHBV on day 3 and treated with suraminbeginning on day 3; lanes 10 to 12, sera from three animals infected with DHBV on day 3 and treated with suramin beginning on day 4.

Suramin is concentrated within lysosomes (4). There, itmay directly inhibit the lysosomal enzymes important forvirus internalization or it may change the lysosomal pH,thereby preventing the fusion of viral and lysosomal mem-

branes. Using lysosomotropic agents, such as ammoniumchloride or chloroquine, we previously demonstrated astrong inhibition ofDHBV infection in vitro (15). These dataindicate that DHBV infection, similar to infections with

FIG. 3. (a) Effect of suramin on DHBV infection and replication in vitro determined by Southern blot analysis of DNAs isolated fromprimary duck hepatocytes after 2 weeks in culture. Lanes 1 and 2, primary hepatocytes isolated from DHBV DNA-negative ducks infectedin vitro with DHBV DNA-positive serum in the absence (lane 1) or presence (lane 2) of suramin at a concentration of 100 Fg/ml; lanes 3 and4, primary hepatocytes isolated from DHBV DNA-positive ducks and grown in the absence (lane 3) or presence (lane 4) of suramin at aconcentration of 100 p.g/ml. For further experimental details, see reference 15. (b) Effect of dextran sulfate on DHBV infection and replicationin vitro determined by Southern blot analysis ofDNA isolated from primary duck hepatocytes after 2 weeks in culture. Lanes 1 to 4, primaryhepatocytes isolated from DHBV DNA-negative ducks infected in vitro with DHBV DNA-positive serum in the absence (lane 1) or presence(lanes 2 to 4) of dextran sulfate at concentrations of 100 pLg/ml (lane 2), 250 p1g/ml (lane 3), or 500 pg/ml (lane 4); lanes 5 to 8, primaryhepatocytes isolated from DHBV DNA-positive ducks and grown in the absence (lane 5) or presence (lanes 6 to 8) of dextran sulfate at

concentrations of 100 pg/ml (lane 6), 250 p,g/ml (lane 7), or 500 pg/ml (lane 8). For further experimental details, see reference 15.

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other enveloped viruses, depends on receptor-mediated en-docytosis and involves membrane fusion triggered by lowpH. In vitro experiments demonstrated that the addition ofsuramin 2 h after infection and that the addition of lysoso-motropic agents 3 h after infection still effectively blockedviral infection (17), suggesting that suramin and lysosomo-tropic agents have similar mechanisms of action. The factthat in in vivo experiments suramin given at the time ofinoculation did not prevent DHBV infection may be due tothe pharmacokinetic properties of suramin. In vivo suraminis mostly bound to plasma proteins (99.7%), which mayreduce or delay its biological availability (2). By increasingthe dose of suramin, this lack of effect of suramin given atthe time of infection may be overcome. Furthermore,suramin has been shown to be a P2 purinoceptor antagonistand competitively inhibits P2 purinoceptor agonist-inducedphospholipase C stimulation (10). It is conceivable thatsuramin may act through such a mechanism that leads to ablock of virus entry.

The present study was supported by grants from the DeutscheForschungsgemeinschaft (SFB 154 TP-A5 and TP-A2).The antibodies to DHBpre-S antigen were kindly provided by H.

Will, Hamburg, Germany. The expert technical assistance of BirgitHockenjos and Petra Kary is gratefully acknowledged.

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