Adefovir dipivoxil added to ongoing lamivudine therapy in patients with lamivudine-resistant hepatitis B e antigen-negative chronic hepatitis B

Authors


Dr K. Tziomalos, 63 Solonos Street, Thessaloniki, 54248, Greece.
E-mail: ktziomalos@yahoo.com

Summary

Background : Long-term treatment with lamivudine is required to control viral replication in patients with hepatitis B e antigen-negative chronic hepatitis B, but is associated with a high rate of viral resistance. The role of adefovir dipivoxil in these patients has not been definitively evaluated.

Aim : To address the role of adefovir in the management of patients with lamivudine-resistant hepatitis B e antigen-negative chronic hepatitis B.

Methods : Patients were assigned to receive adefovir 10 mg once daily plus ongoing lamivudine 100 mg once daily for 52 weeks. The primary end point was reduction in serum hepatitis B virus DNA level (hepatitis B virus DNA response). Secondary end points included the proportion of patients with undetectable hepatitis B virus DNA at week 52 (complete virological response) and the percentage of patients with normalization of alanine transferase level at week 52 (biochemical response).

Results : A total of 49 consecutive patients were enrolled in this study. After 52 weeks of treatment, all patients had an hepatitis B virus DNA response and 57.1% had complete virological response. Biochemical response occurred in 75.6% of patients.

Conclusions : Administration of adefovir in patients with lamivudine-resistant chronic hepatitis B results in significant suppression of viral replication. Nevertheless, continuous therapy will probably be needed in order to maintain remission in these patients.

Introduction

Hepatitis B virus (HBV) infection is a global public health problem. It is estimated that there are at least 400 million HBV carriers in the world and that up to 1 million die annually because of HBV associated liver disease.1 Precore mutant [hepatitis B e antigen (HBeAg)-negative] chronic hepatitis B (CHB) represents a potentially severe and progressive form of chronic liver disease with very rare spontaneous remissions, frequent progression to cirrhosis and increased risk of the development of hepatocellular carcinoma (HCC).2 It accounts for 7–30% of patients with chronic HBV infection worldwide and affects 10–15% of patients with CHB in the United States and Northern Europe and as many as 50–80% of those in Southern Europe and the Middle East and 40–55% of those in Asia.3 Therefore, precore mutant infection makes a substantial contribution to HBV infection across the world and its treatment is an important component in the control of hepatitis infection and associated disease.

The development of orally active anti-viral nucleoside analogues has revolutionized the treatment of CHB. Lamivudine, the first of these agents to enter widespread clinical use, effectively suppresses viral replication; the endpoint of lack of detectable HBV DNA and normal serum alanine transferase (ALT) levels is reached in 65–79% at the end of 52 weeks of treatment with lamivudine.4 Unfortunately, sustained off-therapy responses to lamivudine are rare; only 11% of treated patients have a sustained response 24 weeks after end of 12 months of therapy.5 Therefore, long-term continuous therapy rather than a limited course of treatment with lamivudine might be a more appropriate approach to therapy in HBeAg-negative CHB. Nevertheless, long-term treatment is associated with a high rate of viral resistance. Lamivudine resistance increases from 10–27% at 1 year to 40–56% at 2 years and 67% at 3 years.5–9 Although several questions were initially raised about the clinical significance of viral resistance to lamivudine, it has now become clearer that the emergence of resistance has a negative impact on the efficacy of therapy in HBeAg-negative CHB patients.8, 9

The wide use of lamivudine in chronic hepatitis B during the last 5–6 years has resulted in the constant increase of patients with lamivudine-resistant mutants. Their treatment may be the most pressing task for the current anti-HBV strategies in clinical practice.10 The efficacy of interferon (IFN)-α therapy has not yet been evaluated in any well-designed study in these patients, and therefore no conclusions can be drawn. Adefovir dipivoxil (ADV), an acyclic nucleotide analogue, is the only approved agent that has been shown to be effective in this setting.11, 12 Although the available data are encouraging, the specific degree of inhibition of viral replication as well as the level of biochemical improvement after addition of ADV in patients with lamivudine-resistant HBeAg-negative CHB has not been definitively evaluated thus far.

This study was designed to address the role of ADV in the management of patients with HBeAg-negative CHB with lamivudine-resistant HBV.

Patients and methods

Patients eligible for the study were aged 18 years and older, HBsAg positive, HBeAg negative and anti-HBe positive, and were receiving ongoing lamivudine therapy for chronic hepatitis B for at least 12 months at the time of screening. All patients had developed resistance to lamivudine. Most patients had confirmed HBV polymerase gene mutation within the YMDD motif by DNA sequencing (Trugene HBV genotyping; Visible Genetics Inc., Toronto, Canada). In the remaining patients, because of cost restrictions, breakthrough infection was defined as reappearance of serum HBV DNA in two consecutive occasions, at least 3 months apart, after its initial disappearance. The exclusion criteria were as follows: absolute neutrophil count≤1000 cells/mL; haemoglobin ≤10 or ≤9 g/dL (males or females, respectively); platelet count <50 000/μL; a screening calculated creatinine clearance <50 mL/min or a serum creatinine value >1.5 mg/dL; a serum phosphorus level ≤2.4 mg/dL (normal range 2.5–4.5 mg/dL); prior treatment with ADV or other drugs with activity against HBV within the 3 months preceding study screening; serious concurrent medical conditions, including other concurrent liver diseases; documented or suspected HCC; coinfection with hepatitis C virus or hepatitis delta virus or human immunodeficiency virus; current alcohol or substance use and pregnancy or lactation.

Patients were assigned to receive combination therapy with ADV 10 mg once daily plus ongoing lamivudine 100 mg once daily for 52 weeks. After week 52, lamivudine administration was discontinued and all patients went on to receive indefinite ADV monotherapy.

Patients were evaluated every 2 months. At each visit, any untoward medical occurrences, regardless of causality, were recorded as adverse events. Haematology, biochemistry, and vital signs were also assessed. At 4-month intervals blood was also tested for HBsAg, anti-HBs and for HBV DNA levels.

Routine biochemical and haematologic tests were performed using automated techniques. HBsAg and antibodies against HBsAg were measured using routine commercially available enzyme immunoassays. HBV DNA levels were measured with the use of a commercially available quantitative PCR assay (Amplicor HBV-DNA Monitor Test; Roche Diagnostics, Branchburg, NJ, USA) with a sensitivity of 400 copies/mL.

The primary end point of the study was reduction in serum HBV DNA level (HBV DNA response). This was assessed as the proportion of patients with either HBV DNA level ≤105 copies/mL or a ≥2 log10 reduction from baseline HBV DNA level at weeks 52 in patients with ≥105 copies/mL at baseline. Two criteria were used to establish an HBV DNA response to capture HBV DNA reductions that would likely result in clinical benefit in patients with very high levels of serum HBV DNA (≥109 copies/mL) on entry as well as establish a lower threshold for those with much lower HBV DNA levels on entry.4, 13 Secondary end points included the proportion of patients with undetectable HBV DNA at week 52 (complete virological response) and the percentage of patients with normalization of ALT level at week 52 (biochemical response).

All data were analysed using the statistical package SPSS (version 10.0; SPSS Inc., Chicago, IL, USA). The population analysed included all patients who received at least one dose of study medication. The Mann–Whitney test was used for comparisons of quantitative variables between groups. In all cases, a two-tailed P-value <0.05 was considered statistically significant.

The study was approved by the ethics committee of our institution, and all patients provided written informed consent before screening. The study was performed in accordance with the principles of the Declaration of Helsinki.

Results

Between February 2003 and August 2003, a total of 49 consecutive patients [45 males (91.8%)] were enrolled in this prospective study; their median age was 50 years (range, 25–72 years). Table 1 summarizes baseline characteristics of the study population. Patients had received prior lamivudine therapy for a median of 45.5 months (range, 21–84 months). At baseline, 26.5% of patients (13 of 49) had HBV DNA level ≤ 105 copies/mL.

Table 1.  Baseline characteristics of the patients
  1. HBV, hepatitis B virus; ALT, alanine transferase; ULN, .

Median age, years (range)49.8 (25–72)
Male sex (%)45 (91.8%)
Median duration of prior lamivudine therapy, months (range)45.5 (21–84)
Median HBV DNA level, log10 copies/mL (range)6.7 (2.7–7.6)
Median ALT level times the ULN (range)2.7 (0.9–28.1)

Table 2 summarizes the virologic response in the patients. ADV 10 mg once daily resulted in significant reductions in serum HBV DNA that was durable through 52 weeks (Figure 1). After 52 weeks of treatment, all patients had an HBV DNA response, which occurred after a median time of 7 months (range, 3–11 months). The median change from baseline was −6.5 log10 copies/mL (range, −7.6 to −2.2 copies/mL). 57.1% of patients (28 of 49) had complete virological response at week 52, which occurred after a median time of 5 months (range, 3–11 months). Patients who showed complete virological response had significantly lower baseline HBV DNA levels [median 6.2 log10 copies/mL (range, 2.9–7.6 log10 copies/mL) vs. median 7.0 log10 copies/mL (range, 4.9–7.6 log10 copies/mL), P < 0.005] compared with patients without such a response.

Table 2.  Virologic and alanine transferase (ALT) responses at week 52
  1. HBV, hepatitis B virus; HBsAg, hepatitis B surface antigen; ULN, .

  2. * Lower limit of detection of polymerase chain reaction assay is 400 copies/mL.

No. with HBV DNA response (%)49/49 (100)
No. HBV DNA negative by polymerase chain reaction (%)*28/49 (57)
Median change from baseline in HBV DNA level (range)−6.5 (−7.6 to −2.2)
HBsAg loss (%)0/49 (0)
No. with an ALT response (%)34/45 (75)
Median change from baseline in ALT times the ULN (range)−1.6 (−27.4 to 0.4)
Figure 1.

Median values for hepatitis B virus (HBV) DNA over study period.

None of the patients cleared HBsAg neither developed anti-HBs. Table 2 summarizes biochemical responses. At baseline, 45 of 49 (91.8%) patients had ALT levels greater than the upper limit of normal (ULN) (33 IU/L). Biochemical response occurred in 75.6% of patients (34 of 45) after a median time of 5 months (range, 1–9 months). The median ALT level at baseline was 2.7 times the ULN (91 IU/L); by week 52, this had declined to 0.9 times the ULN (31 IU/L) (median change −1.6 times the ULN, range −27.4 to 0.4) (Figure 2). 83.3% of the patients with a complete virological response also showed biochemical response after a median time of 5 months (range, 1–9 months).

Figure 2.

Median values for alanine transferase, expressed as multiples of ULN, over study period.

Patients were followed-up for a median of 4 months (range, 1–8 months) beyond the 52 weeks of study period. During follow-up, HBV DNA and ALT levels further decreased compared to study completion levels. At the end of follow-up, HBV DNA and complete virological response rates were identical with the ones at the end of study period. Interestingly, during follow-up eight more patients showed normalization of ALT levels; thus, biochemical response rate at the end of follow-up rose up to 93.3% (42 of 45 patients). All patients with a complete virological response showed biochemical response at the end of follow-up.

No patients discontinued study treatment due to an adverse event. There were no serious adverse events. There was no instance in which an elevation of serum creatinine level ≥0.5 mg/dL above baseline or decrease of serum phosphorus level <1.5 mg/dL was observed, as confirmed by two consecutive laboratory evaluations.

Discussion

No lamivudine course of finite duration has been shown to achieve sustained off-therapy responses in a sizeable proportion of patients.5 Thus, lamivudine should be used as long-term therapy aimed at inducing a long-term remission or cure. Nevertheless, its virus-suppressive activity is limited in time by the progressive emergence of drug-resistant mutants. Lamivudine resistance is because of mutations within the conserved YMDD motif in the major catalytic region C of the HBV polymerase gene.14 The lamivudine-resistant amino acids are found at position rt180 (rtL180M in conserved region B) and, in the rtYMDD motif, at position rt204 (rtM204V/I in conserved region C).15 Viral resistance is clinically expressed by the virological breakthrough phenomenon, defined as the reappearance of serum HBV DNA after an initial clearance of viraemia despite the continuation of therapy.10 The emergence of resistance has a negative impact on the efficacy of therapy in CHB patients as virological breakthroughs are almost invariably followed by increasing viraemia levels, culminating in biochemical breakthroughs, which ultimately have an adverse effect on liver histology.8

It is clear that the possible adverse effects of YMDD mutants do cast a concern. Rescue therapies for patients with worsening liver disease caused by lamivudine-resistant mutants are being evaluated. Until recently, treatment options for these patients have been limited to continuation or cessation of lamivudine therapy. Continuation of lamivudine aims to further suppress or to prevent the return of wild-type HBV which is more replicative competent than the YMDD mutant.16 However, this strategy seems ineffective. Lamivudine withdrawal results in re-emergence of wild-type HBV within 3–4 months.17 Therefore, acute exacerbations of liver disease might ensue and could, although uncommon, result in hepatic decompensation or acute liver failure.18 Finally, there is a paucity of data regarding the role of IFN in the treatment of lamivudine-resistant HBV. According to our experience, administration of pegylated IFN-α-2b (100 μg s.c. once weekly) for 12 months in 20 patients with lamivudine-resistant CHB resulted in complete virological end-treatment response in 35% of them and in partial virological end-treatment response in 45% of them; nevertheless, soon after cessation of treatment all but one patient presented virological recurrence (T. Vassiliadis, K. Patsiaoura, K. Tziomalos, T. Gkiourtzis, O. Giouleme, N. Grammatikos, D. Rizopoulou, N. Nikolaidis, E. Orfanou-Koumerkeridou, N. Eugenidis).

Adefovir dipivoxil, a diester prodrug, is rapidly converted after oral administration to the acyclic nucleotide analogue adefovir (9-[2-(phosphonomethoxy)ethyl]-adenine). Adefovir requires intracellular conversion by cellular kinases to its virologically active metabolite, adefovir diphosphate, which is an analogue of deoxyadenosine-5′-triphosphate but without a 3′-hydroxylic root and therefore results in competitive inhibition of DNA synthesis by DNA polymerases and reverse transcriptases.19 Adefovir has broad-spectrum anti-viral activity against hepadnaviruses, retroviruses and herpesviruses.20 Furthermore, it appears to enhance natural killer cell activity and immune responsiveness through the production of endogenous IFN-α.21 Finally, long-term ADV therapy significantly decreases cccDNA levels by a primarily non-cytolytic, cytokine-induced ‘curing’ of infected cells.22 In pivotal studies in HBeAg-negative patients with CHB and patients with lamivudine-resistant HBV, ADV has been shown to significantly reduce HBV DNA levels in most treated patients and to produce improvement in serum alanine aminotransferase levels and liver histology.11, 12, 23

In our study, the drug was highly effective in reducing serum HBV DNA levels with a median change of −6.5 log10 copies/mL at week 52. All patients had an HBV DNA response and in 57.1% of them HBV DNA was undetectable with PCR at the end of the study. Furthermore, the addition of ADV to lamivudine led to normalization of ALT levels in 75.6% of the patients. These results compare favourably with the ones from the two studies reported this far in patients treated with ADV for lamivudine-resistant HBeAg-positive CHB; in these studies, complete virological response rates were 20 and 31% respectively while biochemical response rates were 31 and 53% respectively.11, 12 It must be emphasized that we used broader inclusion criteria in our study, as 26.5% of our patients had baseline HBV DNA level ≤105 copies/mL and 9.2% had normal ALT levels. In contrast, in both aforementioned studies, patients were required to have a screening HBV DNA level ≥106 copies/mL and ALT levels >1.2 times the ULN. This difference could possibly account for the improved results we noticed; indeed, in our study, lower baseline HBV DNA levels predicted for the occurrence of complete virological response.

In contrast to the experience with lamivudine, the emergence of resistance to ADV appears to be delayed and infrequent. A low potential for resistance development with adefovir could be related to its close structural relationship with the natural substrate, which limits the potential for steric hindrance as a mechanism of resistance. In addition, adefovir contains a flexible acyclic linker that may allow adefovir to bind to HBV polymerase with different conformations, and thus, further subvert steric hindrance.24 Moreover, adefovir has a phosphonate bond rather than a phosphate bond as in other nucleoside analogues and might therefore be less susceptible to chain terminator removal.25, 26 Finally, because adefovir already contains an α-phosphorous atom, it only requires two phosphorylation steps to become activated, in contrast to the three phosphorylation steps required by nucleosides such as lamivudine. This structural feature of adefovir may lead to more efficient anabolism to the active triphosphate form and consequently improved activity in a broader range of cells such as bile duct epithelial cells and thus reduce the HBV replication reservoirs from which resistant virus may emerge.27 During the 48-week course of ADV, no patients developed evidence of viral resistance to therapy.23, 28 Adefovir-resistant mutants have been identified only in 1.6% of HBeAg-positive patients at 96 weeks’ follow-up.29 Unfortunately, because of financial reasons, we did not evaluate our patients for the development of ADV-resistant mutants; nevertheless, we must point out that HBV DNA levels decreased steadily during treatment and follow-up in all patients and no case of reappearance of serum HBV DNA after an initial clearance of viraemia was observed.

The most notable adverse effect associated with high-dose ADV in clinical studies was nephrotoxicity, which was typically the primary therapy-limiting drug effect. ADV-related nephrotoxicity occurs at doses ≥30 mg daily as a proximal renal tubular dysfunction, and is primarily manifested by the onset of gradual increases in serum creatinine and decreases in serum phosphorus after 20 weeks of therapy.30 These changes are usually mild to moderate in severity and can be accompanied by changes in serum potassium, bicarbonate, uric acid, glycosuria and proteinuria. The incidence of these changes appears to be dose related. Recent studies using the currently recommended dosage of ADV for HBV infection (10 mg/day) have reported no increases in serum creatinine over 48 weeks of treatment.31 It is noteworthy that even in a population of patients with severe liver disease, wait-listed for liver transplantation, only a limited number of cases with creatinine elevation were observed.32 In our study, there was no instance in which an elevation of serum creatinine level ≥0.5 mg/dL was observed.

This study shows that 52 weeks of treatment with ADV when added to ongoing lamivudine therapy in patients with lamivudine-resistant CHB results in significant suppression of HBV replication. As there is no evidence that sustained off-therapy responses can be achieved with ADV therapy of whatever duration, continuous therapy will probably be needed in order to maintain remission in patients with HBeAg-negative CHB.

Ancillary