Adefovir rapidly suppresses hepatitis B in HBeAg-negative patients developing genotypic resistance to lamivudine

Authors

  • Pietro Lampertico,

    1. Department of Gastroenterology and Endocrinology, A. M. and A. Migliavacca Center for Liver Disease, IRCCS Maggiore Hospital, Fondazione Policlinico, Mangiagalli e Regina Elena, University of Milan, Milan, Italy
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  • Mauro Viganò,

    1. Department of Gastroenterology and Endocrinology, A. M. and A. Migliavacca Center for Liver Disease, IRCCS Maggiore Hospital, Fondazione Policlinico, Mangiagalli e Regina Elena, University of Milan, Milan, Italy
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  • Elena Manenti,

    1. Department of Gastroenterology and Endocrinology, A. M. and A. Migliavacca Center for Liver Disease, IRCCS Maggiore Hospital, Fondazione Policlinico, Mangiagalli e Regina Elena, University of Milan, Milan, Italy
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  • Massimo Iavarone,

    1. Department of Gastroenterology and Endocrinology, A. M. and A. Migliavacca Center for Liver Disease, IRCCS Maggiore Hospital, Fondazione Policlinico, Mangiagalli e Regina Elena, University of Milan, Milan, Italy
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  • Giovanna Lunghi,

    1. Division of Hygiene, IRCCS Maggiore Hospital, Fondazione Policlinico, Mangiagalli e Regina Elena, University of Milan, Milan, Italy
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  • Massimo Colombo

    Corresponding author
    1. Department of Gastroenterology and Endocrinology, A. M. and A. Migliavacca Center for Liver Disease, IRCCS Maggiore Hospital, Fondazione Policlinico, Mangiagalli e Regina Elena, University of Milan, Milan, Italy
    • Department of Gastroenterology and Endocrinology, IRCCS Maggiore Hospital, University of Milan, Via F. Sforza 35-20122, Milan, Italy
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    • fax: (39) 0250320410


  • Potential conflict of interest: Nothing to report.

Abstract

Progression of hepatitis B in patients with lamivudine-resistant strains is slowed down by adefovir dipivoxil (ADV). Whether the time point of ADV administration (genotypic vs. phenotypic resistance) influences the outcome of therapy is unknown. We compared the outcome of ADV therapy in hepatitis B e antigen (HBeAg)-negative chronic hepatitis B patients with genotypic and phenotypic resistance to lamivudine. Ten milligrams of ADV was administered daily for 2 years to 46 HBeAg-negative patients at the time of phenotypic resistance (group A, >6 log10 copies/mL of hepatitis B virus [HBV] DNA and high alanine aminotransferase [ALT] levels) and 28 patients at the time of genotypic resistance (group B, 3-6 log10 copies/mL of HBV-DNA and normal ALT). HBV DNA was assessed every 2 months using Versant 3.0 assay, and lamivudine resistance was confirmed via INNO-LiPA assay in all patients. By month 3, HBV DNA tested negative in all patients from group B compared with only 20 (46%) in group A (P < .0001). The 2-year rates of virological response were 100% in the former patients and 78% in the latter ones (P < .0001). ALT levels remained persistently normal in all group B patients, whereas in group A patients they normalized at rates of 50% at month 6 (P < .0001), 72% at month 12 (P < .01), and 93% at month 24. None of the patients developed ADV resistance or ADV-related side effects. In conclusion, to optimize antiviral treatment in HBeAg-negative patients selecting resistant strains to lamivudine, ADV should be added to lamivudine as soon as genotypic resistance is detected. (HEPATOLOGY 2005.)

The analog nucleoside lamivudine has long been the only alternative to interferon α in the treatment of hepatitis B e antigen (HBeAg)-negative patients with chronic hepatitis B; however, the clinical benefit of prolonged therapy with this drug has often been eroded by the emergence of resistant mutant strains leading to recurrence and deterioration of hepatitis B.1, 2 The initially marginal hepatitis damage associated with low levels of serum hepatitis B virus (HBV) DNA (genotypic resistance) inexorably inflates as mutated HBV strains accumulate to elicit substantial liver injury accompanied by high serum ALT values (phenotypic resistance).3, 4 The clinical implications of phenotypic resistance to lamivudine are many, because it may herald hepatitis progression to cirrhosis, and, in patients with cirrhosis, clinical decompensation, hepatocellular carcinoma (HCC), and delisting from liver transplantation.5, 6 The therapeutic gap caused by the emergence of lamivudine-resistant strains of HBV has been recently filled by the advent of the oral nucleotide analog ADV, whose efficacy in clearing mutated HBV DNA via polymerase chain reaction assay may be as high as 57% in 1-year therapy,7–10 to further increase with treatment prolonged beyond 1 year.11 Higher doses of ADV would be required to accelerate HBV DNA clearance in highly viremic patients, but this strategy is hampered by the relatively narrow therapeutic index of the drug due to significant nephrotoxicity appearing at doses higher than 10 mg.12 The existence of a dose–response relationship for anti-HBV therapy has already emerged from studies with interferon, where response rates in minimally viremic patients were significantly stronger than those obtained in highly viremic patients.13, 14 The 1-year response rates to ADV in HBeAg-negative patients, who had a median of 7.1 log10 copies/mL of HBV DNA, were twice those (21%) obtained in HBeAg-positive patients having viremia that was greater by more than 1 log10, on average.12, 15 Conceptually, treating HBeAg-negative patients who have chronic hepatitis B with ADV upon development of genotypic resistance—when serum levels of HBV DNA are still modest and ALT levels are normal—would be a better strategy than treating patients during the phenotypic resistance phase, when serum HBV DNA and ALT levels are peaking.

This prospective study of 74 HBeAg-negative patients with chronic hepatitis B compared the efficacy of ADV administered upon the onset of genotypic resistance with that of ADV given when phenotypic resistance developed.

Abbreviations:

ADV, adefovir dipivoxil; HBeAg, hepatitis B e antigen; HBV, hepatitis B virus; ALT, alanine aminotransferase; HCC, hepatocellular carcinoma.

Patients and Methods

Study Profile.

In 1997, we started lamivudine treatment in all HBeAg-negative patients with extensive fibrosis or cirrhosis who either did not meet the criteria for interferon therapy or did not respond to interferon.14 All patients on lamivudine therapy were subjected to surveillance for lamivudine resistance with quantitative assessment of serum HBV DNA performed every 2 months. Development of lamivudine resistance comprised the identification of molecular variants of the rt pol gene, rtM204V and/or rtM204I, in patients with a rebound of serum HBV DNA greater than 1 log compared with on-treatment nadir as was confirmed in two separate controls. Genotypic resistance was the presence of moderate levels of HBV DNA (3-6 log10 copies/mL) and persistently normal ALT levels. Phenotypic resistance was the presence of high levels of HBV DNA (>6 log10 copies/mL) and high ALT levels. The study included carriers of HBsAg (≥18 years of age) who tested negative for HBeAg and positive for anti-HBe in the previous 12 months, had a histological or clinical diagnosis of chronic hepatitis or cirrhosis, and developed either genotypic or phenotypic resistance to lamivudine. Two cohorts of patients with lamivudine resistance received ADV. The first cohort was composed of patients who developed genotypic resistance between May 1998 and March 2001, when ADV was not available, and therefore these patients were left on lamivudine alone. In May 2001, when these patients started adding ADV to lamivudine, genotypic resistance had already turned into phenotypic resistance in all of them. The second cohort was composed of patients who developed genotypic resistance between March 2001 and March 2003 and could receive ADV accordingly. In all patients, a daily dose of 10 mg ADV per os was added to an ongoing 100 mg dose of lamivudine.

Patients with impaired renal function (serum creatinine >1.5 mg/dL), antibody to hepatitis C virus, antibody to hepatitis delta virus, or antibody to HIV were excluded. Additional criteria for exclusion were pregnancy, lactation, and drug and alcohol abuse (>40 g/d ethanol). Patients were monitored at monthly intervals with clinical, biochemical, and virological assessments during the first 3 months of treatment and every 2 months thereafter. Surveillance for HCC was conducted with repeated abdominal ultrasound and serum α-fetoprotein measurements every 12 months in patients with chronic hepatitis and every 6 months in patients with cirrhosis.16 All patients gave their written informed consent. Details of the study were approved by the local Institutional Review Committee.

Serum Assays.

Serum aspartate aminotransferase, serum ALT, albumin, bilirubin, creatinine, and international ratio of prothrombin time were measured using standard laboratory procedures. Hepatitis B surface antigen, antibody to hepatitis surface antigen, serum HBeAg, and anti-HBe were detected via microparticle enzyme immunoassay (AXSYM, Abbott Laboratories, North Chicago, IL). Antibody to hepatitis delta virus was assessed via ELISA (Sorin Biomedica, Saluggia, Italy). Antibody to HIV was detected via HIV-1 third-generation assay (AXSYM HIV 1/2, Abbott Laboratories), and antibody to hepatitis C virus was detected via a second-generation test system (Ortho DS, Raritan, NJ). Serum HBV DNA was assessed every 2 months using Versant 3.0 assay (bDNA, Bayer Corp., Tarrytown, NJ), with a sensitivity limit of 2000 copies/mL or 3.3 log10 copies/mL. Serum α-fetoprotein was measured using a standard assay (IRMA, Abbott Laboratories). Lamivudine-associated mutations were determined with a line probe assay for YMDD mutants (INNO-LiPA HBV DR, Innogenetics NV, Ghent, Belgium).17 Adefovir-resistant mutations (i.e., rtN236T and rtA181V) and HBV genotypes were assessed via direct sequencing.18, 19

Endpoints.

The primary endpoints of the study were HBV suppression to undetectable serum HBV DNA levels using Versant 3.0 assay, normalization of ALT levels, and onset of resistance to ADV. The latter was defined as a greater than 1 log rebound of serum HBV DNA compared with on-treatment nadir as confirmed via molecular analysis. Secondary end points were: change in Child-Turcotte-Pugh score of at least 2 points, clinical decompensation (ascites, encephalopathy, jaundice, and gastrointestinal bleeding defined according to internationally agreed criteria), and HCC.15 Monitoring of drug safety included renal toxicity defined by an increase in serum creatinine level of 0.5 mg/dL or more compared with baseline values in two consecutive checkups 2 months apart.

Statistical Analysis.

Data were expressed as the median and range for discrete variables and as counts and percentages for qualitative variables. Significance of differences in the distribution of quantitative and qualitative variables was assessed via Student t test and Fisher exact test, respectively. The 2-year cumulative probability of achieving undetectable HBV DNA was assessed via the Kaplan-Meier method. The difference between the cumulative curves was tested using a log-rank test. Cox's regression model was used for univariate and multivariate analysis of predictors of virological response (i.e., undetectable serum HBV DNA). The following pretreatment variables were evaluated: age, sex, ALT, HBV DNA, genotype, body weight, serum creatinine, creatinine clearance, and cirrhosis. All P values were two-tailed, and a value of .05 was considered statistically significant. Statistical analysis was performed with Stata Statistical Software version 7.0 (Stata Corp., College Station, TX).

Results

Patient Characteristics.

Most of the 74 patients who participated in the study were men, had compensated cirrhosis, and circulated the genotype D of HBV (Table 1). Seven patients (10%) had HCC at baseline, 5 of whom fulfilled the Milan criteria for liver transplantation.20 Forty-six patients had developed phenotypic resistance to lamivudine, and 28 were identified during the asymptomatic phase of genotypic resistance. ADV was added 41 and 33 months, on average, after initiation of lamivudine in genotypic- and phenotypic-resistant patients, respectively. The former group of patients had higher HBV DNA and ALT levels and significantly different mutation patterns at rt204 and rt180 compared with the latter group (Table 1). At baseline, 1 patient in the genotypic-resistant group had moderate levels of HBV DNA (4.5 logs) but higher than normal ALT levels (70 IU/mL [upper limit of normal: 40 IU/mL]). This patient was a middle-aged, overweight man with histological/ultrasound features of steatosis in addition to hepatitis B. He had experienced mild elevation of his ALT levels during the 3 years preceding enrollment, despite a persistent virological response to lamivudine with undetectable HBV DNA via sensitive assay. We think that this elevation was caused by a metabolic syndrome rather than phenotypic resistance to lamivudine. One patient in the phenotypic-resistant group had elevated ALT levels but moderate HBV DNA levels (4.3 logs) at enrollment. In the 2 years preceding enrollment, this patient had experienced persistently elevated ALT levels and HBV DNA levels greater than 6 logs (which dropped to 4.3 logs following an ALT flare at enrollment).

Table 1. Pretreatment Demographic, Clinical, and Virological Characteristics of Patients Treated With ADV Upon Development of Genotypic or Phenotypic Resistance to Lamivudine
Pretreatment FeaturesAll (n = 74)Lamivudine-Resistant Phase
Genotypic (n = 28)Phenotypic (n = 46)
  • NOTE. a versus b, c versus d, P < .001; e versus f, P < .01; g versus h, P < .05 (P value not significant for all other comparisons).

  • *

    HBV genotype was determined in 56 patients (17 with genotypic and 39 with phenotypic resistance to lamivudine).

  • 7 with HCC at baseline.

Male: no. (%)67 (90)25 (89)42 (91)
Age, yrs: median (range)56 (36-77)60 (45-71)53 (36-77)
ALT, IU/L: median (range)67 (20-2870)38 (20-70)a145 (42-2870)b
HBV-DNA, log10copies/mL: median (range)6.4 (3.4-9.3)4.5 (3.4-5.9)c7.3 (4.3-9.3)d
rtM204V mutation: no. (%)40 (54)10 (36)e30 (65)f
rtM204I mutation: no. (%)34 (46)18 (64)e16 (35)f
rtL180M mutation: no. (%)52 (72)15 (52)g37 (80)h
Genotype D of HBV: no. (%)52 (93)*16 (94)*36 (92)*
Cirrhosis: no. (%)56 (76)23 (82)33 (72)
Serum creatinine, mg/dL: median (range)0.9 (0.5-1.3)0.8 (0.5-1.0)0.9 (0.6-1.3)

Virological and Biochemical Response.

By month 3, serum HBV DNA became undetectable in all 28 patients treated at the time of genotypic resistance, compared with 20 treated during the phase of phenotypic resistance (100% vs. 46%; P < .001). Serum levels of HBV DNA remained persistently undetectable in all the former patients throughout the 21 months of therapy that followed. HBV became undetectable in 10 additional phenotypic-resistant patients by month 12 (62%) and in 6 additional patients by month 24 of therapy (36 patients total [78%]). Among the 10 patients who maintained detectable HBV DNA levels despite long-term therapy with ADV, the HBV DNA levels at 2 years were greater than 5 logs in 3 cases, between 5 and 4 logs in 4 cases, and between 4 and 3 logs in 3 cases. Time to undetectable serum HBV DNA was significantly shorter in patients receiving ADV upon development of genotypic resistance than in those receiving ADV at the phase of phenotypic resistance (P < .0001) (Fig. 1).

Figure 1.

Two-year cumulative probability of clearing serum HBV DNA (<3.3 log10 copies/mL) in patients receiving ADV therapy upon onset of genotypic (n = 28) or phenotypic (n = 46) resistance to lamivudine. Patients are stratified for pretreatment viremia. HBV, hepatitis B virus.

Patients receiving ADV during the phase of genotypic resistance showed persistently normal values of ALT throughout the study period. Among patients treated during the phase of phenotypic resistance, ALT normalization was time-dependent: the rates of ALT normalization were 37% at month 3 (P < .0001), 50% at month 6 (P < .0001), 72% at month 12 (P < .01), and 93% at month 24 (P value not significant) (Fig. 2). The 3 patients with persistently high ALT levels at month 24 circulated detectable levels of serum HBV DNA, as well. Overall, 9 patients with phenotypic resistance compared with none of those with genotypic resistance had ALT levels higher than 5 times the upper limit of normal during the first 3 months of ADV therapy (20% vs. 0%; P < .01). In these patients, ALT levels returned within the normal range in 4 to 11 months (median: 5 months).

Figure 2.

Rates of ALT normalization in patients receiving ADV upon onset of genotypic (n = 28) or phenotypic (n = 46) resistance to lamivudine. *P < .001. **P < .01. ALT, alanine aminotransferase.

Only low pretreatment levels of HBV DNA (HR: 0.63; 95% CI: 0.53-0.76; P < .0001) were associated to a higher likelihood of a virological response as determined via univariate analysis. In patients treated at the time of phenotypic resistance, low pretreatment HBV DNA (HR: 0.58; 95% CI: 0.42-0.78; P < .0001) and high ALT levels (HR: 1.001; 95% CI: 1.0003-1.002; P < .01) were independently associated to a higher likelihood of a virological response.

Outcome of Hepatitis B.

Five patients with phenotypic resistance and decompensated cirrhosis showed an improvement in Child-Turcotte-Pugh score equal to or greater than 2 points following 6 to 12 months of treatment with ADV. Child-Turcotte-Pugh score remained unchanged throughout the study period in 3 patients and deteriorated in none. One of 49 HCC-free patients with cirrhosis developed de novo HCC. None of the patients developed liver decompensation, except 2 who died of HCC. Among the 7 patients with HCC prior to starting therapy with ADV, 4 underwent liver transplantation.

Resistance to ADV.

None of the patients had a rebound of serum HBV DNA that was greater than 1 log compared with on-treatment nadir. None of the 10 patients with detectable serum HBV DNA at the end of the study exhibited ADV-related mutations (i.e., rtN236T and rtA181V).

Safety and Tolerability.

None of the patients had to reduce the dose or discontinue therapy with ADV. The median serum creatinine level was 0.9 mg/dL (range: 0.5-1.3) at baseline and 0.9 mg/dL (range: 0.6-1.5) at the end of study (P value not significant).

Discussion

HBV was more rapidly and consistently suppressed in patients who received ADV early during the phase of genotypic resistance to lamivudine than in patients treated later during the phase of phenotypic resistance. By month 3, in fact, HBV DNA was universally cleared from the former patients compared with only half of the latter ones. More importantly, the 2-year clearance rates of HBV DNA were absolute in patients who anticipated ADV therapy at the time of genotypic breakthrough compared with 78% in those receiving ADV during the phase of phenotypic resistance. The stronger anti-HBV activity in the former group of patients could reflect the fact that a fixed dose of ADV is fully active only in minimally viremic patients, as are those identified during the genotypic breakthrough, as suggested also by multivariate analysis. Cross-analysis of the currently available studies on ADV therapy in patients with phenotypic resistance to lamivudine suggests an inverse relationship between pretreatment levels of viremia and antiviral response. The 1-year response rates to ADV were 20% to 35% in the highly viremic HBeAg-positive patients having 8 to 9 log10 copies/mL of HBV DNA, compared with 57% for HBeAg-negative patients with lower baseline viremia (6.7 log10 copies/mL of HBV DNA).7, 8, 10 The power of pretreatment viremia in predicting a response to ADV is also confirmed by the robust response to ADV in lamivudine-naive patients circulating low pretreatment levels of HBV DNA.12, 15 Thus, anticipating ADV administration upon onset of genotypic resistance is a realistic approach for improving therapy of HBeAg-negative patients with chronic hepatitis B who select lamivudine-resistant mutants. Treatment of these patients, in fact, cannot be improved by administrating ADV in dosages higher than 10 mg because of its relatively narrow therapeutic index.12 Anticipated therapy with ADV would also fulfill the goal of HBV suppression by preventing accumulation of liver fibrosis and attenuating the risk of liver decompensation that follows ALT flares.21 In our study, patients receiving ADV therapy upon development of genotypic resistance had less risk of hepatitis recrudescence than patients who received the drug at the time of phenotypic resistance to lamivudine. Among the 9 patients with elevated ALT levels within the first months of ADV therapy, none developed clinical decompensation. However, because of the small size of the study—which primarily enrolled patients with well-compensated hepatitis B—we could not assess whether ADV therapy prevented clinical decompensation as well. However, ALT elevations in phenotypic-resistant patients receiving ADV were short-lived and early in appearance, suggesting that ADV therapy attenuates hepatitis severity.

Further support for our therapeutic choice was demonstrated by the lack of genotypic or phenotypic resistance to ADV over 2 years of combined lamivudine and ADV therapy, which contrasts with the 18% of HBeAg-negative patients who developed ADV resistance following 4 years of monotherapy.11 Combination therapy with lamivudine is also likely to protect against ADV resistance in patients with high levels of lamivudine-resistant strains, such as HIV/HBV-coinfected patients.22 Although the low risk of ADV resistance in patients receiving lamivudine–ADV combination therapy might reflect the lack of cross-resistance between the two drugs,23 this point could be better clarified by a comparative study of long-term ADV monotherapy versus combination therapy with lamivudine.

Because the time lag between genotypic and phenotypic resistance to lamivudine ranges from 3 to 24 months,3 a strategy for monitoring lamivudine-treated patients based upon surveillance at 3-month intervals would enable timely identification of patients who develop genotypic resistance and therefore may benefit from early ADV therapy. We acknowledge that this strategy—which requires frequent monitoring of patients with sequential assessments of viremia and anticipated ADV treatment upon development of genotypic resistance—is expensive. These costs, however, should be weighed against the clinical benefit of preventing hepatitis deterioration that may follow treatment at the phase of phenotypic resistance. With regard to cost-effectiveness, patients with advanced hepatitis B who are at a greater risk of clinical decompensation upon development of ALT elevations would probably better fit our strategy of anticipated ADV treatment.

In conclusion, to maximize the efficacy of treatment in HBeAg-negative patients selecting resistant strains to lamivudine, ADV should be added to lamivudine as early as genotypic resistance is detected.

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