icon star paper   Hepatitis B Articles (HBV)  
Back grey_arrow_rt.gif
 
 
Adefovir-lamivudine combination therapy and hepatitis B viral kinetics
 
 
  Nucleoside and nucleotide analogues are potent inhibitors of HBV replication. Lamivudine (LAM) and adefovir (ADV) are licensed for the treatment of patients with chronic HBV infection, and monotherapy with each agent effects profound reduction of serum HBV titre. However, prolonged therapy is associated with the selection and emergence of resistant HBV species. Adopting the paradigm of HIV treatment, studies are now exploring the potential benefit of combination nucleos(t)ide therapy for HBV. Likely combinations will include drugs that have complementary HBV resistance profiles. For example, LAM-resistant virus (YMDD variant) has the M204V/I substitution. Recent data suggest that A181V/T and N236T substitutions are important for HBV resistance to ADV. Consistent with these observations, LAM-resistant HBV is effectively suppressed by ADV, and ADV-resistant HBV appears to be sensitive to LAM [1,2]. In addition, preliminary data show that combination LAM/ADV, in comparison with LAM monotherapy, can reduce the rate of emergence of YMDD virus [3]. However, it will prove more difficult to show that the combination reduces the rate of emergence of ADV-resistant virus. During ADV monotherapy, resistant species emerge at a very low rate (1-2% per annum) during the early years of treatment. Very large controlled studies with prolonged follow-up will be required to demonstrate with statistical significance that the addition of LAM to ADV can reduce that failure rate. Hopefully, those studies will be carried out.
 
As an alternative to large controlled studies with important and clinically relevant end-points, it may be informative to examine the kinetics of serum HBV titres during combination nucleos(t)ide therapy. In this issue of the Journal, Mihm and colleagues examine the viral kinetics observed during ADV treatment of a small cohort of patients with LAM-resistant HBV infection [4]. As a comparison group that received ADV as monotherapy, they chose a cohort of patients treated by Tsiang and colleagues [5]. The small size of the index cohort and the selection of a comparison cohort with fundamentally different baseline virological characteristics (and who received a different dose of ADV) limit the conclusions that can be drawn from their data. A larger study with a design that incorporated an appropriate control group(s) would have been more informative. Moreover, the authors' aim was to describe short-term viral kinetic data. The relevance of their observations for the development of clinical protocols for antiviral treatment of HBV-infected patients remains speculative. The authors acknowledge the limitations of their data and analysis.
 
The two cohorts can be compared and contrasted, and the kinetic data require close examination. Mihm added ADV (10mg/day) to LAM for 8 patients with documented LAM-resistant HBV infection, including patients with HBeAg-positive (n=5) or HBeAg-negative (n=3) infection. Tsiang describes the treatment with ADV (30mg/day) of 15 patients with HBeAg-positive infection, none of whom had received antiviral therapy during the 6 months before ADV treatment. Both studies apply well-recognised mathematical methods to describe the viral kinetics during ADV treatment. They examined the clearance rate of free virus (c), the drug's efficiency for blocking virus production (e), and the rate of infected cell loss (d).The principal observations are that there was dose-dependent efficacy in blocking viral production (lower e observed for ADV 10mg compared with 30mg/day), and that a greater rate of loss of infected hepatocytes (d)was observed during combination therapy. For those who do not have a degree in advanced mathematics the formulae are a bit frightening. However, study design can be scrutinised, and the inclusion in both publications of actual data points enables critical analysis.
 
The observed differences between combination therapy and monotherapy are described, but the impact on the comparison of inclusion of HBeAg-negative patients in the index cohort, and the administration of a higher dose of adefovir to the comparison group needs to be considered. In addition, the observations need to be reconciled with other published comparisons of combination versus monotherapy. The registration studies for ADV examined the effect of ADV for treatment of HBeAg-positive [6] and HBeAg-negative [7] treatment-naive patients. Though detailed kinetic analysis was not reported, the decline in serum HBV titre at monthly intervals during treatment of these cohorts was presented. The population data showed that ADV 30mg/day effected greater reduction of serum HBV titre than did ADV 10mg/day in HBeAg-positive patients. The same observation has been made by Werle and colleagues [8]. Consistent with these studies, Mihm and colleagues report a lowe e (efficacy for blocking viral production) for the 10mg, than for the 30mg treatment dose. In other words, there is dose-dependent inhibition of viral replication. Of course, in larger pre-registration studies, nephrotoxicity was more commonly observed at the higher dose. Thus, the dose of 10mg/day was chosen for licensing. Of interest, Tenofovir (300mg/day, the licensed dose for treatment of HIV infection) was recently shown to have significantly greater efficacy than ADV (10mg/day) for suppression of HBV infection [9]. Indeed, the effect of Tenofovir on serum HBV titre appears quite similar to that of ADV 30mg/day.
 
Though not subjected to statistical comparison in the phase 3 studies, it appears that greater reduction of HBV titre was achieved during ADV (10mg/day) treatment of HBeAg-negative infection than was observed for HBeAg-positive infection [6,7]. In this context, it is useful to review the actual data, not just an average of deltas, presented by Mihm and colleagues. The highest rates of infected cell loss (d)were observed for patients 6 and 7, both HBeAg-negative. That correlates with the steep second phase decline observed for both patients (in Fig. 1 of the manuscript). If the comparison made by Mihm and colleagues had been confined to the HBeAg-positive patients (n=5 only), then the d values may not have differed from those described by Tsiang and colleagues. That there is no significant difference is consistent with the observations made by Peters and colleagues who examined the response to ADV (10mg/day) of LAM-resistant HBeAg-positive infection [1]. In that double-blind randomised study, comparison could be made between the combination of LAM/ADV and ADV monotherapy. During a 48-week observation period, the decline of serum titre observed during combination treatment was not different from the decline effected by ADV monotherapy. Therefore, the higher d observed by Mihm and colleagues for the combination treatment group may be partly explained by their inclusion in the study cohort of patients with both HBeAg-positivity and HBeAg-negativity at baseline (whereas the comparison cohort were entirely HBeAg-positive).
 
Taken together, published studies show that, during treatment of HBeAg-positive infection, the reduction of titre achieved by ADV is dose-dependant, is probably unaffected by the presence of YMDD, is not enhanced by continuation of LAM in patients with YMDD, and may be inferior to the reduction observed during treatment of HBeAg-negative infection.
 
However, it cannot be concluded from the cited studies that LAM should be discontinued at time of commencement of ADV for YMDD infection. As stated above, more important than a detailed analysis of viral kinetics during short-term therapy is the impact of LAM maintenance as a component of combination therapy on the subsequent emergence of ADV resistance during long-term therapy. In my opinion, this is a likely justification for the combination LAM/ADV instead of ADV monotherapy for treatment of YMDD infection. Also, the cited studies tell us nothing of the likely differences between LAM/ADV combination and ADV monotherapy given ab initio to treatment-naive patients. Preliminary data presented at last year's AASLD annual meeting imply that, in comparison with monotherapy, the combination may enhance the infected cell loss rate (d), and may effect a greater decline of serum HBV titre during short-term therapy [10].
 
In conclusion, I think that the differences between the population kinetics observed by Mihm and Tsiang probably reflect simple differences between quite small cohorts at baseline, and do not necessarily imply a beneficial effect of LAM on second phase kinetics during ADV treatment of YMDD infection. In this case, the raw data are more informative than the population delta. Nevertheless, other published and presented data (and the paradigm of HIV treatment) make me favour the maintenance of LAM after commencement of ADV for treatment of YMDD. For treatment-naive patients, the combination probably retards the emergence of YMDD species and may delay or prevent the emergence of ADV-resistant virus. Perhaps, trials in progress will show that combination treatment for HBV is superior to monotherapy, and will confirm that the treatment paradigm for HIV patients is relevant to the treatment of our patients with chronic hepatitis B infection.
 
References
 
1. Peters MG, Hann HW, Martin P, Heathcote EJ, Buggisch P, Rubin R, et al.. CL for the GS-00-461 study Group. Adefovir Dipivoxil alone or in combination with lamivudine in patients with lamivudine-resistant chronic hepatitis B. Gastroenterology. 2004;126:91-101.
 
2. Villeneuve JP, Durantel D, Durantel S, Westland C, Xiong S, Brosgart CL, et al.. Selection of a hepatitis B virus strain resistant to adefovir in a liver transplantation patient. J Hepatol. 2003;39:1085-1089.
 
3. Sung JJY, Lai JY, Zeuzem S, Chow WC, Heathcote E, Perrillo R, et al.. A randomised double-blind phase 2 study of lamivudine compared to lamivudine plus Adefovir dipivoxil for treatment-naive patients with chronic hepatitis B: week 52 analysis. J Hepatol. 2003;38:25-26.
 
4. Mihm U, Ga¨rtner BC, Faust D, Hofmann WP, Sarrazin C, Zeuzem S, et al. Viral kinetics in patients with lamivudine-resistant hepatitis B during adefovir-lamivudine combination therapy. J Hepatol. 2005;43:217-224.
 
5. Tsiang M, Rooney JF, Toole JJ, Gibbs CS. Biphasic clearance kinetics of hepatitis B virus from patients during Adefovir Dipivoxil therapy. Hepatology. 1999;29:1863-1869.
 
6. Marcellin P, Chang TT, Lim SG, Tong MJ, Sievert W, Shiffman ML, et al.. Cl for the Adefovir Dipivoxil 437 study group. Adefovir dipivoxil for the treatment of hepatitis B e antigen-positive chronic hepatitis B. N Engl J Med. 2003;348:808-816.
 
7. Hadziyannis SJ, Tassopoulos NT, Heathcote EJ, Chang TT, Kitis G, Rizzetto M, et al.. CL for the Adefovir Dipivoxil 438 Study Group. Adefovir Dipivoxil for the treatment of hepatitis B e antigen-negative chronic hepatitis B. N Engl J Med. 2003;348:800-807.
 
8. Werle B, Cinquin K, Marcellin P, Pol S, Maynard M, Trepo C, et al. Evolution of hepatitis B viral load and viral genome sequence during adefovir dipivoxil therapy. J Viral Hepatitis. 2004;11:74-83.
 
9. Van Bommel F, Wunsche T, Mauss S, Reinke P, Bergk A, Schurmann D, et al.. Comparison of adefovir and tenofovir in the treatment of lamivudine-resistant hepatitis B virus infection. Hepatology. 2004;40:1421-1425.
 
10. Lau G, Cooksley H, Ribeiro RM, Powers KA, Bowden S, Mommeja-Marin H, et al.. Randomised, double-blind study comparing adefovir dipivoxil plus emtricitabine combination therapy versus adefovir alone in HBeAg+chronic hepatitis B: efficacy and mechanisms of treatment response. Hepatology. 2004;40:272A.
 

 
Viral kinetics in patients with lamivudine-resistant hepatitis B during adefovir-lamivudine combination therapy
 
Ulrike Mihma, Barbara Christine Gärtnerb, Dominik Faustc, Wolf Peter Hofmanna, Christoph Sarrazina, Stefan Zeuzema, Eva Herrmannde
 
ABSTRACT
 
Background/Aims: Mathematical analysis of viral kinetics during lamivudine-adefovir combination therapy has not yet been performed in patients with lamivudine-resistant hepatitis B.
 
Methods: In 8 patients with lamivudine-resistant hepatitis B, adefovir dipivoxil (10mg/day) was added to ongoing lamivudine. Viral decay during the first 8 weeks of combination therapy was described by a biphasic model to determine the efficacy: ε, of blocking viral production, the clearance: c, of free virus, and the loss of infected cells: δ.
 
Results: Median ε was 98%, median c was 0.7/day, and median δ was 0.07/day. No significant association was found between viral kinetic and baseline parameters and virologic and biochemical treatment response. When compared with viral kinetic constants reported for higher dose adefovir dipivoxil monotherapy, ε was lower (P=0.026) and δ was higher (P=0.008) in this study whereas c did not differ between both studies.
 
Conclusions: Although a recent study did not show any differences in the reduction of HBV DNA comparing monotherapy with adefovir dipivoxil to adefovir-lamivudine combination therapy in patients with lamivudine-resistant chronic hepatitis B, mathematical analysis of early viral kinetics suggests an additional effect of lamivudine on the infected cell loss during adefovir-lamivudine combination therapy.
 
Discussion
 
The present study is the first to mathematically analyze viral kinetics during combination therapy with adefovir dipivoxil added to ongoing lamivudine in patients with lamivudine-resistant chronic hepatitis B. Viral decline was described convincingly by a biphasic model as used before in patients with hepatitis B and nucleoside/nucleotide analogue therapy [23,26-28,30,33] but also more complex staircase decay profiles had been reported [24]. Motivated from a recent in vitro study suggesting only an incomplete inhibition of de novo infection during therapy with adefovir and lamivudine [34], viral kinetics were modeled with both, the assumption of incomplete (η=0.5) and, as in most previous studies, of complete (η=1) inhibition of de novo infection. However, differences between the resulting curve fits and between estimated individual kinetic constants were negligible.
 
Patients' characteristics at the beginning of combination therapy and viral kinetic constants were investigated for an association with treatment outcome after 3 and 6 months of combination therapy. For treatment with lamivudine in patients with HBeAg chronic hepatitis B, it has previously been reported that high pretreatment ALT levels are favorable with respect to HBeAg seroconversion [38,39]. Different patient races, HBV genotype, and HBeAg status did not lead to differences in HBV DNA decline during therapy with adefovir dipivoxil [40]. In the present study, no association of ALT levels or HBeAg status at the time of addition of adefovir dipivoxil to ongoing lamivudine with treatment outcome was observed. Moreover, no significant association of viral kinetic parameters with treatment outcome after 3 or 6 months of combination therapy was established either. Note that the sample size may not provide enough statistical power to detect underlying differences in the viral kinetic or patients' baseline characteristics with respect to different treatment outcomes. Correlation of higher infected cell loss δ with lower HBV DNA levels at month 3 of combination therapy suggest some associations and the patient with the lowest δ never fell below 1.9×104IU/ml of HBV DNA and later developed resistance to adefovir dipivoxil.
 
In the present study, the viral kinetic constants determined from mathematical analysis of viral decay in the first 8 weeks during combination therapy of adefovir dipivoxil and lamivudine were compared to the viral kinetic parameters reported by Tsiang et al. [30] in 10 patients treated 84 days with adefovir monotherapy. Whereas in the present study adefovir was added to ongoing lamivudine in patients with lamivudine resistance, patients in the study of Tsiang et al. [30] had not received anti-HBV treatment 6 months prior to therapy with adefovir. Furthermore, the patients in the present study were given 10mg adefovir dipivoxil daily, whereas the patients in the study of Tsiang et al. [30] took 30mg adefovir dipivoxil daily. Clearance rates c of free virus were highly comparable in both studies. Efficacies ε of inhibition of new virus production were significantly higher in the study of Tsiang et al. [30] than in the present study and rates of infected cell loss δ were significantly higher in the lamivudine-adefovir combination treatment cohort compared to the patients receiving adefovir monotherapy [30]. Higher efficacies ε of inhibition of new virus production in the study of Tsiang et al. [30] result most likely from the higher daily adefovir dose in this patient group as rising efficacies ε of inhibition of new virus production with rising doses of adefovir were described before [31]. Higher rates δ of infected cell loss in the lamivudine-adefovir combination treatment cohort compared to those in patients receiving adefovir monotherapy are more difficult to understand. Of course it cannot be excluded that the differences in the two patient cohorts contribute to this effect. Nevertheless, patients in the adefovir monotherapy group [30] had a mean baseline HBV DNA level of 8.9×107IU/ml compared to a mean HBV DNA level of 2.3×108IU/ml prior to addition of adefovir dipivoxil to lamivudine in the adefovir-lamivudine combination therapy group, indicating an equivalently high disease activity in both patient groups. Thus, a potentially supposed higher disease activity or a flare due to lamivudine resistance cannot explain the differences in the rates of infected cell loss during the adefovir [30] or adefovir-lamivudine therapy. For both, lamivudine [41-44] and adefovir [45,46], effects on the host immune system have been reported. In chronic hepatitis B, hyporesponsiveness of T cells was demonstrated in vitro [44,47,48] and is believed a major mechanism for viral persistence [49]. At least a transient restoration of anti-viral CD4 and a longer lasting restoration of HBV-specific CD8 reactivity was demonstrated following lamivudine treatment [41-44]. HBV-specific CD8 T cells can eliminate intracellular HBV by destruction of infected hepatocytes as well as by non-cytolytic mechanisms, thereby reducing the number of infected cells [50,51]. Taking this into account, it is tempting to speculate that a restoration or stimulation of the host immune system by lamivudine leads to an enhanced clearance of infected hepatocytes during lamivudine-adefovir combination therapy compared with adefovir monotherapy. Note also that such a hypothesis corresponds well with the observation of significantly faster infected cell loss reported for monotherapy with 600mg lamivudine (n=10, median δ: 0.13/day) compared to monotherapy with 150mg lamivudine (n=11, median δ: 0.09/day), even so that difference lost significance for a multivariate piecewise linear model [28]. Nevertheless, such a trend was also suggested by another recent study [52]. Recently, monotherapy with adefovir dipivoxil (n=19) was compared with adefovir dipivoxil added to ongoing lamivudine (n=20) for 48 weeks in patients with lamivudine-resistant chronic hepatitis B [19]. In that study no differences in HBV DNA suppression after 48 weeks could be observed between the two treatments. Nevertheless, in the present study, results of a different methodological approach—mathematical modeling of viral kinetics—imply that there may be advantages in long-term outcome in patients with lamivudine-adefovir combination therapy compared with adefovir monotherapy due to an additional effect of lamivudine on infected cell loss.
 
In conclusion, the data of the present study strongly suggest further evaluation of possible correlations between infected cell loss δ and treatment response and further investigation of potential benefits of a lamivudine-adefovir combination therapy compared with an adefovir monotherapy for long-term outcome in patients with chronic hepatitis B.
 
Introduction
 
Chronic hepatitis B is a major health problem due to its high prevalence and association with severe liver disease and hepatocellular carcinoma. A recent study reports sustained HBeAg loss 26 weeks after 52-week of peginterferon-α-2b in 36% of patients with HBeAg-positive chronic hepatitis B [1]. For patients with HBeAg-negative chronic hepatitis B sustained HBV DNA suppression below 2×104c/ml 24 weeks after the end of a 48-week treatment was observed in 43% of patients treated with peginterferon-α-2a and in 29% of patients with lamivudine monotherapy [2]. Despite these encouraging results, treatment with (peg)interferon-α is limited by its side effects. By treatment with the nucleoside analogue lamivudine, seroconversion rates of HBeAg-positive patients to anti-HBe from 17 to 47% depending on the treatment duration (1-4 years) can be obtained [3-8]. In HBeAg-negative patients with chronic hepatitis B, treatment with lamivudine leads to suppression of HBV replication in the majority of patients. However, in this patient group, viral relapse is observed in almost all patients after discontinuation of therapy [9]. A major problem with prolonged lamivudine therapy is the increasing rate of viral resistance due to mutations within the HBV polymerase [4,6-8,10]. Treatment with adefovir dipivoxil remains effective in patients with resistance against lamivudine [11-19]. However, recently, resistance against adefovir dipivoxil has been observed as well [20,21], however, without cross-resistance towards lamivudine [20,21].
 
Therefore, the question arises if, in patients with lamivudine resistance, adefovir-lamivudine combination may be superior to adefovir monotherapy by avoiding further liver damage during potential development of adefovir resistance. Furthermore in vitro and in vivo investigations suggest a higher anti-viral potency of nucleos(/t)ide combination therapies compared with monotherapies [22,23]. Nevertheless, no differences in HBV DNA suppression after 48 weeks of monotherapy with adefovir (n=19) and adefovir-lamivudine combination therapy (n=20) in patients with lamivudine resistance have been reported in a recent study [19].
 
Mathematical modeling of viral decay summarizes additional information about the course of disease during anti-viral therapy by determination of individual viral kinetic parameters as the clearance rate c of free virus, a drug's efficiency ε of blocking virus production, and the rate δ of infected cell loss. These viral kinetic parameters can be used, e.g. for efficient comparison of treatment groups even for relatively small sample sizes. Whereas viral kinetic models have been applied to lamivudine [23-29] and adefovir monotherapy [30,31] in patients with chronic hepatitis B, mathematical analysis of adefovir plus lamivudine combination therapy has not been done so far.
 
Therefore, in this study, mathematical viral kinetic analysis was performed in patients with lamivudine-resistant chronic hepatitis B who received adefovir dipivoxil added to ongoing lamivudine. Individual viral kinetic parameters were determined and compared to those previously reported for monotherapy with adefovir dipivoxil [30]. Furthermore, patients' characteristics at the beginning of combination therapy and viral kinetic constants were investigated for an association with treatment outcome.
 
2. Patients and methods
 
2.1. Patients
 
In the present study, viral kinetics were analyzed in 8 patients (Table 1) with lamivudine-resistant chronic hepatitis B. Diagnosis of chronic hepatitis B was based on elevated serum aminotransferase (ALT) levels, the consistent detection of hepatitis B surface antigen (HBsAg) for at least 6 months and positive HBV DNA (Amplicor™ HBV Monitor, Roche Diagnostics, Mannheim, Germany). After an initial response to lamivudine monotherapy, in the course of treatment all patients developed lamivudine-resistant hepatitis B, indicated by a rise in HBV DNA to levels ≥1.9×105IU/ml and ALT levels ≥1.2×upper limit of normal (ULN) despite ongoing lamivudine therapy for at least 6 months and good therapy compliance. Lamivudine resistance was confirmed by detection of drug-induced mutations in the HBV polymerase in all patients (Table 1; INNO-LiPA HBV DR, Innogenetics, Gent, Belgium). All patients tested negative for antibodies to human immunodeficiency virus 1 and 2, hepatitis C and D.
 
Adefovir dipivoxil (10mg/day p.o.) was added to ongoing lamivudine (100-150mg/day p.o.). Patients were treated in the outpatient clinics of the Saarland University and University Hospital in Frankfurt/Main. The study was approved by both local Ethics Committees of Medical Research in accordance with the 1975 Declaration of Helsinki and written informed consent was obtained from each patient prior to enrollment.
 
2.2. HBV DNA measurement and genotyping
 
Serum HBV DNA was measured at baseline and daily in the first week of combination therapy, then weekly in the first 4 weeks and then monthly. HBV DNA in each sample was quantified independently with different assays, the Cobas Amplicor™ HBV Monitor test (lower detection limit: 57IU/ml) and with the Cobas TaqMan™ (lower detection limit: 6IU/ml). Some samples were also quantified with the Amplicor™ HBV Monitor test (lower limit: 190IU/ml; all assays: Roche Diagnostics, Mannheim, Germany. For Cobas TaqMan™ 1IU/ml=5.82c/ml, for Cobas Amplicor™ and Amplicor™ HBV Monitor 1IU/ml=5.26c/ml was used for conversion of c/ml to IU/ml). For mathematical modeling, results of all assays were used in parallel taking into account the correlation of the quantification results for the same sample.
 
Genotyping of HBV was performed by the INNO-LiPA HBV GENOTYPING assay (Table 1; Innogenetics, Gent, Belgium).
 
2.3. Mathematical modeling of viral dynamics
 
Viral decline during the first 8 weeks (including day 58 in two patients with no quantifications at day 56) of lamivudine-adefovir combination therapy was described by a biphasic model as used previously [23,24,26-28,30,32,33]
 

vt-1.gif

where V denotes serum viral load, I productively infected cells, ε the efficiency factor of blocking virus production, p the viral production rate, c the viral clearance rate, η the efficiency factor of blocking de novo infection, β the de novo infection rate, T uninfected target cells, and δ the rate of infected cell loss. Previous works [23,26-28,30,33] assumed a complete block of de novo infection of susceptible cells during anti-viral therapy, resulting in η=1 and
 

vtVo-2.gif

However, a more recent in vitro study suggests an incomplete inhibition of de novo infection during therapy with adefovir and lamivudine [34]. Therefore, besides using the assumption η=1 the assumption η=0.5 was used for comparison (see also Ref. [24]) where the differential equation for the compartment of uninfected target cells was modeled as in [35] in the latter case. Solution of differential equations and non-linear least squares fitting was performed with Matlab software (MathWorks Inc., Natick, MA, USA) and estimates for c, δ, and ε were determined.
 
2.4. Statistics
 
Data were analyzed by Mann-Whitney U-test, Spearman and Pearson correlation, respectively. All tests were two-tailed. P-values less than 0.05 were considered significant.
 
3. Results
 
3.1. Viral kinetic constants
 
In 8 patients with lamivudine-resistant hepatitis B, adefovir dipivoxil was added to ongoing lamivudine. Viral load was monitored daily in the first week of combination treatment, then weekly in the first month and monthly thereafter. The majority of samples was quantified with both, the Cobas Amplicor™ HBV Monitor test and the Cobas TaqMan™. The two assays correlated well with a linear correlation coefficient of 0.971 (n=83). For some samples, quantifications with the Amplicor™ HBV Monitor (correlation coefficient with Cobas Amplicor™ HBV Monitor: r=0.97; n=183; Ref. [36]) were also available and included in the analysis. Serum HBV DNA declined in a similar biphasic pattern in all patients and was modeled well with the proposed mathematical model (Fig. 1).
 
Efficiency of blocking viral production ε, clearance rate c of free virus, and rate for infected cell loss δ were estimated for each patient using serum HBV DNA data of the first 8 weeks of combination therapy (Table 2). The efficiency ε of the combination therapy of adefovir plus lamivudine of blocking viral production ranged from 86.3 to 99.7% with a median of 97.8%. For the clearance rate c of free virus, a median of 0.7/day (range: 0.6-1.1/day) was calculated resulting in a median half-life of free virus of 1 day (range: 0.6-1.2 days). The median rate of infected cell loss δ was 0.07/day (range: 0.01-0.16/day) resulting in a median half-life of infected cells of 9.5 days (range: 4.5-92.7 days). Kinetic constants derived under the alternative assumption η=0.5 were highly similar (Table 2) and the resulting fits did not differ visibly (data not shown).
 
3.2. Association of patient baseline characteristics and viral kinetic constants with treatment outcome
 
After 3 months of combination therapy with adefovir and lamivudine HBV DNA fell below 1.9×104IU/ml (Cobas Amplicor™ HBV Monitor or Amplicor™ HBV Monitor) in 5 of 8 patients. None of the initially five HBeAg-positive patients lost HBeAg at this time. ALT levels had normalized after 3 months of combination therapy in 2 of 8 patients (Table 1).
 
After 6 months of combination treatment 5 of 8 patients showed a virologic treatment response with suppression of HBV DNA below 1.9×104IU/ml (Cobas Amplicor™ HBV Monitor or Amplicor™ HBV Monitor). Two of the five initially HBeAg-positive patients lost HBeAg, one of those became positive for anti-HBe. ALT levels lay within normal ranges in 6 of 8 patients after 6 months of combination therapy (Table 1). On the base of monthly ALT quantifications during the first 6 months of combination therapy, 6 of 8 patients showed a decreasing ALT pattern while 2 patients showed a transient increase of ALT at month 1 of combination therapy [37].
 
No significant association of patient characteristics (gender, age, weight, HBeAg-status, HBV genotype, viral load, ALT, AST, GGT, bilirubin, plasmatic coagulation) at the time of addition of adefovir to lamivudine with treatment response (HBV DNA below 1.9×104IU/ml, HBeAg loss, ALT within normal ranges at months 3 and 6 of combination therapy) was observed.
 
Similarly, no association was observed between viral kinetic parameters and ALT decline pattern or treatment response after 3 or 6 months of anti-viral treatment with adefovir plus lamivudine. Nevertheless, Spearman correlation coefficients of -0.68 and -0.59 (P=0.06 and 0.13) between infected cell loss δ and HBV DNA levels at month 3 and 6 of combination treatment, respectively, suggest a possible association here. Patient 5 received adefovir-lamivudine combination therapy for 20 weeks and was set to adefovir monotherapy thereafter. Interestingly, this patient, who showed the lowest rate δ of infected cell loss and consequently the highest half-life of infected cells (Table 2) never fell below 1.9×104IU/ml HBV DNA during adefovir-lamivudine combination therapy or adefovir monotherapy and developed resistance against adefovir dipivoxil after 9 months of adefovir monotherapy.
 
Viral kinetic constants were additionally examined for an association or correlation with baseline parameters at the time of addition of adefovir. Female patients seemed to show slightly lower clearance rates of free virus than male patients (P=0.046). However, no association or correlation was found between viral kinetic constants ε, c, and δ and patients' characteristics like age, weight, duration of infection or lamivudine pretreatment, HBV viral load, viral genotype or HBeAg status. Parameters of liver inflammation or excretory function did not correlate with viral kinetic constants either, only normal plasmatic coagulation may be associated with higher efficacies ε of blocking viral production compared to impaired plasmatic coagulation (P=0.046).
 
3.3. Comparison of viral kinetic constants in patients with adefovir-lamivudine combination therapy versus adefovir monotherapy
 
The viral kinetic constants ε, c, and δ of this study were compared with those reported by Tsiang et al. [30] for monotherapy with adefovir dipivoxil 30mg/day for 12 weeks in 10 patients who had not received anti-viral therapy within 6 months prior to starting adefovir (Fig. 2). The clearance rates of free virus in the study of Tsiang et al. [30] were highly comparable with those derived here (Fig. 2A). Therapy with 30mg/day adefovir dipivoxil seemed to be more effective in blocking viral production (median 99.6%; range 97.9-99.9%; n=10; Ref. [30]) than combination therapy with 10mg/day adefovir dipivoxil plus 100-150mg/day lamivudine (Fig. 2B). The rate of infected cells loss δ, however, was higher in the patients of this study and consecutively half-life of infected cells was lower than in the patients treated with adefovir monotherapy (δ: median 0.04/day; range 0.02-0.06/day; t1/2: median 17.1 days; range 10.8-30.1 days; n=10; P=0.008; Fig. 2C).
 
 
 
 
 
  icon paper stack View Older Articles   Back to Top   www.natap.org