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Screening of antiviral drugs for treatment of hepatitis B
Journal of Hepatology, 1986;3 (Suppl. 2): $45-$48
$45
Elsevier HEP 000169
Screening of Antiviral Drugs for Treatment of Hepatitis B*
K . N . T s i q u a y e 1, P. Collins 2 a n d A . J . Z u c k e r m a n 1 t Department of Medical Microbiology, London School of Hygiene and Tropical Medicine, Keppel Street, London WCI E 7HT and 2Biochemical Virology Department, The Wellcome Research Laboratories, Beckenham, Kent ( U. K.)
Infections with human hepatitis B virus (HBV) constitute a public health problem of major epidemiological significance. The disease is endemic throughout the world and up to 15% of infected adult individuals become persistent carriers. These chronic carriers serve as a reservoir and their numbers are amplified by perinatal infection of 80% or more among infants born to carrier mothers in some regions of the world [1,2], and in whom a carrier state becomes established. The enormity of the problem is stressed by the fact that it is estimated that there are at least 210 million carriers (about 5% of the world's population) of markers of hepatitis B virus [2]. The carrier state cannot be terminated by immunization with vaccines. Effective antiviral drugs are therefore required, firstly for the treatment of chronic hepatitis B infection and the associated liver damage, secondly to arrest progression to more severe forms of liver disease such as cirrhosis and primary liver cancer, and thirdly to reduce the reservoir of carriers of hepatitis B and to limit spread of the virus to contacts. The remarkable species specificity which characterizes hepatitis B virus infection has severely limited
studies on the treatment of persistent infection. Man and the great apes are the only hosts permissive for viral replication, but non-human primates are inappropriate for the evaluation of large numbers of antiviral drugs. Consequently few drugs have been examined for the treatment of persistent HBV infection. However, three animal models (two mammalian and one avian) of HBV infection have been described recently. The natural host of the avian virus (duck hepatitis B virus) is the Pekin duck in which the virus is transmitted congenitally [3,4]. The duck virus shares common virion architecture and genomic size and organization with the human prototype, HBV. The surface antigens of the two viruses do not share antigenic determinants, and the avian virus is not pathogenic for man. However, the duck hepatitis B virus, like its human counterpart, attacks the liver, produces a persistent infection in all the offspring of carrier ducks [4], and may progress to cirrhosis and hepatocellular carcinoma in adult ducks [5]. The domestic duck is therefore an ideal model of the human disease for in vivo evaluation of antiviral compounds for the treatment of carriage of hepatitis B and for
This research programme is supported by a grant from the Wellcome Research Foundation (K.N.T.), and by a grant from the Department of Health and Social Security. * Presented in part at the Symposiumorganized by the British Society for Antimicrobial Chemotherapy in Bath, July 1985, Journal of Antimicrobial Chemotherapy (in press). 0168-8278/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
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Fig. 1. Serum levels of DNA polymerase activity in Pekin ducks given foscarnet (PFA) intraperitoneally at a dose of 168 mg/kg twice daily for 3 weeks.
Fig. 2. Results of combined acyclovir (ACV) and suramin therapy in Pekin ducks. Oral acyclovir was administered for 2 weeks. On day 7, 9, 14 and 21 of acyclovir therapy, suramin was administered intravenously at a dose of 20 mg/kg.
studies of chronic liver disease associated with this infection. Three compounds, acyclovir (acycloguanosine), foscarnet (phosphonoformate) and suramin were tested for antiviral activity in Pekin ducks persistently infected with the duck hepatitis B virus ( D H B V ) . Although foscarnet inhibits HBV-specific D N A polymerase ( D N A P ) activity in vitro, it had no demonstrable effect on the replication of D H B V when it was given intraperitoneally twice daily for 3 weeks using a dose of 168 mg/kg (Fig. 1). However, acyclovir, which is inactive in vitro against H B V and D H B V polymerase (Tsiquaye, KN, unpublished
data) and suramin, an inhibitor of polymerase activity of D H B V [6] reduced significantly but transiently the activity of the enzyme in persistently infected ducks. A combination of oral acyclovir (20 mg/kg/ day for 2 weeks) and suramin administered intravenously (20 mg/kg on day 7, 9, 14 and 21 of acyclovir therapy) proved effective in suppressing D N A P activity (Fig. 2). Cessation of treatment was followed by reduced levels of D N A P activity which were maintained for about 3 weeks longer than observed with either of the two drugs administered alone. The data indicate that the duck is a particularly suitable model for screening and testing antiviral drugs and synthetic analogues.
References
1 Zuckerman AJ. Perinatal transmission of hepatitis B. Arch Dis Childh. 1984; 59:1007-1009. 2 World Health Organisation. Prevention of Liver Cancer (Technical Report Series, No. 691), WHO, Geneva, 1983. 3 O'Connell AP, Urban MK, London WT. Naturally occurring infection of Pekin duck embryos by duck hepatitis B virus. Proc Nat Acad Sci (USA) 1983; 80:1703-1706. 4 Tsiquaye KN, McCaul TF, Zuckerman AJ. Maternal transDiscussion
Dr RS Tedder (London, U.K.): What is the state of the nucleic acid in these d u c k s - - is it both free and in-
mission of duck hepatitis B virus in pedigree Pekin ducks. Hepatology 1985; 5:46-52. 5 Omata M, Uchiumi K, Yoshimi I, Yokosuka O, Mori J, Terao K, Wei-Fa Y, O'Connell AP, London WT, Okuda K. Duck hepatitis B virus and liver diseases. Gastroenterology 1983; 85:260-267. 6 Tsiquaye K, Zuckerman A. Suramin inhibits duck hepatitis B virus DNA polymerase activity. J Hepatol 1985; 1:663-669.
tegrated? Is it known?
Professor AJ Zuckerman: It is both free and integrated. We are doing the D N A studies now.
$47
Dr RS Tedder: If we are going to try to prevent virus replication, we will have to go very far upstream, at least in the human hepatitis B infections. To prevent integration, people will probably need to be treated as soon as they have been infected, rather than the 200 million people in the world (to whom Professor Zuckerman alluded) who are already established hepatitis B carriers, who are the major source of all secondary infections that will occur from whatever day we look at that problem onwards. Professor AJ Zuckerman: Of course, I agree with you entirely. We are trying to emphasize that at long last there is a readily available model which is available for experimentation. The question of when to give the drug is very relevant. I think this has already been referred to by Dr Williams. That is something that we can now study. Professor A L W F Eddleston (London, U.K.): I think we shall hear later that.probably in man there may be an interaction between the immune system and the drugs concerned. How valid is the duck model? I ask out of ignorance, because I know nothing about it. What does the liver disease look like in the ducks? Do they show spontaneous seroconversion (as happens in man), and this phasic illness (as also happens in man)?
Professor Zuckerman: Spontaneous seroconversion does not take place in these ducks. They are persistently infected in ovo. It is quite easy to infect the eggs, the ducks hatch and we have persistently infected ducks - - large flocks of them, I may add (to which I think Dr Tsiquaye will testify). We are now studying liver damage, but there is a lot of evidence from other groups that, in addition to persistent infection, there is chronic liver disease and it also progresses to hepatocellular carcinoma. The model is therefore highly appropriate, but I am not aware of any spontaneous seroconversion. Dr Tsiquaye would probably be able to reassure me on that. Is that right, Dr Tsiquaye? Dr Tsiquaye (London, U.K.): I think that is right.
We have not so far found any seroconversion in any of the ducks .that we have studied. These are both experimentally infected animals and congenitally or maternally-transmitted infections.
Dr Tedder: Can we define what we mean by 'seroconversion'? Are we talking about surface antigen or equivalent or analogue of e antigen? Dr Tsiquaye: In the ducks we are looking at viral markers - - D N A polymerase, the actual particles in the serum - - and so far no loss of viral particles has been observed. Dr Tedder: So we are talking here about the loss of the duck surface antigen and of the whole virus. (Dr Tsiquaye: Yes.) Dr M Omata (Chiba, Japan): With regard to the liver disease, we have found integrated viral D N A in the Pekin duck. This was reported i~n the Proceedings of the National Academy of Sciences (September 1985). We also found cirrhotic changes. There is certainly liver damage in the infected duck, especially the Chinese duck. Dr DA Shafritz (New York, U.S.A.): I am very excited about this finding with the ducks. The ability to infect them in utero might provide an excellent model for trying to reverse the integration state. In fact, that may be the reason why there is integrated viral D N A in these ducks. It could be related to liver cancer, regardless or independent of liver disease activity. This could be a very exciting model to try various modes. I would also hasten to add that the types of things at which we would look would be most appropriate for viral replication in free virions. It may not be of help in clearing surface antigen from an integrated template.
Dr JH Hoofnagle (Bethesda, U.S.A.): First, with regard to Professor Zuckerman's presentation, the exciting thing about the duck is that it provides a way to screen drugs to find out whether they inhibit hepa-
$48 titis B virus-like agents. That is why it is an excellent model. However, it wil not provide the answer whether those drugs will work in humans because of the enormous difference between chronic hepatitis in
these animals and in humans. That difference was pointed out by Dr Perrillo and the others. It is that in the humans there is a very major contribution of the immune response in the response to antivirals.
$45
Elsevier HEP 000169
Screening of Antiviral Drugs for Treatment of Hepatitis B*
K . N . T s i q u a y e 1, P. Collins 2 a n d A . J . Z u c k e r m a n 1 t Department of Medical Microbiology, London School of Hygiene and Tropical Medicine, Keppel Street, London WCI E 7HT and 2Biochemical Virology Department, The Wellcome Research Laboratories, Beckenham, Kent ( U. K.)
Infections with human hepatitis B virus (HBV) constitute a public health problem of major epidemiological significance. The disease is endemic throughout the world and up to 15% of infected adult individuals become persistent carriers. These chronic carriers serve as a reservoir and their numbers are amplified by perinatal infection of 80% or more among infants born to carrier mothers in some regions of the world [1,2], and in whom a carrier state becomes established. The enormity of the problem is stressed by the fact that it is estimated that there are at least 210 million carriers (about 5% of the world's population) of markers of hepatitis B virus [2]. The carrier state cannot be terminated by immunization with vaccines. Effective antiviral drugs are therefore required, firstly for the treatment of chronic hepatitis B infection and the associated liver damage, secondly to arrest progression to more severe forms of liver disease such as cirrhosis and primary liver cancer, and thirdly to reduce the reservoir of carriers of hepatitis B and to limit spread of the virus to contacts. The remarkable species specificity which characterizes hepatitis B virus infection has severely limited
studies on the treatment of persistent infection. Man and the great apes are the only hosts permissive for viral replication, but non-human primates are inappropriate for the evaluation of large numbers of antiviral drugs. Consequently few drugs have been examined for the treatment of persistent HBV infection. However, three animal models (two mammalian and one avian) of HBV infection have been described recently. The natural host of the avian virus (duck hepatitis B virus) is the Pekin duck in which the virus is transmitted congenitally [3,4]. The duck virus shares common virion architecture and genomic size and organization with the human prototype, HBV. The surface antigens of the two viruses do not share antigenic determinants, and the avian virus is not pathogenic for man. However, the duck hepatitis B virus, like its human counterpart, attacks the liver, produces a persistent infection in all the offspring of carrier ducks [4], and may progress to cirrhosis and hepatocellular carcinoma in adult ducks [5]. The domestic duck is therefore an ideal model of the human disease for in vivo evaluation of antiviral compounds for the treatment of carriage of hepatitis B and for
This research programme is supported by a grant from the Wellcome Research Foundation (K.N.T.), and by a grant from the Department of Health and Social Security. * Presented in part at the Symposiumorganized by the British Society for Antimicrobial Chemotherapy in Bath, July 1985, Journal of Antimicrobial Chemotherapy (in press). 0168-8278/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
$46
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f
16
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DBW (cP)
8//"~/
%-
~ / a/ /,/
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20r~,, .,~.
x 16
\
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/
2 WEEKS
tl
111o~ 115 9
8
z:
-1
0
1
2
3
tl
S 6 WEEKS
7
8
9
10
II
12
Fig. 1. Serum levels of DNA polymerase activity in Pekin ducks given foscarnet (PFA) intraperitoneally at a dose of 168 mg/kg twice daily for 3 weeks.
Fig. 2. Results of combined acyclovir (ACV) and suramin therapy in Pekin ducks. Oral acyclovir was administered for 2 weeks. On day 7, 9, 14 and 21 of acyclovir therapy, suramin was administered intravenously at a dose of 20 mg/kg.
studies of chronic liver disease associated with this infection. Three compounds, acyclovir (acycloguanosine), foscarnet (phosphonoformate) and suramin were tested for antiviral activity in Pekin ducks persistently infected with the duck hepatitis B virus ( D H B V ) . Although foscarnet inhibits HBV-specific D N A polymerase ( D N A P ) activity in vitro, it had no demonstrable effect on the replication of D H B V when it was given intraperitoneally twice daily for 3 weeks using a dose of 168 mg/kg (Fig. 1). However, acyclovir, which is inactive in vitro against H B V and D H B V polymerase (Tsiquaye, KN, unpublished
data) and suramin, an inhibitor of polymerase activity of D H B V [6] reduced significantly but transiently the activity of the enzyme in persistently infected ducks. A combination of oral acyclovir (20 mg/kg/ day for 2 weeks) and suramin administered intravenously (20 mg/kg on day 7, 9, 14 and 21 of acyclovir therapy) proved effective in suppressing D N A P activity (Fig. 2). Cessation of treatment was followed by reduced levels of D N A P activity which were maintained for about 3 weeks longer than observed with either of the two drugs administered alone. The data indicate that the duck is a particularly suitable model for screening and testing antiviral drugs and synthetic analogues.
References
1 Zuckerman AJ. Perinatal transmission of hepatitis B. Arch Dis Childh. 1984; 59:1007-1009. 2 World Health Organisation. Prevention of Liver Cancer (Technical Report Series, No. 691), WHO, Geneva, 1983. 3 O'Connell AP, Urban MK, London WT. Naturally occurring infection of Pekin duck embryos by duck hepatitis B virus. Proc Nat Acad Sci (USA) 1983; 80:1703-1706. 4 Tsiquaye KN, McCaul TF, Zuckerman AJ. Maternal transDiscussion
Dr RS Tedder (London, U.K.): What is the state of the nucleic acid in these d u c k s - - is it both free and in-
mission of duck hepatitis B virus in pedigree Pekin ducks. Hepatology 1985; 5:46-52. 5 Omata M, Uchiumi K, Yoshimi I, Yokosuka O, Mori J, Terao K, Wei-Fa Y, O'Connell AP, London WT, Okuda K. Duck hepatitis B virus and liver diseases. Gastroenterology 1983; 85:260-267. 6 Tsiquaye K, Zuckerman A. Suramin inhibits duck hepatitis B virus DNA polymerase activity. J Hepatol 1985; 1:663-669.
tegrated? Is it known?
Professor AJ Zuckerman: It is both free and integrated. We are doing the D N A studies now.
$47
Dr RS Tedder: If we are going to try to prevent virus replication, we will have to go very far upstream, at least in the human hepatitis B infections. To prevent integration, people will probably need to be treated as soon as they have been infected, rather than the 200 million people in the world (to whom Professor Zuckerman alluded) who are already established hepatitis B carriers, who are the major source of all secondary infections that will occur from whatever day we look at that problem onwards. Professor AJ Zuckerman: Of course, I agree with you entirely. We are trying to emphasize that at long last there is a readily available model which is available for experimentation. The question of when to give the drug is very relevant. I think this has already been referred to by Dr Williams. That is something that we can now study. Professor A L W F Eddleston (London, U.K.): I think we shall hear later that.probably in man there may be an interaction between the immune system and the drugs concerned. How valid is the duck model? I ask out of ignorance, because I know nothing about it. What does the liver disease look like in the ducks? Do they show spontaneous seroconversion (as happens in man), and this phasic illness (as also happens in man)?
Professor Zuckerman: Spontaneous seroconversion does not take place in these ducks. They are persistently infected in ovo. It is quite easy to infect the eggs, the ducks hatch and we have persistently infected ducks - - large flocks of them, I may add (to which I think Dr Tsiquaye will testify). We are now studying liver damage, but there is a lot of evidence from other groups that, in addition to persistent infection, there is chronic liver disease and it also progresses to hepatocellular carcinoma. The model is therefore highly appropriate, but I am not aware of any spontaneous seroconversion. Dr Tsiquaye would probably be able to reassure me on that. Is that right, Dr Tsiquaye? Dr Tsiquaye (London, U.K.): I think that is right.
We have not so far found any seroconversion in any of the ducks .that we have studied. These are both experimentally infected animals and congenitally or maternally-transmitted infections.
Dr Tedder: Can we define what we mean by 'seroconversion'? Are we talking about surface antigen or equivalent or analogue of e antigen? Dr Tsiquaye: In the ducks we are looking at viral markers - - D N A polymerase, the actual particles in the serum - - and so far no loss of viral particles has been observed. Dr Tedder: So we are talking here about the loss of the duck surface antigen and of the whole virus. (Dr Tsiquaye: Yes.) Dr M Omata (Chiba, Japan): With regard to the liver disease, we have found integrated viral D N A in the Pekin duck. This was reported i~n the Proceedings of the National Academy of Sciences (September 1985). We also found cirrhotic changes. There is certainly liver damage in the infected duck, especially the Chinese duck. Dr DA Shafritz (New York, U.S.A.): I am very excited about this finding with the ducks. The ability to infect them in utero might provide an excellent model for trying to reverse the integration state. In fact, that may be the reason why there is integrated viral D N A in these ducks. It could be related to liver cancer, regardless or independent of liver disease activity. This could be a very exciting model to try various modes. I would also hasten to add that the types of things at which we would look would be most appropriate for viral replication in free virions. It may not be of help in clearing surface antigen from an integrated template.
Dr JH Hoofnagle (Bethesda, U.S.A.): First, with regard to Professor Zuckerman's presentation, the exciting thing about the duck is that it provides a way to screen drugs to find out whether they inhibit hepa-
$48 titis B virus-like agents. That is why it is an excellent model. However, it wil not provide the answer whether those drugs will work in humans because of the enormous difference between chronic hepatitis in
these animals and in humans. That difference was pointed out by Dr Perrillo and the others. It is that in the humans there is a very major contribution of the immune response in the response to antivirals.