Hepatitis B virus and primary hepatocellular carcinoma: treatment of HBV carriers with Phyllanthus amarus B.S. Blumberg**, I. Miilman*, P.S. Venkateswaran* and S.P. Thyagarajan t A viricide capable of eliminating hepatitis B virus ( l i b V) from chronic carriers should, theoretically, decrease the risk of primary hepatocellular carcinoma. Extracts of Phyllanthus amarus have been shown to inhibit the DNA polymerase of HBV and woodchuck hepatitis virus (WHV) in vitro. Three of four recently infected WHV carriers treated i.p. with P. amarus extract lost WHV, animals infected for >~3 months showed a decrease in virus levels. Preliminary results in human carriers treated orally with P. amarus for 1 month indicated that ~ 60% of the carriers lost HBV during the observation period. Keywords: Phyllanthus amarus; hepatitis B virus; woodchuck hepatitis virus; hepatocellular carcinoma

Introduction There are ~ 300 million carriers of hepatitis B virus (HBV) in the world. Based on a substantial body of data, they are known to be at high risk of developing primary hepatocellular carcinoma (PHC) and/or chronic liver disease. The main thrust of current preventive efforts is to provide vaccination, particularly of the newborn. An additional potential focus for prevention and treatment is the carriers of HBV. Humans appear to be the main reservoir for HBV and infectious carriers are the primary source for the spread of infection in a community. The development of a treatment for HBV carriers could have an enormous beneficial effect on morbidity and mortality and also make a major contribution to the control and possible eradication of HBV. Many individuals who develop PHC are infected at an early age. In Japan and East Asia, chronic infection from a carrier mother may begin within the first year, but rarely before three months of age. In Africa, persistent infection rarely occurs before ~ 12-18 months of age but is common after that and may be due to horizontal transmission from the mother, siblings or other household members. Cancer, however, does not develop until much later - about 40 and 50 years of age in Africa and Asia, respectively; there is a very long incubation period for development of PHC and consequently, an extensive period during which treatments may be undertaken to mitigate the high and tragic risk of developing PHC. A model developed in 1982 by London and Blumberg I can be used not only to explain certain interesting features of the relation between P H C and HBV (Figure I), but to formulate a strategy for eventual treatment of chronic HBV carriers. *Fox Chase Cancer Center, Philadelphia, PA, USA. *Postgraduate Institute of Basic Medical Sciences, University of Madras, India. **To whom correspondence should be addressed. This work will also appear in Cancer Detection and Prevention 1990, and is printed here with permission from CRC Press, Inc., Boca Raton, FL, USA 0264-410x/90/s10086-07 ,~~ i _ 1990 Butterworth & Co. (Publishers) Ltd $86 Vaccine, Vol. 8, Supplement 1990

A model for HBV and the pathogenesis of PHC The long 'incubation' period between HBV infection and the development of P H C has already been mentioned; any model has to include time as a major component. Hepatitis B surface antigen (HBsAg) and hepatitis Be antigen (HBeAg), indicative of viral replication, are present in the sera in high levels within the first few years of infection, but the amount of virus and viral replication decreases as infection continues; HBsAg may be present in very low amounts or be undetectable when P H C is advanced. In cases where the early tumour develops, ~-fetoprotein may often be detected and its level can increase as the tumour progresses; ferritin and other 'markers' of cancer may also increase. While tow levels of HBV or replicating virus occur in the cancerous hepatocytes of a patient with PHC, much more virus and replication can be detected in the surrounding cells. Integrated viral genomes are usually found both in the cancerous and non-cancerous cells; in some cases, integration may have occurred early in infection but is not detectable when the tumour is diagnosed. There is no evidence of an oncogene in the virus or of consistent integration adjacent to a host oncogene. The London-Blumberg model t postulates the existence of two kinds of cells with respect to HBV infection and replication. One cell type, a fully differentiated liver cell would, when infected, sustain active replication of HBV. Integration of HBV DNA may also occur. This has been designated as an 'S" cell. i.e. susceptible to replication. The second kind of cell was postulated to be an undifferentiated cell, common in the fetus, frequent in the newborn but rare in adults. This has been termed an 'R' cell, i.e. resistant to replication. Upon infection, replication does not occur in the R cell, but HBV DNA may integrate into the host DNA. The fate of these two cells following infection is very different. In the actively replicating S cell, HBV antigens, particularly the hepatitis B core antigen (HBcAg), are expressed in the liver cell. They are recognized as 'foreign' by the host and the response of the immune system destroys the infected S cells. In the R cells, replication

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does not occur or is reduced and viral antigens are not expressed on the liver cell surface. R cells are relatively protected and at a selective advantage when compared with the S cells. The death of the S cells stimulates the division of the advantaged R cells, which can maintain some semblance ofliver function. This is reflected in the decreased amount of HBsAg and replicating virus as infection proceeds. The rapid division of the R cells leads to the increased production of ~-fetoprotein and other fetal proteins, and also increases the probability that a mutational event will take place. This, in turn, can result in the generation of a favoured clone (perhaps because of the activation of a growth factor). This may give rise to uncontrolled growth and the development of a perceptible tumour which will grow, metastasize and lead to the eventual death of the patient. Based on recent observations using restriction fragment length polymorphisms (RFLP) to study the chromosomes of liver tumour and normal liver tissue from the same individual, it has been postulated that the mutational event required by the model is the removal of a hypothesized second anti-oncogene allele located on chromosome 4. There may be similar events on other chromosomes. This model has several interesting consequences. Essentially, all treatments for cancer are directed at the destruction or removal of cancer cells by surgery, radiation, chemicals, immune modifiers, etc. The inherent disadvantage of these approaches is that they also kill

or damage normal cells, since cancer and normal cells have many similarities. The use of these treatments is limited by the therapeutic index, which is often low. This model for HBV-induced PHC suggests that treatment should be directed at the S cells in which HBV is replicating and not at the cancer cells. Such treatment can be started years, even decades, before the cancer may arise. Since the carcinogenic process is slow, the treatment need not be instantaneous; low doses of the postulated medication can be used and toxicity minimized. Even if the medication does not totally eliminate the HBV that is steering the cancer process, it could conceivably slow down HBV replication and the destruction of liver cells. The asymptomatic period could thereby be extended and the carrier remain free of chronic liver disease and liver cancer to live out his or her expected life-span. Search for an appropriate medication

An understanding of this concept encouraged the search for an antiviral agent which would be effective against HBV. Since most medications used in modem medicine are derived from natural substances, a search was begun in the 1970s to screen plants for activity against HBV. A description of the laboratory and animal studies conducted in Philadelphia and the clinical trial performed in Madras are given here. Additional laboratory investigations have been described elsewhere 2. The first screening strategy used was the ability of plant

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T r e a t m e n t o f H B V c a r r i e r s with P. amarus:

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antiviral agents could mitigate the problem, often encountered when developing therapies from natural products, that a material which appears to be effective in vitro or in tissue cultures is ineffective in animals and/or human patients and vice versa. It also deals with another problem encountered in the discovery of drugs derived from natural products. A selection of the major active component is often made early in an investigation; later it is realized that it may not be the active principle in patients or that several active principles acting together may be required.

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extracts to coat viral HBsAg and thereby inhibit the reaction with antibodies to HBsAg (anti-HBs). The rationale was that such an inhibition might have an effect on pathogenesis in uivo. Using this approach, > 200 out of ~ 1000 plants screened were found to be strong inhibitors. Subsequently, as more was learned about the replication mechanism of HBV, screening for the ability of plant extracts to inhibit the DNA polymerase (DNAp) of HBV in vitro was carried out. Extracts of a marine alga of the Fucaceae family, which strongly inhibited the HBsAg/anti-HBs interaction3 were studied. P.S. Vankateswaran, a natural products chemist, joined the group to carry out the isolation and characterization studies on the Fucaceae. He also initiated studies on a plant used for the treatment of jaundice in India. Extracts of this plant, originally classified as Phyllanthus niruri but now known as P. amarus, strongly inhibited HBV DNAp as well as the HBsAg/anti-HBs interaction. Before proceeding with further work on Phyllanthus, the available world's literature on ethnobotany was reviewed, with the help of T. Sheppherd of Oxford University, to identify plants that had been used for the treatment of jaundice and other symptoms of liver disease; nearly 1000 such plants were found. The species names were entered in a data base management system and sorted alphabetically. All plants for which species of the same genus had been used in three or more global regions as a treatment for jaundice and other liver symptoms were placed on a shortlist, which eventually included < 100 species. Phyllanthus species were prominent on this list. They had been used in India, China, Burma, Pakistan, the Philippines, Guam, the West Indies, South America, East and West Africa, and elsewhere in the tropics and subtropics for the treatment of jaundice and other liver abnormalities. It was decided to focus activities on P. amarus, which could be collected in the Madras area. The strategy was initially to test the whole extract (i.e. not fractions) in animals and, only after appropriate safety tests, in humans. If effectiveness was demonstrated, the fractions containing the active principle (or principles) would be determined and then characterized chemically. This procedure of bioassay-directed purification of

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D N A p o f H B Y and similar viruses. As noted, extracts of P. amarus inhibit the DNAp of human HBV and the closely related woodchuck hepatitis virus (WHV) and duck hepatitis B virus (DHBV). As seen in Figure 2, there is a linear dose-inhibition relationship4. The ability of extract of P. amarus to inhibit DNAp from other sources was also determined. The concentration of the extract of P. amarus required to produce a 50% inhibition of the DNAp activity was determined by adding serial dilutions of the extract to assay mixtures containing appropriate 3H-labelled nucleotide triphosphates, DNA template and endogenous or, where necessary, exogenous DNAp. Assay mixtures without the inhibitor and those without the enzyme served as controls for normal reaction and blank reading. A comparison of the concentrations required for 50% inhibition of the DNAp from viral (AMV RT), bacterial (Escherichia coli DNAp, DNAp 1 and the Klenow fragment) and mammalian sources (calf thymus) are given in Table I. The results indicate that the concentrations required to inhibit DNAp from other sources (115-460 #g m1-1) are 2-8 higher than the concentrations required for HBV DNAp. From this it is inferred that the extract is relatively specific for HBV DNAp. HBsAg. The extract also inhibits the binding of HBsAg or woodchuck hepatitis surface antigen (WHsAg) with anti-HBs in assays using Ausria II kits (Abbott, Chicago, IL, USA) but at concentrations ~ 10 times higher than those required for DNAp inhibition. P. amarus, at a concentration of 5mgml-~, inhibits the HBsAg-antiHBs reaction by ~60% (Table 2). To determine the relative specificity of inhibition, the inhibitory effects of

Table 1

Inhibition of HBV DNAp and other DNAp by P. amarus Concentration for 50%

inhibition Enzyme

(#g ml - 1)

Reference

HBV DNAp E. coli DNAp 1 E. coli DNAp

59 460 387

Venkateswaran"

E. coli DNAp Klenow fragment

120

Calf thymus DNAp :(

115

AMV RT (with DNA template)

295

Venkateswaran" Unpublished results a Unpublished results a Unpublished results a Unpublished results a

aLoeb, L., Venkateswaren, P.S., Millman, I. and Blumberg, B.S., unpublished results

Treatment of HBV carriers with P. amarus: B. Blumberg et al. Table 2

Effect of P. amarus extract on the binding of anti-HBs to HBsAg or WHsAg

Inhibition of anti-HBs binding (%) P. a m a r u s

extract

(mg ml - 1)

WHsAg

HBsAg

5.00 2.50 1.25 0.63 0.50 0.31 0.10

61 45 25 11 13 13 5

63 35 18 9 0 0 0

Adapted from Ref. 4

extracts of P. amarus were tested on other antigenantibody interactions. These included human thyroglobulin, lactoferrin, IgM, a-fetoprotein and HBcAg. In each case, the reaction mixture, including the plant extract, was compared with a reaction mixture containing only phosphate-buffered saline; the results were internally consistent. In all comparisons of plant extract with buffered saline, there was no evidence of significant inhibition.

Studies in woodchucks Woodchucks chronically infected with WHV are a remarkable 'model' for the development of chronic liver disease and PHC in humans infected with I-IBV5'6, allowing tests of new drugs giving results analogous to those in human clinical trials. HBV and WHV are very similar. There is substantial immunological crossreactivity~-9 and ~79% DNA homology1° between WHV and HBV. In both hepadnaviridae, endogenous DNAp is required for replication. The pathogenesis of PHC and chronic liver disease is similar in both humans and woodchucks, including the carrier state, development of several forms of chronic hepatitis and progression to PHC. The likelihood that an animal found to be WHsAgpositive at the time of capture would remain a carrier was estimated. Using a life table analysis11, the probability that a woodchuck positive for WHsAg at the time of receipt from the trapper would remain positive until death was determined. This retrospective analysis was based on 192 animals that were WHsAg-positive at the time ofcapture, tested at least twice subsequent to capture and had not received medication in a treatment experiment. During the first 90 days of captivity, 68 animals died while remaining WHsAg-positive and 27 animals became WHsAg-negative. An additional 28 WHsAgpositive animals died during the next 90 days, while no animals became WHsAg-negative. Between days 180 and 270, two animals became WHsAg-negative and 12 WHsAg-positive animals died. After day 270, no animals became WHsAg-negative; the remaining 55 animals all died while still WHsAg-positive with 29 animals surviving for 1 year or more. Based on these data, woodchucks that are trapped when WHsAg-positive and remain carriers for as little as 4 months have >95% probability of remaining carriers. The chronically infected animals used in our P. amarus trials (with some exceptions) were observed to be carriers for 4 months or more. In a series of studies of long-term carrier woodchucks, it was found that the P. amarus extracts generally

eliminated or decreased WHV levels; this was not the case in controls (Figure 3). Similar results were obtained in additional studies using P. tenellus, a species of Phyllanthus collected in the US (Figure 4). In one experiment, animals that had been infected for ~ 1 month were treated. Three of four treated animals lost WHsAg, which did not return; they also lost DNAp, while the controls did not. In another experiment, the animals were initially treated s.c. and then i.p. 4. The results of all woodchuck in vivo studies, both acute and chronic, are summarized in Table 3. In each of the studies, more treated than control animals responded and the overall response was significantly different (p < 0.001).

Implications for development and prevention of PHC The previous studies on chronically infected woodchucks were combined and analysed retrospectively using the presence of PHC at the time of death as the outcome variable. The results were summarized in a proportional hazards analysis (Figure 5) showing the probability of dying without evidence of PHC (by examination at autopsy) versus the number of days observed. Three groups were considered including all animals with WHsAg at the time of capture, surviving for at least 100 days and for which liver pathology was available. The first group contained the untreated and nonexperimental cohort and originally included 90 animals. As animals were entered into the experiments they were removed from this group and entered into the second or third groups. The second group included untreated animals selected as controls in the chronic experiments summarized in Table 3. The third group included animals treated with P. amarus. The final outcome for the 90 animals meeting with criteria was as follows: 58 animals died as unselected controls, 28 with tumours; 9 died as selected controls, 6 with tumours; and 23 died as selected and treated, 4 with tumours. Not all the animals included in the experiments described met the criteria for inclusion in this retrospective analysis. The PHC rate (~20%) in treated animals was lower than the other two groups (~ 85%) and was significantly different from the unselected controls (p=0.01). Although these results imply that P. amarus is effective as a 'preventive treatment' for cancer of the liver, they must be interpreted with circumspection. They were derived from a retrospective analysis of data taken from studies designed for quite different reasons; the results are subject to a range of biases that are difficult to quantify. Nevertheless, this analysis encouraged us to design prospective studies, now in progress, which could directly test the hypothesis that treatment of woodchuck carriers using P. amarus can significantly decrease the risk of PHC.

Clinical trials in humans As noted above, Phyllanthus has been used for centuries for the treatment of jaundice in India and elsewhere but, to our knowledge, there have been no reports of a scientifically designed randomized double-blind trial in hepatitis B carriers. Collaboration was initiated with our colleague, S.P. Thyagarajan, of the Postgraduate Institute of Basic Medical Sciences, University of Madras, India, in the design and analysis of such a trial for which the preliminary results recently became available 12.

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A total of 79 human carriers of HBV were included in the study. Of these, 40 were given 200mg of dried, powdered whole plant (P. amarus) three times daily. The remaining 39 carriers were given lactose placebo at the same dosage and frequency. Carriers were assigned randomly to the treatment or control groups and neither

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Vaccine, Vol. 8, S u p p l e m e n t 1990

the carriers nor their physicians were told of the assignment. The drug or placebo was administered for 30 days then stopped; the carriers were then tested monthly for up to 9 months after cessation of treatment or placebo. Thirty-seven of the treated carriers and 23 of the controls returned for follow-up.

Treatment of H B V carriers with P. a m a r u s : B. B / u m b e r g et al. Table 3 Summary of in vivo woodchuck studies No. of responders =

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Experiment

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being treated for longer periods to determine whether active replication of virus requires longer duration of therapy. There was no evidence of toxicity in the treated individuals, but these observations were based primarily on clinical examination. Deficiencies of this study are described in the published report t2. Additional control trials in Europe, Asia and elsewhere are currently being designed to validate (or reject) the observations in South India.

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Table 4 Preliminary human clinical trial: loss of HBsAg in treated and

A large number of our current studies are designed to identify active principles, which mediate what appears to be an effective action of P . amarus in animal and human carriers. The principles which inhibit the DNAp of HBV are currently under investigation, but it is recognized that other factors may also be of importance. The goal is to identify these active principles, characterize them chemically and eventually provide a medication that can be used in the treatment of many of the 300 million carriers in the world. It can be inferred that this would greatly decrease their risk for chronic liver disease and/or PHC; follow-up studies will be required to test this inference. The availability of such a treatment would contribute greatly to the control of HBV in conjunction with the vaccination programmes now in progress.

placebo group at first follow-up visit after a one-month treatment with P. amarus

Acknowledgements

Loss of HBsAg Treatment

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This work was supported by USPHS Grants CA-40737, RR-05895 and CA-06927 from the National Institutes of Health and by an appropriation from the Commonwealth of Pennsylvania. For the initial studies, P. amarus was collected by P.S. Srinivasan.

References The results of the trial are summarized in Table 4. Of the treated subjects 59%, but only 4% of 23 control subjects, became HBsAg-negative after 30 days and remained so until the end of the follow-up period. Thirteen out of 14 carriers with HBsAg but without HBeAg became negative, while only five out of 17 HBsAg-positive, HBeAg-positive subjects lost the surface antigen. HBsAg did not reappear in any of these subjects. The individuals who remained HBsAg-positive are now

1 London, W.T. and Blumberg, B.S. A cellular model of the role of hepatitis B virus in pathogenesis of primary hepatocellular carcinoma. Hepatology 1982, 2, 10S 2 Thyagarajan, S.P., Thiruneelakantan, K., Subramanian, S. and Sundaravelu, T. In vitro inactivation of HBsAg by Eclipta alba Hessk and Phyllanthus niruri Linn. Ind. J. Med. Res. 1982, 76, 124 3 Venkateswaran, P.S., Millman, I. and Blumberg, B.S. Interaction of fucoidan from Pelvetia fastigiata with surface antigens of hepatitis B and woodchuck hepatitis virus. Plant, Med. 1989, 55, 235 4 Venkateswaran, P.S., Millman, I. and Blumberg, B.S. Effects of an

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extract from Phyllanthus niruri on hepatitis B and woodchuck hepatitis viruses: In vitro and in vivo studies. Proc. Natl Acad. Sci. USA 1987, 84, 274 Summers, J., Smolec, J.M. and Snyder, R. A virus similar to human hepatitis B virus associated with hepatitis and hepatoma in woodchucks. Proc. Natl Acad. Sci. USA 1978, 75, 45,33 Summers, J., Smolec, J.M,, Werner, B.G., Kelly, T.J., Tyler, G.V. and Snyder, R.L. Hepatitis B virus and woodchuck hepatitis virus are members of a novel class of DNA viruses. In: Viruses in Naturally Occurring Turnouts (Eds Essex, M., Todaro, G. and zur Hausen, H.) Cold Spring Harbor Conferences on Cell Proliferation, Vol. 7, Cold Spring Harbor Laboratory, Cold Spring Harbor. NY, USA, pp. 45~ 47O Werner, B.G., Smolec, JM., Snyder, R. and Summers, J. Serological relationship of woodchuck hepatitis virus to human hepatitis B virus. J. Virol. 1979, 32, 314

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Millman, I., Halbherr, T. and Simmons, H. Immunological crossreactivities of woodchuck and hepatitis B viral antigens. Infect. Immun. 1982, 35, 752 9 Millman, I., Southam, L., Halbherr, T., Simmons, H. and Kang, CM. Woodchuck hepatitis virus: experimental infection and natural occurrence. Hepatology 1984, 4, 817 10 Galibert, F., Chert, T.N. and Mandart, E. Localization and nucleotide sequence of the gene coding for the woodchuck hepatitis virus surface antigen: comparison with the gene coding for the human hepatitis B virus surface antigen. Proc. Natl Acad. Sci. USA 1981, 78, 5315 ~1 Kaplan, E.L and Meier, P Nonparametric estimation from incomplete observations. J. Am. Stat. Assoc. 1958, 53, 457 12 Thyagarajan, S.P,, Subramanian, S., Thirunalasundari, T. et al. Effect of Phyllanthus amarus on chronic carriers of hepatitis B virus. Lancet 1988, li, 764