Foscarnet decreases serum and liver duck hepatitis B virus DNA in chronically infected ducks

Foscarnet decreases serum and liver duck hepatitis B virus DNA in chronically infected ducks

GASTROENTEROLOGY 1986;91:618-24 Foscarnet Decreases Serum and Liver Duck He atitis B Virus DNA in Chronica Ply Infected Ducks AVERELL H. SHERKER, KAT...

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GASTROENTEROLOGY 1986;91:618-24

Foscarnet Decreases Serum and Liver Duck He atitis B Virus DNA in Chronica Ply Infected Ducks AVERELL H. SHERKER, KATSUTARO and KUNIO OKUDA

HIROTA, MASAO

OMATA,

First Department of Internal Medicine, Chiba University School of Medicine, Chiba, Japan

Foscarnet (trisodium phosphonoformate) is a new antiviral compound with in vitro inhibitory effects against the DNA polymerases of hepadna viruses. To study the eflects of the drug in chronic hepadna virus infection, we treated ducks chronically infected with duck hepatitis B virus for 10 days with either low-dose foscarnet [5O mgfkg i.p. b.i.d.), highdose foscarnet (250 mg/kg i.p. b.i.d.), or sterile water injections. Serum duck hepatitis B virus DNA and intrahepatic replicative forms of the virus were measured using molecular biological techniques with both a double-stranded radiolabeled DNA probe and a plus-strand (noncoding) specific RNA probe. We found a dose-related decrease in serum and intrahepatic duck hepatitis B virus DNA during treatment, with a rapid return toward baseline values after the cessation of treatment. There was a disproportionate decrease in the plus strand of viral DNA with treatment. We conclude that foscarnet exerts its effect in hepadna virus infection through inhibition of viral DNA polymerase. Further study is necessary to determine whether foscarnet, by itself or in combination with other treatment modalities, has a role to play in the treatment of chronic hepatitis B infections in humans. Hepatitis B virus (HBV) infection is a major worldwide public health problem. It is estimated that >lO% of the population of China, Southeast Asia, Received February 3, 1986. Accepted April 11,1986. Address requests for reprints to: Masao Omata, M.D., First Department of Medicine, Chiba University School of Medicine, Chiba 280, Japan. Dr. Sherker’s present address is: Department of Internal Medicine, Mount Sinai Hospital, Toronto, Canada. This research was supported in part by Japanese Ministry of Education grant (B) 58480215. This study was presented in part at the Falk Symposium on New Trends in Hepatology, Tokyo, Japan on January 25, 1986. 0 1986 by the American Gastroenterological Association 0016-5085/86/$3.50

and sub-Saharan Africa are chronic HBV carriers. In Western countries, chronic HBV carriers are frequently found among members of high risk groups such as male homosexuals and intravenous drug abusers (1). The chronic carrier state is associated with a high rate of morbidity and mortality due to chronic active hepatitis, liver cirrhosis, and hepatocellular carcinoma (2). Various treatments have been used in an attempt to alter the natural course of the carrier state. These include interferon (3-8), antiviral agents (6,9-15), and immunomodulatory regimens (14-201, either alone or in combination. To date, no treatment has proved highly efficacious and some have been limited by significant toxicities (21). Foscarnet (trisodium phosphonoformate) is a new antiviral compound which has been shown to have in vitro inhibitory activity against a number of reverse transcriptases of retroviruses and the DNA polymerases of herpesviruses, HBV, and woodchuck hepatitis virus (22). It has been used in immunocompromised patients with life-threatening infections due to herpesviruses, and has been found to have little associated toxicity (23,24). Duck hepatitis B virus (DHBV) is a member of the group of hepadna viruses and shares many properties, including a unique replicative pathway, with HBV (25). We report here the results of a trial of foscarnet administered to chronically infected, DHBV carrier ducks. Materials and Methods Ducks and Treatment

Protocol

Forty-six newborn White Pekin ducks were inoculated with 50 ~1 of serum pooled from DHBV carrier ducks. At 14 wk of age the ducks were screened for serum levels Abbreviation used in this paper: DHBV, duck hepatitis B virus.

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FOSCARNET IN CHRONIC DHBV INFECTION

of DHBV DNA, and 6 were chosen with moderate and approximately equal amounts of circulating viral DNA (data not shown). Two ducks were randomized to each of three groups: high-dose foscarnet, low-dose foscarnet, and control. Foscarnet sodium anhydrous crystals were provided by Professor Bo Oberg (Astra Lakemedel AB, Sodertalje, Sweden) and were made up to a 2% wtlwt solution in sterile water. The solution was stored at +4’C and adjusted to pH 7.4 with 1 N HCl immediately before use. Ducks in the low-dose group were given foscarnet, 50 mg/kg b.i.d., by intraperitoneal injection for 10 days. Ducks in the high-dose group were given foscarnet, 250 mg/kg i.p. b.i.d., for 10 days, and ducks in the control group were given i.p. injections of 10 ml of sterile water twice daily for 10 days. Serum samples were obtained twice from each duck before the initiation of treatment; on days 3, 6, and 10 of the treatment period; and 4 and 10 days after the cessation of treatment. Wedge liver biopsy specimens were obtained under local anesthesia on the day before starting treatment and on the final day of treatment. The ducks were killed 10 days after completing treatment and a liver tissue sample was obtained at that time. Liver specimens were immediately frozen and stored at -80°C until use.

Radiolabeled

Probes

Two different radiolabeled probes were used to detect DHBV DNA in serum and liver tissue. The first was made by the method of nick translation using cloned DHBV DNA and [cY-32P]deoxycytidine 5’-[cw-32P]triphosphate as previously described (26). A plus-strand (noncoding) specific RNA probe was also synthesized. pSPDHBV5.2 was a generous gift of Dr. Jesse Summers and Jan Tuttleman (Fox Chase Cancer Center, Philadelphia, Pa.). It consists of a clone of DHBV DNA inserted into the Eco RI site of the pSP65 vector and grown in Escherichia coli. The orientation of the viral DNA is such that its transcripts are of minus-strand polarity. The plasmid was linearized with Pst I and transcribed in the presence of SP6 grade [cw-32P]UTP (Amersham Japan Limited, Tokyo, Japan) using the Amersham SP6 RNA polymerase kit according to a modification of the method of Melton et al. (27). The specificity of the probe was tested using cloned DHBV DNA, as well as Ml3 vector cloned, singlestranded, plus-strand and minus-strand DNA, spotted directly onto nitrocellulose filter paper. A Northern blot containing DHBV mRNA transcripts was also used. The RNA probe hybridized only to cloned DHBV DNA, plusstrand Ml3 DNA, and mRNA, proving its plus-strand specificity (data not shown). Detection

of DHBV DNA

To detect DHBV DNA in serum, the dot blot hybridization technique was used. Aliquots of 5 ~1 each of serum were blotted on nitrocellulose filter paper (Schleicher and Schull, Dassel, West Germany, BA 85) using the BRL Hybridot Manifold (Bethesda Research

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Laboratories, Gaithersburg, Md.) with weak vacuum suction. Pooled serum from DHBV carrier ducks and serum from an uninfected duck were included as positive and negative controls, respectively. The filter was air-dried and then submerged in 0.1 M NaOH and 1.5 M NaCl for 5 min, followed by 0.1 M Tris-HCl (pH 7.4) and 1.5 M NaCl for 3 min, and then baked at 80°C under vacuum for 2 h. The filter was then hybridized with the nick-translated, radiolabeled, cloned DHBV DNA probe as described by Wahl et al. (28). Briefly, the paper was prehybridized at 42°C for 90 min in 10 ml of 50% formamide, 5x standard saline citrate, 0.1% polyvinyl pyrrolidone, 0.1% Ficoll400, 0.1% bovine serum albumin, 0.5 mgiml denatured calf thymus DNA, 1% glycine, and 50 mM sodium phosphate buffer (pH 6.5) in a polyethylene bag. The filter was then hybridized with radiolabeled DHBV DNA at 42°C for 24 h in 10 ml of 50% formamide, 5x standard saline citrate, 0.02%) polyvinyl pyrrolidone, 0.02% Ficoll 400, 0.02% bovine serum albumin, 0.1 mgiml denatured calf thymus DNA, 25 mM sodium phosphate buffer (pH 6.5), and 10% sodium dextran sulfate 500. It was then washed three times (20 min per wash) at room temperature with 0.1% sodium dodecyl sulfate in 2x standard saline citrate buffer for a total of 20 min, followed by three exchanges at 56°C of 0.1% sodium dodecyl sulfate in 0.1~ standard saline citrate buffer. The filter was then dried and autoradiographed at -80°C with Kodak X-Omat film (Eastman Kodak, Rochester, N.Y.) using a Du Pont lightening plus screen (Du Pont Instruments, Wilmington, Del.). To detect DHBV DNA in liver tissue, 150-250 mg of liver tissue was homogenized in 5 ml of ice-cold 0.01 M Tris-HCl (pH 7.4) and 0.01 M ethylenediaminetetraacetic acid, followed by the addition of 5 ml of 0.2 M NaCl, 0.02 M Tris-HCl, 0.02 M ethylenediaminetetraacetic acid, and 1% sodium dodecyl sulfate containing 1 mgiml Pronase and incubation at 37°C for 30 min. The nucleic acids were deproteinized by two extractions with Tris-buffered (pH 7.4) phenol-chloroform (l:l), then precipitated and washed with -20°C absolute ethanol. Total nucleic acids were determined by measuring the optical density at 260 nm (Hitachi 220A spectrophotometer, Hitachi Ltd., Tokyo, Japan), and l-pg aliquots of nucleic acid were diluted in a total of 5 ~1 of distilled water and blotted on nitrocellulose filter paper, hybridized with either the nick-translated, cloned DHBV DNA probe or the strand-specific RNA probe, and autoradiographed as outlined above. Liver DNA samples (25 pg) were also separated by horizontal slab gel electrophoresis on 1.5% agarose and transferred to nitrocellulose filter paper by the method of Southern (29) as modified by Wahl et al. (28). Hybridization and autoradiography were performed as outlined above. To quantitate DHBV DNA, dot blot and Southern blot autoradiographs were analyzed with a Sakura PDS 15 densitometer (Sakura Co., Tokyo, Japan) and peak heights were measured.

Results All ducks survived the treatment protocol and wedge liver biopsies without any apparent ill effects.

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820 SHERKEXETAL.

we low 1

treatment

post

a 10s

low

c

1 2

2 high 3

b

Pas

leg high 3 4

4 control 5 6 Figure

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1. Serum DHBV DNA demonstrated by the dot blot hybridization technique. Serum samples were obtained twice before the initiation of treatment [pre), three times during the course of treatment, and twice after the cessation of treatment (post) for each duck. Ducks 1 and z were treated with foscarnet 50 mg/kg b.i.d., ducks 3 and 4 with foscarnet 250 mg/kg b.i.d., and ducks 5 and 6 with sterile water injections. Radiolabeled, cloned DHBV DNA is used as the probe. A marked reduction in DHBV DNA is seen during the treatment period in the 2 ducks treated with the higher dose. A lesser reduction is seen in duck 1 of the low-dose group. The serum DHBV DNA tends to return to baseline values after the cessation of treatment. pos, positive control; neg, negative control.

In the 2 ducks treated with foscarnet 250 mg/kg b.i.d. (high dose), there was an almost total elimination of circulating DHBV DNA during the treatment period. In the follow-up period after the cessation of treatment, DHBV DNA once again became detectable. In 1 of the 2 ducks treated with foscarnet 50 mg/kg b.i.d. (low dose, duck No. l),there was a less marked decline in serum DHBV DNA during treatment. There was some variability in the amount of circulating viral DNA in the control ducks, but it never fell to the level seen in the high-dose ducks and there was no consistent decline observed during the treatment period (Figure 1). There was a gradual decline in serum DHBV DNA concentration with time, and the effect of foscarnet might be superimposed on this general trend. In the dot blot hybridization of DNA extracted from liver using the double-stranded DNA probe, there was a marked decline in DHBV DNA during treatment in the high-dose group. Again, viral DNA tended to return to its baseline value after the cessation of treatment. There was little change seen in the low-dose ducks, and the control ducks did not exhibit the variability observed in serum (Figure 2). When the plus-strand specific RNA probe was used for dot blot hybridization of liver DNA, a decline was seen in the level of viral plus strand during treatment in the low-dose ducks, whereas in the high-dose ducks the decline persisted through the follow-up period (Figure 3).

w control Figure

2. Intrahepatic DHBV DNA by the dot blot hybridization technique. Deoxyribonucleic acid was extracted from liver tissue obtained by wedge biopsy before treatment [a) and at the end of the treatment period (b). Ducks were killed 10 days after the cessation of treatment and liver tissue was obtained (c). Denatured, radiolabeled, cloned DHBV DNA is used as the probe. Intrahepatic DHBV DNA is shown to decrease markedly in the ducks treated with high-dose foscarnet but to return to baseline levels after treatment is stopped.

Densitograms were done of the liver dot blot autoradiograms, and DHBV DNA was quantitated by measuring the peak heights of the dots. The ratio of treatment and posttreatment densities to pretreatment values for each individual duck was calculated for each of the two probes. In the high-dose group there was an almost complete decline in DHBV DNA

a low

b

c

1 2 Pas

high

control

5 6

Figure

3. Intrahepatic DHBV DNA as in Figure 2, using a plusstrand specific RNA probe. With this probe, the decrease in DHBV DNA in the ducks treated with highdose foscarnet appears more marked and is sustained through the posttreatment period in duck 4. In the ducks treated with low-dose foscarnet there is a decrease in DHBV DNA that was not apparent using the double-stranded DNA probe. This suggests that foscarnet preferentially inhibits the formation of the plus strand of DHBV DNA.

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FOSCARNET IN CHRONIC DHBV INFECTION

changes were specimens.

observed

in posttreatment

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biopsy

Discussion

Figure 4. DHBV DNA obtained from densitograms of liver tissue dot blot hybridizations, expressed as a percentage of the pretreatment value for each individual duck. Mean results for low dose (A], high dose (C!), and control (0) group ducks are shown. Mean values of DHBV genomeequivalent per cell before treatment were 48 copies in low-dose group, 60 copies in high-dose group, and 65 copies in control group. Both double-stranded DNA probe (solid line) and plus-strand specific RNA probe (broken line] results are shown. Using the doublestranded DNA probe, a marked decline in DHBV DNA is observed in the high-dose group. With the plusstrand specific probe, this decline is more sustained and a moderate decline is seen during treatment in the low-dose group as well.

observed during treatment, which was noted to persist after treatment when the plus-strand specific probe was used. In the low-dose group an appreciable decrease in viral DNA was seen only with the plus-strand specific probe (Figure 4). Southern blot hybridizations of liver DNA were analyzed. When the double-stranded probe was used, a marked decrease in the replicative forms of DHBV DNA was observed in the ducks that received high-dose foscarnet and a lesser decrease was seen in the low-dose ducks. There was a tendency for all replicative forms to return to their baseline levels after the cessation of treatment (Figure 5). When the plus-strand specific probe was used, the decrease in DHBV DNA replicative forms was better appreciated in both low-dose and high-dose ducks. Hybridization with the plus-strand specific probe results in a marked attenuation of the signal due to the partially double-stranded species of DNA. This tends to “unmask” the supercoiled form, which is Seen to persist during low-dose treatment (Figure 6). Liver wedge biopsies revealed mild nonspecific abnormalities in all ducks before treatment, and no significant

According to the mechanism proposed by Summers and Mason (25), the hepadna viruses replicate through a unique pathway. The minus (coding) strand of viral DNA codes for a full-length, plus-strand RNA transcript known as the “pregenome.” Pregenomic RNA is then reverse-transcribed into minus-strand DNA, using viral DNA polymerase. This results in the degradation of the “pregenome.” Minus-strand DNA codes for the synthesis of plus-strand DNA using DNA polymerase. The plus strand is usually incomplete, resulting in a partially double-stranded circular DNA in circulating virions. Foscarnet has been shown in vitro to be a potent inhibitor of a number of viral RNA polymerases, reverse transcriptases, and DNA polymerases (22). Among these are woodchuck hepatitis virus DNA polymerase (30) and HBV DNA polymerase (31). Foscarnet has previously been studied in vivo in two animal models of chronic hepadna virus infection. In DHBV infection, the drug had no demonstrable effect on viral replication as measured by serum DNA polymerase activity (Tsiquaye KN, Collins P, Zuckerman AJ, abstract presented at the Interna-

Figure 5. Southern blot hybridization of DNA extracted from liver tissue using the double-stranded, cloned DHBV DNA probe. Ducks 1 and 2 were treated with low-dose foscarnet, ducks 3 and 4 were treated with high-dose foscarnet, and duck 5 is a representative control group duck. Liver tissue was obtained before the initiation of treatment (lane a], at the end of the treatment period (lone b), and 10 days after the cessation of treatment (lane c). A moderate decrease in DHBV DNA is seen during treatment in the low-dose ducks and a more marked decrease is seen in the high-dose ducks. Lane v is cloned, linear DHBV DNA. RC, relaxed circular viral DNA; PDS, partially double-stranded DNA; SC, supercoiled DNA; and SS, single-stranded DNA.

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Figure 6. Southern blot hybridization of liver tissue DNA using the plus-strand specific RNA probe. Ducks 1 and 2 are low dose; 8 and 4, high dose; and 5, control. Lanes a, b, and c are pretreatment, end of treatment period, and 10 days posttreatment, respectively. Because of the marked attenuation of the signal due to partially double-stranded DNA, it can be appreciated that the supercoiled form of viral DNA (SC] is relatively unaffected by treatment with low-dose foscarnet. Lane v is cloned DHBV DNA. RC, relaxed circular DNA; L, linear DNA; and SC, supercoiled DNA.

tional Symposium on Antiviral Agents in Chronic Hepatitis B Infection, London, November 11,1985). In woodchuck hepatitis virus, a 2-wk course of foscarnet was shown to have no effect on DNA polymerase activity (32). The present study differs from the two previous studies in several important respects. The dose of foscarnet used in woodchucks was comparable to that used in our low-dose group, so some of our ducks received a considerably higher dose of the drug. In addition, we monitored viral replication using molecular biologic techniques as opposed to the DNA polymerase assays used by the other studies, The DNA polymerase assay has been associated with falsely elevated results due to inadvertent bacterial contamination and difficulty in determining low level activities (33,341. There are also potential species differences. In the present study foscarnet was shown to be effective in reducing circulating DHBV virions, as measured by serum DHBV DNA, in chronically infected ducks. This reduction was dose-related and readily reversed after the cessation of a short course of treatment. Serum DHBV DNA in control ducks was observed to exhibit a moderate degree of variability. This phenomenon has been observed by us (unpublished data) and others (Dr. Patricia Marion, personal communication) repeatedly, especially in young carrier ducks. A waxing and waning pattern of DNA polymerase activity in viremic ducks has also been

GASTROENTEROLOGY Vol. 91. No. 4

observed (35). This variability is far less marked when intrahepatic viral DNA is monitored, suggesting that it may be related to a varying rate of release of virions into the serum or clearance of circulating viral particles. During the course of treatment, intrahepatic viral DNA was shown to fall in a dose-related manner. After treatment was stopped, DNA tended to return toward its baseline value. These changes were most marked and prolonged when the plus-strand specific probe was used, suggesting that the drug has a disproportionate inhibitory effect on plus-strand synthesis using the minus-strand DNA template. This is consistent with the in vitro observation that foscarnet is a potent inhibitor of the DNA polymerase of hepadna viruses. In addition, DNA polymerase is thought to catalyze the reverse transcription of the pregenome, and foscarnet is known to inhibit the reverse transcriptases of several retroviruses (22). Thus, the drug may exert its effect at two sites in&the hepadna virus replicative pathway: reverse transcription of the pregenome to form minus strand, and plus-strand synthesis. The drug is not viricidal in the dosages and treatment conditions used in this study. When intrahepatic replicative forms of the virus were studied using Southern blot hybridization, the supercoiled form was shown to be least affected by treatment. In viral transmission studies, it has been shown that the supercoiled form of DNA is the first form detected in the liver and may be responsible for the initiation .of the viral replicative cycle (36,37). We have shown that the supercoiled form tends to persist after treatment with interferon (8) and in patients with mild chronic liver disease due to HBV, who exhibit “inactive” viral replication (38). The failure to eliminate the supercoiled species of viral DNA is likely responsible for the resumption of high levels of viral replication after foscarnet is withdrawn. Foscarnet has been used in immunocompromised patients with life-threatening cytomegalovirus infections (23,24,38,40) with no obvious toxicity and some clinical improvement. However, one should remember that the severe disease of these patients and other medications might have made an evaluation of toxic effects difficult. Toxicity, fertility, and teratogenicity studies in animals have shown no adverse effects of the drug (22). Because of the proposed mechanism of action of the drug, it would not be expected to efficiently eradicate chronic infections with HBV if used alone for a short period of time. Long-term treatment or combination with other therapeutic modalities may, however, be beneficial. In view of the beneficial effects shown in this study and the low clinical and

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experimental toxicity foscarnet in chronic indicated.

FOSCARNET IN CHRONIC DHBV INFECTION

of the drug, further studies of hapadna virus infection are

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38. Yokosuka 0, Omata M, Imazeki F, Okuda K. Active and inactive replication of hepatitis B virus deoxyribonucleic acid in chronic liver disease. Gastroenterology 1985;89:616-6. 39. Apperley JF, Marcus RE, Goldman JM, et al. Foscarnet for cytomegalovirus pneumonitis (lett). Lancet 1985;i:1151. 40. Ringden 0, Wilezek H, Lomavist B, et al. Foscarnet for cytomegalovirus infections (lett). Lancet 1985;i:503-4.