A viral kinetic study using pegylated interferon alfa-2b and/or lamivudine in patients with chronic hepatitis B/HBeAg negative

Authors


  • Potential conflict of interest: Dr. Hatzakis is a consultant for and has received research and travel grants from Gilead.

Abstract

We studied viral dynamic parameters in 44 chronic hepatitis B/hepatitis B e antigen (HBeAg)(−) patients treated with pegylated interferon alfa-2b (PEG-IFN) 100 or 200 μg weekly or lamivudine 100 mg daily or the combination of PEG-IFN 100 or 200 μg with lamivudine. Patients receiving PEG-IFN monotherapy exhibited viral load oscillations between weekly injections, which were resolved by the addition of lamivudine. The median pharmacological delay was estimated at 4.1, 5.8, and 1.8 hours in PEG-IFN monotherapy, PEG-IFN 100/200 μg + lamivudine, and lamivudine monotherapy, respectively (P = .44). The median half-life of free virus was 12.7 hours (range, 2.4-69.2 hours). The mean antiviral effectiveness of PEG-IFN 100/200 μg monotherapy was lower than that of lamivudine (82.6% vs. 96.4%; P = .005). The mean effectiveness of PEG-IFN 100 μg + lamivudine and PEG-IFN 200 μg + lamivudine was 92.8% and 94.4%, respectively. The half-life of infected cells ranged from 2.7 to 75 days. The median half-life of infected cells in patients receiving the combination regimens of PEG-IFN and lamivudine was similar to that of lamivudine patients (5.0 days vs. 6.0 days, P = .77). In conclusion, the addition of pegylated interferon alfa-2b in lamivudine treatment was found to neither enhance the potency of blocking HBV production nor the decay rates of infected cells. Supplementary material for this article can be found on the HEPATOLOGY website (http://www.interscience.wiley.com/jpages/0270-9139/suppmat/index.html). (HEPATOLOGY 2005;42:77–85.)

Interferon alfa (IFN), lamivudine, and adefovir dipivoxil are the only approved treatments for chronic hepatitis B. IFN treatment has a moderate efficacy and frequent side effects, and is associated with an inconvenient 3-times-weekly dosing regimen. Lamivudine and adefovir have significant antiviral activity, although viral rebound after cessation of therapy and development of resistance after long-term lamivudine therapy are major clinical limitations.

The new pegylated forms of IFN (pegylated interferon alfa-2a or 2b) are currently being evaluated among chronic hepatitis B patients. The benefit of conjugation of IFN with polyethylene glycol is the improvement of delivery of IFN by significantly prolonging its plasma half-life and thereby providing protracted activity, which allows once-per-week dosing. Results from trials among hepatitis B e antigen (HBeAg) (+) or HBeAg (−) chronic hepatitis B patients indicate that treatment with pegylated interferon alfa-2a is superior to conventional interferon alfa-2a or lamivudine monotherapy.1–3 The combination of pegylated interferon alfa-2a or alfa-2b and lamivudine was not found to improve sustained response rates over pegylated interferon monotherapy in HBeAg (−) or HBeAg (+) chronic hepatitis B patients, respectively.3, 4

Mathematical modeling of the dynamics of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infection during antiviral therapy has provided useful insight into viral replication, host cell death rate, and treatment efficacy.5–7 In the last years, similar studies have been carried out to assess the viral dynamics of hepatitis B virus (HBV) during the administration of lamivudine, adefovir dipivoxil, or entecavir.8–16 Studies of viral dynamics among chronic hepatitis B patients receiving pegylated interferon are currently underway.4, 17 However, methodological issues arise when analyzing viral load decay among patients receiving pegylated interferons. The once-weekly dosing of the drug is associated with a decline in its concentration between injections, possibly resulting in decrease in efficacy and subsequent rebound of viral levels. Thus, an appropriate model that accounts for the decreasing efficacy of pegylated interferon between weekly doses has to be used to obtain estimates of the viral dynamics parameters for these patients.18

We conducted a study to examine the dynamics of HBV in hepatitis B surface antigen (HBsAg)(+)/HBeAg(−) patients during the first 4 weeks of treatment with pegylated interferon alfa-2b (PEG-IFN), alone or in combination with lamivudine. We investigated the viral decay patterns after treatment administration in order to provide a refined estimate of the virion clearance and to assess the antiviral efficacy of the regimens and the decline of infected cells.

Abbreviations

IFN, interferon alpha; HCV, hepatitis C virus; HBV, hepatitis B virus; PEG-IFN, pegylated interferon alfa-2b; ALT, alanine aminotransferase; LMV, lamivudine; QD, daily; QW, once weekly.

Patients and Methods

Patients.

HBeAg(−)/anti-Hbe(+) patients previously untreated for chronic hepatitis B, 18 to 70 years of age, with body weight >65 kg, elevated alanine aminotransferase (ALT), and HBV DNA > 106 copies/mL were eligible for enrollment. By the middle of the study, the HBV DNA level entry criterion was modified to >105 copies/mL to facilitate accrual. Patients were excluded if they were positive for antibodies to hepatitis C or HIV. HBV genotype was not tested, but almost all Greek chronic hepatitis B patients are of genotype D. The study was approved by the institutional review boards of participating clinical sites and was conducted according to the Declaration of Helsinki. All patients enrolled in the study provided written informed consent.

Study Design.

The administered regimens included lamivudine and pegylated interferon α-2b (PEG INTRON, Schering Plough, Kenilworth, NJ). More specifically, patients were randomized into 5 treatment groups, Arm A: PEG-IFN 100 μg once per week for 48 weeks, Arm B: PEG-IFN 200 μg once per week for 4 weeks followed by PEG-IFN 100 μg once per week for 44 weeks (PEG-IFN 200 μg), Arm C: PEG-IFN 100 μg once per week and lamivudine 100 mg once per day for 48 weeks (PEG-IFN 100 μg + LMV), Arm D: PEG-IFN 200 μg once per week and lamivudine 100 mg once per day for 4 weeks followed by PEG-IFN 100 μg once per week and lamivudine 100 mg once per day for 44 weeks (PEG-IFN 200 μg + LMV) and Arm E: lamivudine 100 mg once per day for 48 weeks (LMV).

One hundred twenty-five patients were originally screened to enter the study. Of those patients, 47 fulfilled the inclusion criteria and signed the informed consent. Three patients were subsequently excluded from the analysis; two patients with HBV DNA >105 copies/mL at screening had <105 copies/mL at baseline, and one patient was finally diagnosed as acute hepatitis B. In total, we analyzed the viral dynamics of 44 patients.

HBV DNA kinetics analysis was based on measurements up to week 4. A screening sample, a baseline sample (within 14 days before initiating therapy), as well as samples at day 0 (hours 0, 4, 8, 16, and 20), day 1 (hours 24, 28, and 36), day 2 (hour 48), and at days 4, 5, 7, 9, 11, 12, 14, 21, and 28 were taken for HBV DNA testing. During that period, PEG-IFN was given on days 0 (hour 0), 7, 14, 21, and 28. The frequent blood sampling scheme during the first 2 days of treatment was considered necessary to determine pharmacological and intracellular delays in viral decline and, thus, to obtain a more accurate estimate of the viral clearance rate. After the enrollment of the first 29 patients, whose data were adequate to proceed to the estimation of the viral clearance rate, blood samples were collected less frequently during the first 2 days of treatment for the remaining 15 patients (hours 0, 24, and 48). ALT testing was performed at days 0, 1, 2, 3, 4, 5, 7, 9, 11, 12, 14, 21, and 28.

Quantification of HBV DNA in Plasma.

A highly sensitive real-time polymerase chain reaction (PCR) assay was used to quantify HBV DNA.19 This method has a cutoff of 250 copies/mL, and its ability of quantification lies in a 10-log10 dynamic range. The measurements obtained by the PCR assay can be converted to copies/mL of Amplicor HBV Monitor values using the following formula: log10[real-time PCR (copies/mL)] = 0.38 + 0.92 × log10[Amplicor HBV Monitor (copies/mL)].19

Exploratory Analysis of Viral Decline.

The presence of weekly oscillations in viral decline among patients receiving PEG-IFN, as monotherapy or in combination with lamivudine, was initially assessed by examining the individual scatter plots of HBV DNA levels versus time. Given that the highest serum concentration of PEG-IFN levels is observed approximately 2 days after dosing,20 we estimated the slope of viral decline or rebound at the end of the week (days 3-7) to conclude on the presence of weekly oscillations.

This initial examination of the 4-week data showed that viral decline was biphasic in patients receiving lamivudine monotherapy or in combination with PEG-IFN. To estimate the breakpoint location, in other words, the number of days after which the initial rapid viral decline slowed down, a piecewise linear model with unknown breakpoint was fitted to the data of each patient from these treatment groups.21

Mathematical Model for Viral Dynamics.

After this exploratory examination of the viral load data, HBV viral dynamics were studied by using a standard model of viral infection.7 This model consists of three differential equations describing the change in the number of uninfected cells (target cells T), the number of infected cells (I), and viral load (V), in other words:

equation image(1)

Target cells T are produced at rate s, die with a death rate constant d and become infected with an infection rate β. Infected cells are produced at rate β and are lost at a rate δ. Virions are produced from infected cells at a rate p and are cleared with a virion clearance rate c. Treatment may result in viral decline either by reducing the production of virions by a fraction (1 − ε(t)) and/or by blocking the de novo rate of infection by a fraction (1 − η(t)). To solve the model equations, we assumed that: (i) the viral load and the number of infected cells are in quasi-steady state before treatment initiation, (ii) the number of uninfected target cells remains approximately constant during the period of analysis (4 weeks), and (iii) treatment effectiveness in blocking the de novo rate of infection is constant over time (η(t) = η(t)). We also took into account of the possibility of a delay t0 in viral decay after treatment administration. When antiviral effectiveness is assumed to be constant, that is, ε(t) =ε, the solution of the system becomes:

equation image(2)

with t > t0, where λ1, λ2 are the first-phase and second-phase slopes of viral decline, with λ1,2 = ½ {(c + δ) ± [(c − δ)2 + 4(1 − ε)(1 − η)cδ]1/2}, A = (εc − λ2)/(λ1 − λ2) and Vo the baseline viral load.7 We performed the analysis assuming partial blocking with η = 0.5 and, as a sensitivity analysis, we also considered the assumptions of η = 1 and η = 0 to obtain minimal and maximal estimates for δ. Because data from previous studies indicate that infected cells live much longer than 7 days, we assumed that their number remains constant during the first 7 days of treatment. Under this assumption, the solution of the model for the viral kinetics in the first week of therapy takes the form7:

equation image(3)

In the case of PEG-IFN, the once-weekly dosing scheme may result in a decline in the concentration of the drug between doses and, thus, in a reduction in the drug effectiveness in blocking virion production. To accommodate the case of nonconstant ε(t), we used a model proposed by Powers et al.,18 in which the concentration C of PEG-IFN is assumed to decay exponentially after a delay tp, that is,

equation image(4)

where C0 is the drug concentration at time tp, in other words, the maximum serum concentration, and k is the rate constant for drug elimination. Then, ε(t) is assumed to depend on drug concentration as:

equation image(5)

where r = C0/IC50 and IC50 is the concentration at which the drug's effectiveness is half its maximum. Incorporating equations 4 and 5 into equation 1 results in a model appropriate to analyze viral dynamics under PEG-IFN treatment.18

Data Fitting.

Using nonlinear regression analysis, we fitted the viral load data up to day 7 for each patient separately by using the appropriate model for treatment effectiveness and obtained estimates of c, delay in viral decline (t0), or in the occurrence of PEG-IFN maximum serum concentration (tp) and either ε (for constant effectiveness) or r, k (for nonconstant effectiveness). Thus, maximum effectiveness can be calculated as εmax = r/(1 + r) whereas minimum effectiveness, that is, drug efficacy just before the second weekly injection, is εmin = re −7k/(1 + re−7k).18 For comparison purposes, we also fitted the constant-effectiveness model in patients exhibiting weekly oscillations using the parameters c, t0 obtained from the nonconstant effectiveness model to estimate the fixed effectiveness. The reported estimates of c, t0, and tp were based on the subset of patients with very frequent sampling during the first days of treatment on whose data the appropriate model could be fit.

Next, we substituted in equation 2 the parameters c and ε already obtained for each patient and used non-linear least-squares fitting of the viral load up to day 28 to estimate δ. In the case of patients with weekly oscillations, we substituted in equation 2 the parameter c already obtained from the nonconstant effectiveness model and the fixed ε estimated by the constant effectiveness model to estimate δ. In this analysis, we used only weekly viral load data to avoid bias caused by oscillations within each week. For the estimation of δ, four patients with first phase decline < 0.5 log10 (nonresponders) were not included in the analysis (3 from PEG-IFN 100 μg and 1 from PEG-IFN 200 μg). For 3 patients with initial response and subsequent rebound in the second phase of viral decay, we assumed that δ was equal to 0 when performing non-parametric testing (2 from PEG-IFN 200 μg, 1 from PEG-IFN 200 μg + LMV). These patients, however, were not included in the reported medians of δ and half-life of infected cells.

Once we obtained estimates for c and δ, we calculated the half-life of free virus and of productively infected cells as ln2/c and ln2/δ, respectively.

Other Statistical Analyses.

The t test, the one-way analysis of variance, or the nonparametric Wilcoxon rank-sum test were used to assess the presence of differences in continuous variables (baseline characteristics, viral decline, and viral dynamics parameters) among the different treatment groups, as appropriate.

Results

Baseline Characteristics of the Patients.

Table 1 lists the demographic and baseline characteristics of the patients by treatment group. Patients in the 5 treatment groups were comparable with regard to age, sex, and weight. However, there were statistically significant differences in baseline ALT and HBV DNA levels (P = .038 and P = .024), being higher in the lamivudine group.

Table 1. Demographic and Baseline Characteristics of 44 Patients According to Treatment Group
 PEG-IFN 100 μgPEG-IFN 200 μgPEG-IFN 100 μg + LMVPEG-IFN 200 μg + LMVLMV
N99998
Gender     
 Male/Female7/29/09/07/26/2
Age (years)     
 Mean (SD)48.7 (13.1)38.8 (14.4)48.1 (11.5)51.1 (8.5)46.0 (15.0)
Baseline ALT levels (IU/L)     
 Median (25th, 75th)91 (53, 117)122 (75, 337)122 (78, 188)83 (68, 93)352 (214, 442)
 Range35–14944–81857–23763–21068–715
Baseline HBV DNA (log10 copies/mL)     
 Mean (SD)6.17 (0.88)7.14 (1.18)7.26 (1.02)7.00 (1.52)7.99 (0.66)
 Range5.01–7.215.51–9.135.42–8.615.09–9.946.53–8.85
Weight at study entry (kg)     
 Median (25th, 75th)71 (60, 83)74 (70, 84)85 (70, 88)89 (70, 90)82 (70, 84)

Viral Decline.

The average viral load decline from baseline levels at week 4 was 3.0 log10 copies/mL for LMV, 1.1 log10 copies/mL for PEG-IFN 100 μg, 1.3 log10 copies/mL for PEG-IFN 200 μg, 3.2 log10 copies/mL for PEG-IFN 100 μg + LMV, and 2.9 log10 copies/mL for PEG 200-IFN μg + LMV (P < .001).

The individual scatter plots of HBV DNA levels during the first week of treatment versus time showed the possible presence of weekly oscillations only among patients receiving PEG-IFN monotherapy (Fig. 1A). These patients exhibited an initial decline in viral load levels after the injection and then had a rebound toward the end of the week. Patients who received the combination regimens of PEG-IFN and lamivudine exhibited a pattern of decay similar to that of lamivudine group: after an initial delay, serum viral load declined rapidly (first phase), and then viral decay slowed down without presenting oscillations between weekly injections (second phase). We estimated the slope of viral decline or rebound at the end of the first week (from day 3 to day 7). The median slope was 0.007 log10 copies/mL per day (range, −0.233-0.127) in PEG-IFN 100/200 μg monotherapy, −0.121 (range: −0.447-0.092) in PEG-IFN 100/200 μg + LMV arms and −0.209 (range, −0.395 to −0.039) in LMV monotherapy (Fig. 2A). The slope of PEG-IFN 100/200 μg + LMV differed from that estimated for PEG-IFN 100/200 μg monotherapy (P = .005) and was similar to the LMV monotherapy estimate (P = .20).

Figure 1.

Observed viral decline during (A) the first week and (B) the first 4 weeks of treatment for a subset of patients. The squares indicate observed HBV DNA levels, while the solid lines are non-linear least-squares fits. (Log10 HBVDNA are measured in copies per mL.)

Figure 2.

Boxplots of parameter estimates obtained for viral and infected cells dynamics and antiviral efficacy, according to treatment group; (A) slope of viral decline or rebound at the end of the first week, (B) virion clearance rate, (C) pharmacological delay, (D) antiviral effectiveness, (E) half-life of infected cells, (F) slope λ2 of 2nd phase viral decline (PEG-IFN: pegylated interferon alpha-2b, LMV: lamivudine).

We further examined whether viral decline from 1 week to the next, disregarding interval changes in viral load in every week, was constant from week 1 on, as assumed by the biphasic decline model of equation 2. The average slopes of viral decline during days 14-21 and 21-28 were similar (−0.027 and −0.033 log10 copies/mL per day, P = .73). They differed, however, from the slope estimated for days 7-14 (−0.067 log10 copies/mL per day). This increased slope of viral decline during the second week may be attributed to the fact that in some patients the first phase decline was prolonged. Overall, no evidence of staircase decline patterns was found. The median estimate of the location of the change-point from the first to the second phase viral decline was 3.1 days (25th, 75th: 2.8, 3.9 days).

Viral Clearance and Pharmacological Delay.

An estimate of the viral clearance rate was obtained by using data from patients with frequent blood sampling in the early phase of therapy and the appropriate model for treatment effectiveness. The viral clearance rate estimates ranged from 0.240 to 6.847/day, with a median of 1.305/day (25th, 75th: 0.772, 2.218/day) and did not differ significantly between the treatment groups (P = .19) (Fig. 2B). The corresponding half-life of free virus ranged from 2.4 to 69.2 hours, with a median of 12.7 hours.

The median pharmacological delay was estimated as 4.1 hours (range, 0-32.1 hours). Patients receiving PEG-IFN monotherapy or in combination with lamivudine had a more prolonged delay in viral decay compared with lamivudine monotherapy; this difference, however, was not statistically significant (median, 4.1 hours for PEG-IFN 100/200 μg, 5.8 hours for PEG-IFN 100/200 μg + LMV, 1.8 hours for LMV, P = .44) (Fig. 2C).

Antiviral Efficacy.

The estimated antiviral effectiveness in patients receiving PEG-IFN monotherapy is presented in Table 2. For comparison purposes, the estimates obtained from the constant effectiveness model are also presented. It was not possible to fit the viral load data of 2 nonresponders receiving PEG-IFN 100 μg with either model (not included in Table 2). These patients are not considered further. Eight patients receiving PEG-IFN monotherapy did not exhibit viral load rebound at the end of the first week (viral load slope at days 3-7 < 0: patients 83, 113, 115 from PEG-IFN 100 μg and 23, 29, 43, 60, 92 from PEG-IFN 200 μg). We therefore used the constant effectiveness model to fit the data of these patients. It was not possible to fit the non-constant effectiveness model to the data of 2 patients exhibiting oscillations (patients 63, 110). In the remaining patients with rebound, the mean (SD) εmax of PEG-IFN 100/200 μg was 89.5% (8.7%) and declined to a mean (SD) εmin of 38.1% (32.9%). The fixed ε estimated from the constant effectiveness model fell within the range of (εmin, εmax) in all patients and, with the exception of patient 18, it was similar to the average effectiveness estimated empirically by averaging the effectiveness estimates throughout the week (assuming piecewise-constant ε per 2-hour intervals).

Table 2. Effectiveness of PEG-IFN Monotherapy in Blocking Virion Production
PatientSlope of Viral Decline/Rebound*Non-constant Effectiveness ModelConstant Effectiveness ModelFinal Effectiveness Estimate
k (day−1)εmin (%)εmax (%)Average ε (%)ε (%)ε (%)
  • NOTE. Two nonresponders receiving PEG-IFN 100 μg are not included in the table because it was not possible to fit their viral load data with either model.

  • *

    At days 3–7.

  • Estimated from εmax or from the constant effectiveness model, as appropriate.

  • Empirically estimated by averaging the efficacy estimates obtained per 2-hour intervals throughout the week.

  • §

    NF: no fit of the model to the viral load data of these patients.

  • NA: not applicable (no rebound).

PEG-IFN 100 μg       
 100.1271.250.394.834.126.594.8
 180.0072.160.082.411.969.282.4
 630.015NF§NFNFNF66.7
 1100.060NFNFNFNF91.3
 83−0.172NANANANA89.089.0
 113−0.091NANANANA89.389.3
 115−0.211NANANANA86.786.7
PEG-IFN 200 μg       
 130.1260.6263.599.289.581.999.2
 190.0070.1861.784.766.165.184.7
 640.0440.3129.678.553.151.178.5
 900.0320.3673.497.185.779.097.1
 23−0.005NANANANA57.657.6
 29−0.048NANANANA54.654.6
 43−0.072NANANANA59.059.0
 60−0.233NANANANA98.498.4
 92−0.048NANANANA84.984.9
Mean (SD)−0.029 (0.107)0.81 (0.76)38.1 (32.9)89.5 (8.7)56.7 (30.1)71.9 (19.1)82.6 (15.1)

We computed an overall estimate of the effectiveness achieved by PEG-IFN 100/200 μg monotherapy using the estimate of εmax or of the fixed ε, depending on the most appropriate model for each patient. The mean (SD) estimate was 82.6% (15.1%) and differed significantly from the corresponding estimate of 96.4% (3.0%) obtained for lamivudine monotherapy (P = .005) (Fig. 2D).

The mean (SD) antiviral effectiveness of PEG-IFN 100 μg + LMV and PEG-IFN 200 μg + LMV was 92.8% (7.7%) and 94.4% (5.6%), respectively, and was similar to the efficacy of lamivudine monotherapy (P = .22 and P = .40, respectively) (Fig. 2D).

No association was found between antiviral effectiveness and baseline HBV DNA, baseline ALT levels, patients' age at study entry, sex, or weight (data not shown).

Rate of Loss of Infected Cells.

The median estimate of δ was 0.071/day in PEG-IFN 100/200 μg (25th, 75th: 0.032, 0.126), 0.120/day in PEG-IFN 100μg + LMV (25th, 75th: 0.092, 0.196), 0.146/day in PEG-IFN 200μg + LMV (25th, 75th: 0.102,0.169), and 0.119/day in LMV (25th, 75th: 0.086, 0.186). The median half-life of infected cells in patients receiving PEG-IFN monotherapy (9.8 days) or the combination regimens of PEG-IFN 100/200 μg and lamivudine (5.0 days) was similar to that of LMV patients (6.0 days) (Fig. 2E). Overall, the half-life of infected cells ranged from 2.7 to 75 days, with a median of 6.5 days. The slope λ2 of the second-phase viral decline according to treatment is shown in Fig. 2F.

No association was found between the rate of loss of infected cells and baseline ALT levels, baseline HBV DNA, patients' age at study entry, sex, or weight (data not shown).

The fitted viral load decline during the first 4 weeks of treatment, along with the observed HBV DNA levels, is presented for a representative subset of patients in Fig. 1B.

Discussion

In the current study, HBV viral dynamics were analyzed during the first 4 weeks of treatment with pegylated interferon alfa-2b, alone or in combination with lamivudine, in 44 HBsAg(+)/HBeAg(−) patients. To our knowledge, it is the largest study on HBV dynamics and the first one examining the kinetics of the virus under pegylated-inteferon alfa-2b treatment among HBsAg(+)/HBeAg(−) patients.

The once-weekly dosing of PEG-IFN is associated with a decline in the drug concentration between doses, and this may have an effect on viral load decay. Our data verified the presence of weekly viral load oscillations among patients receiving PEG-IFN monotherapy. The addition of lamivudine to PEG-IFN treatment was found to resolve these oscillations. Based on these findings, we analyzed the viral dynamics of patients receiving PEG-IFN monotherapy using a model proposed by Powers et al.18 that accounts for the decreasing effectiveness of the drug between weekly injections. Viral load decay in patients receiving lamivudine as monotherapy or in combination with PEG-IFN was biphasic, with an initial rapid decline, lasting for approximately 3 days, followed by a slower viral decay or plateau, and was therefore analyzed by using a standard model.7 The slopes of viral decay from 1 week to the next for the first 4 weeks of treatment were constant, indicating that there were no staircase viral decline patterns as those reported by Lewin et al.12 Lewin et al., however, had analyzed viral decline during a longer treatment period (12 weeks).

The frequent blood-sampling scheme during the first 2 days of treatment (9 blood samples obtained the first 48 hours) allowed us to determine pharmacological delay and thus to obtain a more refined estimate of the virion clearance rate. The median pharmacological delay was estimated as 4.1 and 5.8 hours in PEG-IFN monotherapy and PEG-IFN + LMV combination arms, respectively, and 1.8 hours in the lamivudine arm. In the case of PEG-IFN monotherapy, this estimate actually measures the delay in the occurrence of maximum serum concentration of the drug and not the delay until viral load starts declining. Preliminary results from another viral dynamics study suggest that patients receiving pegylated interferon alfa-2a monotherapy exhibited a single-phase decrease of HBV-DNA after a pharmacological delay of 3 days.17 This may be due to the different pharmacokinetic profiles of pegylated interferon alfa-2a and alfa-2b.20

According to our estimates, virion clearance is more rapid than was previously thought, with a median half-life of 12.7 hours, which is consistent with the relatively small virion size. Two other studies with short intervals between blood sample collections in the early phase of therapy reported an average virion half-life of 13 hours.13, 15 Previous studies with less frequent blood sampling reported average estimates for the virion half-life of 24 to 29 hours, which were, in fact, upper estimates of the actual value8, 10, 12 (Table 3). These results suggest that free virus in HBV infection is cleared fast, though still slower compared with HCV and HIV (half-life in HCV and HIV infection: 2.7 hours and 5.8 hours).5, 7 Approximately 30% of the patients had an estimated half-life of free virus less than 9 hours. This finding has been reported also by Lewin et al.,12 who described, however, very rapid viral clearance in only 2 of 14 patients (half-life: 1.0 hour and 5.4 hours).

Table 3. Comparison of Viral Dynamic Parameters and Antiviral Efficacy Estimates With Other Studies
Studies (Reference)TreatmentPatientsNumber of PatientsPeriod Studied (Weeks)Number of Initial Blood Samples (First 2 Days)t1/2 Virus (Hours)t1/2 Infected Cells (Days)Antiviral Efficacy
  1. Abbreviations: PEG-IFN, pegylated interferon alpha-2b; LMV, lamivudine; FCV, famciclovir; GCV, ganciclovir.

Nowak et al.8LMV 20–600 mgHBeAg (+)234224.010–10087%–99%
Lewin et al.12LMV 150 mgHBeAg (+)1512328.52.4–>12095%
LMV 150 mg + FCV99%
Tsiang et al.9AdefovirHBeAg (+)1012126.411–3099%
Lau et al.10LMV 150 mgHBeAg (+)2112348.34394%
LMV 150 mg + FCV99%
Wolters et al.14EntecavirHBeAg (+)/(−)104416.05.2–31.887%–98%
Wolters et al.15LMV 150 mgHBeAg (+)214913.0<0–33192%
LMV 600 mg96%
Wolters et al.13LMV 150 mgHBeAg (+)/(−)124912.73–2693%
LMV 150 mg + FCV95%
LMV 150 mg + GCV86%
Sypsa et al.PEG-IFN 100/200 μgHBeAg (−)444912.72.7–7583%
PEG-IFN 100/200 μg + LMV93%
LMV 100 mg96%

The effectiveness achieved by PEG-IFN 100/200 μg monotherapy using the estimate of εmax or of the fixed efficacy, depending on the most appropriate model for each patient, was on average 82.6% and was significantly lower than that of lamivudine monotherapy (96.4%; P = .005). The combination regimens of PEG-IFN 100 μg + LMV and PEG-IFN 200 μg + LMV had a similar effectiveness (92.8% and 94.4%, respectively). Consequently, the addition of PEG-IFN in lamivudine treatment was not found to increase its potency in blocking new virus production. This finding is in agreement with data from recently reported clinical trials in which the combination of pegylated interferon alfa-2a or alfa-2b and lamivudine was not found to improve sustained response rates over pegylated interferon monotherapy in HbeAg (−) or HBeAg (+) chronic hepatitis B patients, respectively.3, 4

The rate of loss of infected cells exhibited considerable heterogeneity among patients. The associated half-life of infected cells ranged from 2.7 to 75 days, with a median of 6.5 days, and was similar to estimates obtained by other studies (Table 3). An analogous variability has been identified in HCV (range, 1.7-70 days)7 but not in HIV infection (range, 1.0-2.7 days).5 The addition of PEG-IFN in lamivudine treatment was not found to enhance the decay rates of infected cells (median half-life for the combination regimens: 5.0 days vs. 6.0 days for LMV).

In conclusion, the potency of lamivudine treatment in blocking new virus production in HBeAg (−) patients was not found to increase by the addition of pegylated interferon alfa-2b. The analysis of long-term data will elucidate whether the combination regimen of pegylated interferon alfa-2b and lamivudine can induce durable post-treatment remission or whether its effectiveness will be affected by the emergence of lamivudine-resistant HBV.

Acknowledgements

The authors thank the anonymous referees whose comments led to a significant improvement of the manuscript.

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