INTRODUCTION

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More than 350 million individuals around the world, or approximately 5% of the world's population, have chronic hepatitis B virus (HBV) infection (20). In areas where HBV is endemic, it is most frequently transmitted vertically from the mother to her newborn or between close contacts during early childhood. After acute infection, the virus is eliminated in 90 to 95% of infected adults, while in the remaining 5 to 10% a chronic infection persists (19). The progression to chronic hepatitis after an acute infection is inversely related to age. Infants and young children who acquire HBV infection are most at risk for chronic infection and consequently are at highest risk for eventual cirrhosis and primary hepatocellular carcinoma (15).

Alpha interferon has recently been approved for use in the treatment of chronic hepatitis B in children. However, in children, as in adults, two-thirds to three-quarters of the patients do not respond to treatment, stressing the need for alternative and/or more effective treatments (4, 21, 22). In addition, the need for repeated and prolonged parenteral administration, as well as the numerous side effects of interferon, is an important obstacle to treatment acceptance and compliance.

Lamivudine, ()--2',3'-dideoxy-3'-thiacytidine (also known as 3TC and GR109714X), is an oral nucleoside analogue that inhibits viral DNA replication and that has in vitro values for inhibiting viral replication by 50% (IC₅₀) and 90% (IC₉₀) in hepatoma cell lines of about 0.02 µM (5 ng/mL) and 0.3 µM (70 ng/ml), respectively (data on file). Studies with adults have shown that 52 weeks of lamivudine treatment resulted in a significantly higher histologic response rate compared to that achieved with placebo (P < 0.001) and a significant reduction in the progression of fibrosis compared to that achieved with placebo (P = 0.01) (12). In two placebo-controlled studies lamivudine was associated with a significantly higher rate of hepatitis B e antigen (HBeAg) seroconversion (loss of HBeAg and production of anti-HBe antibody), sustained HBV DNA suppression, and sustained normalization of alanine aminotransferase (ALT) levels compared to those achieved with placebo (12; J. Dienstag, E. Schiff, T. Wright, R. Perillo, H.-W. Hann, L. Crowther, M. Woessner, M. Rubin, and N. Brown, Gastroenterology 114:A1235, 1998, abstr. L0148). HBeAg seroconversion in lamivudine-treated patients occurred at a rate similar to that in patients receiving alpha interferon and appeared to be durable for at least 2 to 12 months posttreatment (Dienstag et al., abstr. L0148). Lamivudine is approved for use in human immunodeficiency virus (HIV)-infected children and adults, as well as HBV-infected adults in some countries.

Pharmacokinetic studies conducted with HIV- or HBV-infected patients and healthy volunteers have shown that lamivudine is extensively and rapidly absorbed and exhibits linear kinetics. Renal impairment requires dose modification when creatinine clearance (CL_CR) falls below 50 ml/min, whereas hepatic impairment and transplantation have no significant impact on the level of lamivudine exposure (6-9, 16, 18, 24, 25). A previous pharmacokinetic study with HIV-infected children (13, 17) showed that lamivudine was similarly well absorbed but that the apparent bioavailability was lower and the clearance was higher relative to those for adults. Consequently, the recommended pediatric dose of lamivudine for treatment of HIV infection is twice the adult dose, in milligram-per-kilogram terms.

The study described here was carried out to evaluate the safety profile, antiviral activity, and pharmacokinetics of lamivudine over 4 weeks in order to establish the most appropriate dose for children with chronic hepatitis B.

	MATERIALS AND METHODS

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Patients. Eligible patients included males and females (ages, 2 to 17 years) with detectable hepatitis B surface antigen (HBsAg) in the serum at screening and for 6 months before screening, HBeAg and HBV DNA (determined by the Chiron Quantiplex assay) in the serum at the time of screening, and serum ALT and aspartate aminotransferase (AST) levels below 300 IU/liter. Patients were excluded if they had hepatitis C virus or HIV infection or decompensated liver disease (defined by a serum bilirubin level more than twice the upper limit of the reference range, a serum albumin level less than 32 g/liter, or a history of ascites, variceal hemorrhage, or hepatic encephalopathy). Patients were also excluded if they had received an investigational drug within 30 days before enrollment in the study, any antiviral therapy, biologic modifiers, immunomodulators, or systemic corticosteroids within 6 months of screening.

Study design. Patients aged 2 to 12 years were centrally randomized to receive one of the following doses of lamivudine oral solution: 0.35 mg/kg of body weight twice daily (b.d.), 3 mg/kg once daily (o.d.), 1.5 mg/kg b.d., or 4 mg/kg b.d. The 13- to 17-year-olds all received 100-mg lamivudine tablets orally o.d. Patients were instructed to avoid fatty foods and dairy products on the mornings of days 1 and 28. They were permitted a light breakfast but were instructed to refrain totally from food and drink for 1 h before dosing. They were also asked to refrain from fluids for 1 h after dosing and from food for 4 h after dosing. The patients were stratified into the age groups 2 to 6 years (stratum 1), 7 to 12 years (stratum 2), and 13 to 17 years (stratum 3) to ensure the inclusion of adequate numbers of subjects in each dose group within each age group. The aim was to recruit into the study a total of 50 patients from four centers.

Drug assay and pharmacokinetic evaluations. Samples of blood were collected and processed to provide serum for lamivudine assays. Urine was also collected and was analyzed for lamivudine concentrations but was collected only from the individuals in the group receiving the highest dose (4 mg/kg b.d.). A single group was chosen, as collection of urine from small children is notoriously difficult and the 4-mg/kg b.d. group would be the group most likely to yield practically useful results because large enough amounts of drug would be given to quantify the amount excreted. The assay with serum used at Glaxo Wellcome, , N.C., was a modification of the published high-pressure liquid chromatography assay (5) and used a gas chromatography system with tandem mass spectrometry detection validated to good clinical practice guidelines. The assay with urine was a straightforward application of the high-pressure liquid chromatography method in which direct injection following solid-phase extraction on Bond-Elut cartridge was used. The lower limits of detection were 2 to 5 ng/ml for serum and 1 µg/ml for urine.

Clinical evaluations. Serum was assayed for HBV DNA, HBeAg and anti-HBe antibody, HBsAg and anti-HBs antibody, and ALT levels at each clinic visit. Hematology and biochemistry tests were also performed to determine the safety of the treatment, and the adverse events that had occurred since the previous visit were recorded.

Assays for HBV DNA. The samples were assayed for HBV DNA at the Public Health Laboratory Service, Heartlands Hospital, Birmingham, United Kingdom. The HBV DNA in serum was quantified by the Quantiplex HBV DNA assay (Chiron Corporation, Emeryville, Calif.) (10), which has a lower limit of detection of 2.5 pg/ml and a 25% interassay coefficient of variation. The day 1 and day 28 samples were also assayed by the Roche Amplicor PCR assay (3), which has a lower limit of detection of 400 copies/ml and a 7 to 13% interassay coefficient of variation. HBeAg and anti-HBe antibody were assessed semiquantitatively and qualitatively by the Amerlite Assay (Ortho Clinical Diagnostics, Amersham, United Kingdom). HBsAg was assessed quantitatively by the Amerlite assay (Ortho Clinical Diagnostics), and anti-HBs antibody was assessed quantitatively by the Amerlite Anti-HBs assay (Ortho Clinical Diagnostics).

Statistical analysis. No statistical comparisons of the efficacy or safety parameters were made in this study because of the small numbers of patients. The modified intent-to-treat population (ITTm) was used for the efficacy analyses and was defined as all patients with confirmed chronic hepatitis B who were randomized. The as-treated population was used for the safety analyses and was defined as all patients for whom no clear evidence of failure to take study medication was available.

	RESULTS

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Study population. The study was conducted between June 1997 and January 1998. A total of 58 patients were screened for entry into the study, and of these, 53 patients were considered to meet the study criteria and were randomized at the baseline to a study treatment (as-treated population). The ITTm population comprised 52 patients because 1 patient did not have evidence of HBsAg for 6 months before enrollment. These 52 patients were stratified by age, with 17 patients in stratum 1, 23 patients in stratum 2 and 12 patients in stratum 3.

Pharmacokinetics. All doses of lamivudine, whether given by solution or tablet, showed rapid absorption, with t_max values being, on average, 0.5 to 1 h (Fig. 1). Steady-state parameter estimates showed a small degree of accumulation (AUC and C_max values on day 28 were generally a little higher than those on day 1). The data were generally consistent with profiles seen in adults, although CL/F is increased in pediatric patients relative to that in adults. This function between lamivudine CL/F and age can be seen in Fig. 2. Pharmacokinetic parameter estimates for the different dose and age groups are shown in Tables 2 and 3.

View larger version (14K): [in this window] [in a new window]   FIG. 1.   Steady-state median serum lamivudine concentration-time profiles following the administration of different oral doses of lamivudine. Lamivudine was given as a solution to provide doses of 0.35 mg/kg b.d. (), 1.5 mg/kg b.d. (), 3 mg/kg o.d. (), and 4 mg/kg b.d. () and as a 100-mg tablet (). Note that sampling was continued to 24 h for the o.d. dose cohorts only.

View larger version (11K): [in this window] [in a new window]   FIG. 2.   Relationship between lamivudine clearance and age. CL/F values are for adults () and pediatric or adolescent () patients.

HBV DNA levels. By week 2 all treatments showed considerable antiviral activity, and there was some differentiation of activity by dose, as indicated by rapid falls in median HBV DNA levels. Median percent reductions in HBV DNA levels of at least 99.1 and 99.5% were seen at week 4 (Fig. 3). At week 4, the number of patients that became HBV DNA negative varied across the treatment groups, ranging from 17% (2 of 12) in the 100-mg group to 64% (7 of 11) in the 4-mg/kg b.d. group. By week 8 (4 weeks posttreatment) all patients had again become HBV DNA positive.

View larger version (14K): [in this window] [in a new window]   FIG. 3.   Mean change from baseline in log10 HBV DNA level (in picograms per milliliter) obtained by the Chiron Quantiplex assay for the ITTm population. Lamivudine was given as a solution to provide doses of 0.35 mg/kg b.d. (), 1.5 mg/kg b.d. (), 3 mg/kg o.d. (), and 4 mg/kg b.d. () and as a 100-mg tablet ().

Pharmacokinetics and pharmacodynamics. The relationship between dose, systemic exposure, and antiviral response, assessed as the log₁₀ change in HBV DNA levels measured by the Chiron Quantiplex assay, is shown in Fig. 4. Although some of the samples became negative for HBV DNA by the Chiron Quantiplex assay, outstretching the limits of the assay, this response was also evaluated by a PCR assay, by which a similar functional relationship between dose and response could be seen (Fig. 5). In both Fig. 4 and Fig. 5 it is apparent that beyond a systemic exposure (AUC) of about 4,000 ng · h/ml, assuming that trough levels (i.e., the concentration just before the next dose) are well above the IC₅₀ (4 to 7 ng/ml), there is no appreciable difference in response.

View larger version (14K): [in this window] [in a new window]   FIG. 4.   Pharmacokinetics and pharmacodynamics of lamivudine in pediatric patients (ages 2 to 12 years) as steady-state exposure versus HBV DNA suppression (as determined by the Chiron Quantiplex assay). All pediatric data are by dose group, with group means and standard deviations given. Lamivudine was given as a solution to provide doses of 0.35 mg/kg b.d. (), 1.5 mg/kg b.d. (), 3 mg/kg o.d. (), and 4 mg/kg b.d. ().

View larger version (13K): [in this window] [in a new window]   FIG. 5.   Pharmacokinetics and pharmacodynamics of lamivudine as steady-state exposure versus HBV DNA suppression (as determined by the Roche PCR assay). All data are by dose group. Lamivudine was given as a solution to provide doses of 0.35 mg/kg b.d. (), 1.5 mg/kg b.d. (), 3 mg/kg o.d. (), and 4 mg/kg b.d. (). A 100-mg dose (tablet) was given to adolescents (). The line of best fit (y = 0.13lnx + 1.5). is also shown.

Safety. The number of patients experiencing adverse events was similar across all five treatment groups (36 of 53 [68%] overall). The 2- to 6-year-olds receiving 4 mg/kg b.d. all had at least one adverse event. The percentage of patients experiencing adverse events categorized as drug related was much lower, with an overall incidence of 21% (11 of 53). No patient in the 100-mg group (13- to 17-year-olds) had an adverse event considered by the investigators to be drug related.

	DISCUSSION

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Lamivudine is a well-established part of combination therapy for HIV (as Epivir) and has been extensively studied in adults with HBV infection. This study has shown that similar antiviral efficacy can be seen in pediatric and adolescent patients and that modification of the daily adult dose is required, as for the treatment of HIV infection, to achieve similar levels of systemic exposure in these patients.

All doses of lamivudine, whether given by solution or tablet, showed rapid absorption, with most pharmacokinetic parameter values generally in line with those from previous studies with either adults, adolescents, or children. When broken out by age (Table 3) most of the pharmacokinetic parameters are broadly similar; the exception is CL/F. A small difference in exposure (AUC) between the pediatric population in this and a previous study with HIV-infected children who received the same dose level (13) was seen: Lewis et al. (13) found values of 4,457 ng · h/ml (AUC from the first dose) following the administration of 4-mg/kg b.d. doses. In the present study a comparable dose provided values of 6,540 ng · h/ml. The AUC values in the study of Lewis et al. (13) are predicted from data obtained after administration of a single dose, which probably underestimates the steady-state AUC by about 30%, as their estimate of t_1/2, 1.85 h, is truncated by more limited sampling (see below in Discussion). The AUC values found in the present study are still slightly higher than expected, probably reflecting the difference in disease state and potential effects on absorption, since the HIV-infected children in the study of Lewis et al. (13) had advanced HIV disease and may have absorbed less lamivudine.

The extent of exposure (AUC) was generally proportional to the dose, as seen in previous studies with both adults and (HIV-infected) children.

CL/F estimates were similar to those seen in previous studies (9) and, when stratified by age, demonstrate the same relationship as that seen in HIV-infected patients. CL/F values are at a peak at 2 years of age and decline with age to about 12 years of age, when the values are similar to those seen in adults. This is mirrored by a slightly shorter t_1/2 (Table 3), is independent of disease (HBV or HIV infection), and is consistent with the ratio of organ clearance function and body size, which decreases with age (i.e., organ function increases less rapidly than body size as the child matures) (11). The increase in CL/F observed in children is consistent with these changes and has been observed with many compounds with pharmacokinetic profiles similar to those of lamivudine (2, 23).

The majority of renally cleared lamivudine (44 to 72%) was excreted within 12 h, although a small underestimation of CL_R is possible, as lamivudine was present in the final collection. CL_R values, although they displayed a high degree of variability, were similar to those seen in previous studies in which urine was also analyzed for the primary metabolite, lamivudine sulfoxide (8). This metabolite is present in small quantities (5 to 10%) in urine from adult patients with healthy renal function, but the quantities increase in the urine of patients with significant renal dysfunction (7). Some of the unaccounted for drug material in this study is likely to be present in urine as the sulfoxide metabolite, but this was not ascertained.

In HBV-infected adults, 100 mg of lamivudine (i.e., about 1.4 mg/kg, based on an average 70-kg adult) provides an average AUC at steady state of 4,400 ng · h/ml, which correlates with maximal viral suppression (9). In this study estimates of AUC for the 3-mg/kg/day dose groups were comparable to those for adults. This demonstrates that, as seen in HIV-infected patients, the adult dose (in milligram-per-kilogram terms) should be doubled for children to achieve exposures comparable to those achieved in adults.

A small degree of accumulation (R, ~ 1.2) caused steady-state values to be slightly higher than those on day 1 for AUC and C_max. This degree of accumulation is expected on the basis of t_1/2 values of 6 to 8 h, which was seen in the o.d. dose groups when sampling was continued to 24 h postdosing. The shorter t_1/2 of about 2 h was seen in all groups when sampling was to 12 h postdosing and reflects an underestimate because of the use of points likely to be in the distribution phase to establish the terminal slope. This is consistent with previous data, in which sampling to 12 h generally yielded t_1/2 estimates of 2 h for children (13) and 3 to 4 h for adults (19).

Trough values at steady-state, assessed at day 14 and day 28, showed that for all doses detectable lamivudine concentrations were present in serum. This means that trough concentrations can be maintained at the required level, i.e., above 4 to 7 ng/ml (equivalent to the in vitro IC₅₀) with o.d. dosing. The long t_1/2 of the triphosphate moiety established in vitro (17 to 19 hours [9]) contributes to the duration of the effect and the lack of an observed difference between o.d. and b.d. dosing intervals.

Lamivudine treatment results in a rapid decline in HBV DNA levels in children at all doses studied. The lowest dose used in this study was 0.35 mg/kg b.d., which was by practical necessity when using a 5-mg/ml solution. This equates in exposure (following the doubling of a dose to account for the increased clearance in children) to a 20- to 25-mg dose for adults. At this dose over 90% suppression is seen in adults, and therefore, large differences in response between the doses in this study were not expected or seen. However, plotting of exposure (AUC) against log reduction from the baseline HBV DNA level (Fig. 4) revealed a slightly lower response (~2.4 log₁₀) in the 0.35-mg/kg b.d. group, whereas the effects were equally maximal (~3 log₁₀) in the 1.5- and or 4-mg/kg b.d. groups. At the lowest measured AUC only a 1-log reduction was seen. Once AUC values are 4,000 ng · h/ml or more, i.e., those achieved with doses of 3 mg/kg/day or more, little difference in viral response is observed by the Chiron Quantiplex assay. However, the lower limit of detection (2.5 pg/ml) of the Chiron Quantiplex assay was reached for some of the patients, with the lowest number (n = 2) becoming undetectable by the Chiron Quantiplex assay for the 0.35-mg/kg group, i.e., the lowest-dose group. Consequently, confirmation of the lack of differentiation of activity by dose and the overall magnitude of the drop in log₁₀ achievable at 4 weeks by Roche Amplicor PCR analysis of the same samples is fundamental in understanding the dose-response.

The 100-mg o.d. group (the 13- to 17-year-olds) did not appear to respond as well as the younger groups in this study, with a median drop of 2.1 log₁₀ (range, 1.6 to 3.6 log₁₀). This magnitude of drop has, however, been seen in studies with adults, in whom changes of 2 to 3 log₁₀ at 4 weeks are commonplace (12). The reasons for the difference between the groups in this study are unknown but could likely be due to the variability caused by the small numbers of patients in the study. If it is a true difference, then physiological factors such as hormonal change and immunological status may have had some influence. Compliance may also have been an issue in the group of adolescent patients in the present study, as the younger children were more likely to have been supervised while taking their medication. However, no firm evidence, such as from the tablet count, of this was seen. Initial (baseline) HBV DNA levels were also slightly lower in the adolescent group, which would mean that they had the potential for only a 2 to 2.5 log₁₀ reduction before the limit of detection of the assay was reached.

The effect of lamivudine has not previously been studied in children with HBV infection, so it has not been established whether HBV DNA suppression will correlate to liver histology improvements in the long term. However, studies with adults (12) have shown an association between HBV DNA suppression and improvements in liver histology. Histological abnormalities are usually less severe in children than in adults, probably because of the shorter duration of the disease, the fact that the children may be in an immunotolerant phase, and the absence of superimposed causes of liver damage such as alcohol consumption. On the basis of studies with adults it can be expected that lamivudine will prevent histological progression in children with chronic hepatitis B, active viral replication, and chronic cytolysis. Rapid increases in HBV DNA levels to the baseline levels were seen after the cessation of treatment; this was expected on the basis of results from short-term treatment studies with adults. This increase in HBV DNA levels was not associated with a rise in serum ALT levels with the short, 4-week treatment period, which is also not sufficient to have any effect on HBeAg or HBsAg levels or on seroconversion. It is probable that a longer treatment period would have an impact on HBeAg loss and seroconversion, as seen in adults (12; Dienstag et al., abstr. L0148). Longer-term studies with lamivudine in this age group will also need to address whether rises in ALT levels are seen after the cessation of long-term treatment. A higher proportion of lamivudine-treated adult patients than placebo-treated adult patients have ALT elevations after the cessation of treatment, but this has not been associated with an increased risk of decompensation (N. Leung, J. Deinstag, E. Schiff, M. Sullivan, M. Atkins, R. Grice, M. Woessner, N. Brown, and C. M. Hunt, Hepatology 28:587A, 1998, abstract 1698). Isolated reports of posttreatment flares have, however, been reported (P. Honkoop, R. A. de Man, R. Heijtink, and S. W. Schalm, Letter, Lancet, 346:1156-1157, 1995) but not usually in patients who have seroconverted. In addition, future studies will need to investigate the development of YMDD variant virus in this age group. Among adults who have been studied to date, after 52 weeks of treatment 16 to 32% developed YMDD mutant HBV, which was usually first detected after week 36 M. Atkins, C. M. Hunt, and N. Brown, Hepatology 28:319A, 1998, abstr. 625). These patients nonetheless exhibited continued suppression of HBV DNA (median decrease, 80%) and significantly improved sustained ALT normalization and histologic responses compared to those for the placebo-treated controls.

Lamivudine has previously been shown to be well tolerated by adults, and this study has also shown that a short-term course of lamivudine is well tolerated by children and adolescents infected with HBV. A daily dose of 3 mg/kg is suitable for pediatric patients aged 2 to 12 years.