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Entecavir Treatment for HBV in Woodchucks
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WHERE ARE THE ADVOCATES?
Is HBV a liver disease or a viral disease that should be treated as HIV is treated: suppress HBV early to undetectable. The HBV treatment guidelines are fluid & changing, while new HBV drugs are quickly becoming available and adding to the arsenal of older approved drugs. Drugs approved for HBV therapy are interferon, Pegasys, lamivudine, adefovir, & entecavir; tenofovir is not yet FDA approved but used by doctors & particularly HIV treaters anyway because of its potency against both HIV & HBV & its combination with FTC in a Fixed Dose Regimen (FDC). Often hepatologists take a wait & see approach to treating HBV. Recent studies however report HBV DNA, viral load is associated with development of liver cancer (HCC). So, is it ok to have high viral loads or any viral loads for 10 years before starting HBV therapy? There appears to be a strong difference of opinion about this between virologists and treaters, hepatologists. As in HIV & HCV, the patients are the ones who have to live with these viruses. The patients risk development of liver cancer or cirrhosis, or in the case of HIV full blown AIDS. A more focused discussion needs to be had between the interested parties to address this issue: patients, doctors, researchers, virologists. A Commentary recently published in GUT Nov 5, 2005 by M Sherman says:
"Predicting survival in hepatitis B" Should Targeted HBV DNA Suppression Be the Goal of Therapy?
"..... prolonged and maximal suppression of HBV DNA to levels below the detection limit of PCR based assays may be necessary to reduce the risk of complications......
...........In conclusion, prolonged low level viraemia causing insidious and continual liver damage, as reflected by relatively mild elevations in ALT levels, is the most likely pathway leading to the development of complications for Asian patients with CHB. Long term antiviral therapy aiming at maximal suppression of HBV even after HBeAg seroconversion may be required for Asian patients."
http://www.natap.org/2005/HBV/102105_03.htm
"Long-Term Entecavir Treatment Results in Sustained Antiviral Efficacy and Prolonged Life Span in the Woodchuck Model of Chronic Hepatitis Infection"
The Journal of Infectious Diseases 2001;184:1236-1245
It is believed that effective suppression or elimination of cccDNA may be required for sustained virus suppression and clearance of virus É.Viral DNA and cccDNA levels in the liver were all significantly reduced during ETV treatment, and all treated animals demonstrated a 4-log10 reduction in intrahepatic cccDNA levels relative to levels in controls. Furthermore, intrahepatic expression of viral core and surface proteins during therapy mirrored viral DNA levelsÉ.. ÉÉOf particular importance was the finding that the long-term survival rates in the absence of HCC of the 11 younger animals used in the 14- and 36-month treatment groups were 50% and 80%, respectively, compared with only 4% for historic controlsÉ.Woodchucks chronically infected with WHV closely mimic the human carrier state in regard to viral pathogenesis and represent a particularly challenging model, since nearly all the infected animals will eventually die of HCC within 34 years after infectionÉ.
Authors; Richard J. Colonno,1 Eugene V. Genovesi,1 Ivette Medina,1 Lucinda Lamb,1 Stephen K. Durham,1 Meei-Li Huang,2 Lawrence Corey,2 Margaret Littlejohn,4 Steven Locarnini,4 Bud C. Tennant,3 Burt Rose,1 and Junius M. Clark1
1Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, Connecticut; 2Fred Hutchinson Cancer Research Center, Seattle, Washington; 3Cornell University, Ithaca, New York; 4Victorian Infectious Disease Reference Laboratory, North Melbourne, Australia
Human hepatitis B virus (HBV) infection is widespread and is a major cause of human liver disease [1]. Although primary HBV infections in adults are usually self-limiting, 6%-10% of infected individuals become chronically infected carriers and are at significant risk for the development of liver cirrhosis and hepatocellular carcinoma (HCC). Worldwide, there are nearly 400 million chronic carriers.
The Eastern woodchuck, Marmota monax, chronically infected with the hepadnavirus woodchuck hepatitis virus (WHV), is an established animal model for studies of the pathogenesis of and therapy for HBV infection. In most immunologically immature neonatal woodchucks, WHV infection causes a life-long WHV-carrier state with chronic viremia. A feature of the WHV-carrier state that is common to the HBV-carrier state of humans is the progressive development of primary HCC. It is a particularly challenging animal model in which to observe a prolonged antiviral response, because 96% of these WHV chronically infected carrier woodchucks usually die by the age of 4 years, and all eventually die as a result of HCC.
The objectives of the current study were to evaluate the long-term antiviral efficacy and tolerance of ETV in WHV chronically infected woodchucks over a prolonged treatment period, to determine whether suppression of viral replication could be maintained using a once-a-week dosing regimen. Such a sustained treatment period would allow us to monitor for the emergence of resistant variants over time, to explore the relationship between viral replication and development of HCC, and to study the impact of a highly effective antiviral on the life span of chronically infected animals.
Discussion
Woodchucks chronically infected with WHV are a reliable and predictive model for evaluating the effectiveness and toxicity of antiviral agents. These animals closely mimic the human carrier state in regard to viral pathogenesis and represent a particularly challenging model, since nearly all the infected animals will eventually die of HCC within 34 years after infection. Because progression to HCC is accelerated in these animals, they provide an excellent opportunity to explore the relationship between WHV replication and the development of HCC. ETV is an ideal antiviral for this purpose, because it exhibits excellent potency and pharmacokinetics enabling weekly dosing and is very well tolerated. There were several objectives for the current study, including evaluation of the long-term antiviral efficacy and safety, resistance development, effect on the development of HCC, and overall survival resulting from prolonged therapy with ETV.
As reported elsewhere, oral dosing of ETV at concentrations as little as 20 ug/kg daily can reduce serum viral DNA levels by several logs. Because of the favorable pharmacokinetic properties of ETV and the relatively long intracellular half-life of ETV triphosphate, a strategy of using a weekly dosing regimen was undertaken to enable a long-term study to be more easily conducted. Although not an optimal dosing regimen, a weekly regimen had been efficacious in earlier experiments. All the animals in the current study displayed multilog decreases in serum viral DNA levels that were sustained during the initial 12 months of weekly treatment with ETV, confirming the effectiveness of a weekly treatment regimen. Viral DNA and cccDNA levels in the liver were all significantly reduced during ETV treatment, and all treated animals demonstrated a 4-log10 reduction in intrahepatic cccDNA levels relative to levels in controls. Furthermore, intrahepatic expression of viral core and surface proteins during therapy mirrored viral DNA levels. Three of the animals who were withdrawn from treatment after 14 months showed a sustained response during the 2-year posttreatment period, and all 5 of the younger animals in the 36-month treatment group had nearly undetectable viral DNA for >2 years.
It is believed that effective suppression or elimination of cccDNA may be required for sustained virus suppression and clearance of virus, since this key replicative intermediate serves as template for the transcription of all viral RNAs. Normally present at 1050 copies per infected hepatocyte, cccDNA serves to sustain chronic HBV infection. It is widely viewed as being refractory to nucleoside therapy and, hence, could pose a major barrier to the eradication of HBV infection. (NOTE from Jules Levin: since this publication an adefovir study did show reduced cccDNA by adefovir in patients. ETV treatment reduced cccDNA levels in both DHBV-infected ducks and WHV-infected woodchucks. Examination of liver biopsy samples from ETV-treated animals showed a significant decrease and long-term suppression of this key target species. This suppression likely contributes to the large decreases in viral DNA levels and the sustained response observed in several animals when treatment was withdrawn.
As recently reviewed by BrŽchot et al., it has been well established that HBV plays a role in liver cell carcinogenesis. HBV DNA sequences are integrated into cellular DNA in about 90% of liver tumor samples from hepatitis B surface antigenpositive patients, although integration is not always necessary. Although there does not appear to be a preferential integration site, integration of viral sequences can cause chromosomal DNA instability and insertional mutagenesis, with insertional activation of oncogenes frequently observed in woodchucks. In addition, these integrations usually maintain a functional X open-reading frame, a known transactivator of a wide variety of cellular genes.
A number of animals in this study (4/11) died of HCC, despite the apparent effectiveness of ETV treatment in significantly reducing viral DNA and surface antigen levels. These results appear to support earlier models that propose that random integration events during viral replication will undoubtedly lead to a critical integration event that results in cellular transformation and progression down a pathway to HCC development. Such a model appears to be supported by the relationships observed between HCC development, viral DNA levels, and age of the animal when treatment was initiated. Both of the older woodchucks (infected for 20 months before treatment) died of HCC at about age 3 years, despite having suppressed viral DNA levels. Woodchuck 96-101 developed HCC, despite having undetectable viral DNA levels, as determined by PCR, during several months of treatment. These results suggest an uncoupling of viral replication and progression to HCC and support the concept that integration is a random consequence of viral DNA replication and that an early integration event can result in progression to HCC, regardless of subsequent intervention of viral replication.
Selection of lamivudine-resistant strains, which may lead to a failure of antiviral therapy in individuals with chronic HBV infections, occurs in about 16%-43% of patients within 1 year, appearing as early as 6 months and increasing with duration of treatment. Lamivudine-resistant variants also were observed in the woodchuck after 6 months of treatment, with genotypic resistance appearing months before phenotypic resistance was observed. The apparent absence of any resistance development during the prolonged ETV treatment period is yet another measure of the potent inhibition achieved. Since resistant viruses usually arise through a selection process of advantaged variants containing randomly generated mutations, a minimum threshold level of viral DNA polymerase activity must remain if a sufficient number of mutations are to be generated for selection. ETV, which inhibits all 3 viral polymerase functions (priming, RNA-dependent DNA synthesis, and DNA-dependent DNA synthesis), appears to suppress replication rates below this threshold level. Indeed, viral DNA levels in many of the treated animals were below the PCR cutoff of 200 copies/mL. Supporting this conclusion was the absence of any nucleotide changes from the baseline sequence when viral DNA was isolated and the WHV polymerase genes were sequenced.
The finding that several of the animals had sustained responses and survived to the age of 4 years likely is a reflection of the potent inhibitory activity of ETV. The finding of 1 animal who showed a rebound in viral DNA levels, despite 3 years of significant suppression by ETV treatment, was surprising but not unexpected. This animal displayed viral DNA just above PCR-detectable levels for most of the treatment period, suggesting that low-level viral replication was continuing during treatment. In hindsight, we might have achieved even better antiviral efficacy had we used a higher concentration of ETV or a daily dosing regimen. Nonetheless, it is interesting to consider how the virus managed to persist at low levels during a 36-month treatment period. Earlier studies by Michalak demonstrated a life-long occult persistence of pathogenic virus in woodchucks who had appeared to clear acute WHV infection.
Of particular importance was the finding that the long-term survival rates in the absence of HCC of the 11 younger animals used in the 14- and 36-month treatment groups were 50% and 80%, respectively, compared with only 4% for historic controls. Each of the ETV-treated groups was compared with the uninfected and infected/untreated groups by use of Fischer's exact test. Because repeated testing increases the probability of false results, the 2 P values were corrected by use of Bonferroni's method. The results were statistically significant (P = .0132 for the 14-month treatment group, P = .0004 for the 36-month treatment group, and P = .00002 for the combined groups). Since there was no histopathology indicative of HCC in any of the surviving animals at the end of the study, it is highly likely that these particular animals would have lived much longer. Therefore, long-term ETV therapy in WHV chronically infected woodchucks appeared to extend the life span of the treated animals and delay the onset of HCC.
The results from the current study show that ETV is a very potent inhibitor of WHV, as evidenced by the multilog decrease in viral DNA levels, cccDNA, and viral surface and core antigen expression. ETV was well tolerated, and there was no evidence of viral resistance, despite a treatment period of up to 3 years in some animals. ETV treatment appeared to prolong life in several animals and delay the onset of HCC. These results support the continued human clinical evaluation of ETV in the treatment of chronic HBV infections.
ABSTRACT
Entecavir (ETV) is a guanosine nucleoside analogue with potent antiviral efficacy in woodchucks chronically infected with woodchuck hepatitis virus. To explore the consequences of prolonged virus suppression, woodchucks received ETV orally for 8 weeks and then weekly for 12 months. Of the 6 animals withdrawn from therapy and monitored for an additional 28 months, 3 had a sustained antiviral response and had no evidence of hepatocellular carcinoma (HCC). Of the 6 animals that continued on a weekly ETV regimen for an additional 22 months, 4 exhibited serum viral DNA levels near the lower limit of detection for >2 years and had no evidence of HCC. Viral antigens and covalently closed circular DNA levels in liver samples were significantly reduced in all animals. ETV was well tolerated, and there was no evidence of resistant variants. On the basis of historical data, long-term ETV treatment appeared to significantly prolong the life of treated animals and delay the emergence of HCC.
RESULTS
Nineteen chronically infected woodchucks were dosed daily with ETV (0.5 mg/kg orally) for 8 weeks during the induction phase of the study. In all but 2 of these animals, treatment was initiated at age 8 months; the remaining 2 animals were a year older, and treatment for them was initiated at age 20 months. Serum WHV DNA levels were initially monitored by dot-blot analysis (limit of detection, 30 pg/mL), which showed serum viral DNA levels in all 19 animals reduced to undetectable levels (averaging >4 log10 reduction) by the fifth week of daily therapy. The treatment period required to achieve undetectable levels did not appear to correlate with initial baseline serum viral DNA levels. After completion of the 8-week daily dosing period, 6 of the 19 animals were withdrawn from therapy and were monitored for an additional 28 weeks. All 6 animals exhibited a rebound in their serum WHV DNA levels within 1-8 weeks after completing the daily dosing phase of the study. Again, there was no obvious correlation between initial baseline DNA levels, time needed to achieve undetectable levels by dot-blot analysis, and the time until a rebound was observed. This group of animals served as controls, illustrating that the initial decreases in WHV DNA levels had been directly attributable to ETV treatment.
The 13 remaining animals, including the 2 older animals, were included in the maintenance phase of the study, in which daily oral treatment was replaced by a regimen of 0.5 mg/kg of ETV administered orally once a week for an additional 12 months. In addition to the dot-blot method, DNA levels were subsequently quantitated by PCR (limit of detection, 200 copies/mL). As shown in figures 2 and 3, viral DNA levels decreased in all animals with continued ETV treatment. One animal (no. 96-043) died of HCC before completion of the 12-month treatment (during study week 39), despite experiencing a 4-log10 drop in viral DNA levels during the 10 months of ETV treatment. The remaining 12 animals showed 5-8 log10 reductions in their viral DNA levels during the weekly dosing period. Two woodchucks (nos. 95-012 and 96-043) showed transient increases in viral DNA levels shortly after the switch to weekly treatment, although serum WHV DNA levels continued to decrease with continued weekly therapy. One of these animals (no. 96-043) was the one that died at week 39 after developing HCC.
Viral surface antigen levels, which were also monitored during treatment, showed a concomitant reduction in parallel with viral DNA reductions in all animals for whom this was measured. Despite treatment, there was no measurable anti-WHsAg detected, and the levels of anti-WHcAg remained unchanged. After completion of the maintenance phase (total of 14 months of therapy), liver biopsy specimens were obtained from 10 selected animals and were examined for WHV core antigen. Compared with findings in biopsy samples from untreated animals, viral core antigen was undetectable by immunostaining in the hepatocytes from 9 of the 10 animals and was detected at only low levels in the tenth animal (no. 95-012).
After completion of the maintenance-dosing phase, the remaining 12 woodchucks were divided into 2 groups of 6 each. One group was withdrawn from therapy (designated "14-month treatment group"); the second group continued to receive a weekly maintenance dose of 0.5 mg/kg for an additional 22 months, for a total of 3 years of therapy (designated "36-month treatment group") before being withdrawn from ETV treatment. This group of animals was monitored for an additional 5 months. Each group contained 1 of the 2 older animals (nos. 95-012 and 95-323), both of which died of HCC within 1 month of beginning this phase of the study, despite displaying substantial decreases in serum viral DNA levels. As a result, both groups continued with only 5 woodchucks per group.
In 2 of the 5 animals (nos. 96-044 and 96-061) in the 14-month treatment group, serum WHV DNA levels returned to pretreatment levels within 17 weeks after cessation of therapy. Woodchucks 96-044 and 96-061 both died of HCC at ages 27 and 44 months, respectively. Animal 96-161, who had maintained undetectable serum WHV DNA levels by PCR in the absence of ETV, had to be euthanized at week 163 because of an arterial-venous anastomosis that likely resulted from the numerous blood samples obtained during the study. This animal achieved a nearly 7-log10 viral DNA reduction to undetectable levels (by PCR) within 6 months and maintained that level, despite the discontinuation of ETV treatment. Liver biopsy results for this woodchuck showed no histopathologic evidence of HCC. Two other animals (nos. 96-025 and 96-152) survived to the end of the study with sustained reductions in their viral DNA and surface antigen levels for >2 years after withdrawal of therapy and with no evidence of HCC at necropsy. Both animals had exhibited a transient increase in serum viral DNA and surface antigen levels when treatment was stopped, but the levels again became undetectable without additional therapy.
For >2 years, all animals in the 36-month treatment group had serum viral DNA levels near or below the limit of PCR detection. One (no. 96-101) of the 5 animals died at week 149 of therapy (43 months of age) and had evidence of HCC at necropsy, despite having undetectable serum WHV DNA levels by PCR. The remaining 4 animals survived until the end of the study--5 months after being withdrawn from therapy. Three of the 4 animals had undetectable serum WHV DNA levels by PCR, and 1 (no. 96-162) had a return to pretreatment serum WHV DNA levels 12 weeks after withdrawal of therapy. WHV surface antigen levels paralleled serum viral DNA levels in each of these animals except woodchuck 96-101, in which there was an unexplained increase during the last 18 months of treatment. None of the 4 surviving animals had gross evidence of HCC at necropsy.
At study end, the 6 surviving animals in both treatment groups were about 4 years old. Liver biopsy material from these 6 animals (nos. 96-025, 96-152, 96-012, 96-023, 96-131, and 96-162) was again stained for the presence of viral core antigen: 5 remained negative for viral core antigen, whereas the 1 animal (no. 96-162) that exhibited a rebound in viral DNA levels was also positive for viral core antigen in hepatocytes (data not shown). The livers from these 6 animals were also evaluated for the presence of viral surface antigen: results for animals 96-012, 96-131, and 96-152 were negative. Livers from animals 96-023 and 96-025 were also negative for membrane-associated antigens, but cytoplasmic staining was marginally positive (<1 positive cell/1000). Membrane-associated antigens and cytoplasmic staining of the liver sample from woodchuck 96-162 were positive for WHsAg.
Because cccDNA is thought to play a key role in sustaining chronic HBV infections, the presence of viral DNA and cccDNA in liver samples also was determined by use of a newly developed PCR methodology, as described in Materials and Methods. After completion of the maintenance phase, all 9 woodchuck liver samples examined had achieved at least a 4-log10 reduction in cccDNA levels, with 8 below the level of detection. Similar reductions were also observed for WHV DNA levels in analyzed livers. Liver samples obtained at the conclusion of the study were very similar, with the only increase in cccDNA levels observed in the woodchuck (no. 96-162) who exhibited a rebound in serum WHV DNA to pretreatment levels.
Despite such a long treatment period, there was no evidence of the emergence of resistant WHV variants. To confirm this observation, the sequences of the viral polymerase gene from several of the treated woodchucks were examined at the end of the treatment period. The WHV polymerase domain extends from aa 418-731, with the B domain extending from aa 544568 and the C domain (YMDD) extending from aa 585-591. Mutations associated with resistance to 3TC occur within or near the conserved B and C domains of the polymerase. PCR amplification of the viral polymerase gene was attempted for 10 woodchucks at time points near the end of their ETV treatment: number 96-044 at week 74; 96-061, 96-025, and 96-161 at week 59; 96-152 at week 66; 96-101, 96-023, and 96-131 at week 144; and 96-012 and 96-162 at week 172. Because of the unexpected rebound in viral DNA levels after 3 years of treatment, the WHV polymerase gene of woodchuck 96-162 also was sequenced at week 178. Compared with the sequence of the parent virus, no nucleotide changes in the polymerase gene were identified in any of the 7 animals where a PCR product could be recovered, suggesting that replication may have been reduced to levels too low to generate resistant variants (data not shown).
ETV treatment was well tolerated in this study: no compound related effects were noted during a 3-year treatment period for some animals. Compared with results in a historic woodchuck study, the clinical chemistry and hematology profile results showed no variations from normal values in treated animals. The expected increases in ¡-glutamyl transpeptidase were observed in animals that developed HCC.
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