Introduction

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Pharmacokinetic studies have shown that the concentration-time profile of peginterferon- -2b is marked by rapid absorption, a prolonged concentration peak and delayed elimination; these pharmacokinetic properties result in measurable serum concentrations for several days after administration []. Nevertheless, studies of viral kinetics indicate that administration of peginterferon- -2b does not result in a continuous decline in viral load in patients infected with hepatitis C virus (HCV) genotype 1 [] as is seen with daily administration of standard interferon []. Results of these studies showed a general rebound of viral levels in blood 2-4 days after administration of peginterferon- -2b that seemed to be related to the dose (rebound was greater with a higher dose). Because such a rebound is not typical with daily standard interferon [] or with peginterferon- -2a (40 kDa) [], it appears to be related to the pharmacokinetics of peginterferon- -2b. In fact, the serum concentration of peginterferon- -2b decreases exponentially with almost undetectable levels on days 6 and 7 after dosing []. Thus, the decrease in interferon concentration may result in the rebound of viral levels in blood.

To study the relationship between peginterferon- -2b concentration and viral load, this prospective randomized study was designed to evaluate patients with chronic hepatitis C infected with HCV genotype 1 treated with 1.0  g/kg peginterferon- -2b either once or twice weekly for 4 weeks before starting combination therapy.

Patients

All patients were interferon-naïve, infected with HCV genotype 1, anti-HCV antibody positive and had detectable HCV RNA by PCR, and elevated serum alanine aminotransferase (ALT) levels for at least 6 months. Except in one patient with a coagulation disorder, a liver biopsy was performed within 4 weeks of commencement of study. Necroinflammatory scores and degree of fibrosis were assessed according to the Modified Hepatic Activity Index grading and staging system []. Standard inclusion and exclusion criteria were used []. In particular, patients with decompensated liver disease, coinfection with human immunodeficiency virus or hepatitis B virus, evidence of alcohol abuse, diabetes mellitus requiring insulin therapy, or preexisting psychiatric disorders were excluded from the study.

Study protocol

Before starting peginterferon/ribavirin combination therapy, patients were randomized to receive 1.0  g/kg peginterferon- -2b (PegIntron^®, Essex Pharma, München, Germany) either once weekly (days 0, 7, 14 and 21) or twice weekly (days 0, 3, 7, 10, 14, 17, 21 and 24) for 4 weeks. This dose of peginterferon- -2b was selected because it has been demonstrated to be the most effective in the treatment of HCV when given as monotherapy []. Blood samples to determine HCV RNA levels and peginterferon- -2b concentrations were obtained on days 0, 1, 2, 3, 4, 7, 8, 9, 10, 11, 14, 21 and 28. After completion of the study (day 28) all patients received 180  g/week peginterferon- -2a (40 kDa) (PEGASYS^®, Roche, Basel, CH, Switzerland) in combination with 800 mg daily ribavirin (COPEGUS^®, Roche) for 48 weeks. Treatment was terminated in patients who did not achieve a virological response after 6 months of treatment, in accordance with the European Association for the Study of the Liver consensus conference []. During combination therapy, patients were seen at monthly intervals and a qualitative HCV RNA was measured after 3, 6 and 12 months. The study protocol was approved by the ethics committee of the medical faculty of the University of Vienna. All patients gave signed informed consent. The study was conducted according GCP guidelines and externally monitored by IST GmbH (Mannheim, Germany).

Determinations

Blood was collected in 9 mL tubes (Vacuette, Greiner bio-one GmbH, Kremsmünster, Austria) and placed on ice immediately. Within 30 min after venipuncture, blood was centrifuged at 1500  g at 4 °C for 20 min. Immediately after centrifugation serum aliquots were frozen at -70 °C until further use.

Serum HCV RNA

For quantification of serum HCV RNA, the COBAS AMPLICOR HCV MONITOR^® Test, v2.0 (Roche Diagnostic Systems, Pleasanton, CA, USA) was used according to the manufacturer's package insert. Quantitative limit of detection is between 200-600 IU/mL depending on test performance. Samples with a viral load >850 000 IU/mL were diluted according to the manufacturer's guidelines, but only undiluted values were used for analysis of interferon sensitivity. Qualitative serum HCV RNA was tested by the COBAS AMPLICOR^® HCV test, v2.0 (Roche Diagnostic Systems, the lower level of detection is 50 IU/mL).

HCV genotypes were determined by the Line Probe assay (Innogenetics NV, Zwijnaarde, Belgium).

Peginterferon- -2b concentration in blood

Serum concentrations of peginterferon- -2b were measured by a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) method developed at MDS Laboratories (Wangen, CH, Switzerland). This assay used two distinct mouse monoclonal antihuman interferon antibodies (Li-1 and Li-9) directed towards different epitopes of interferon- -2a. Epitope mapping studies clearly showed that these two antibodies bind to epitopes located on the opposite ends of the interferon- protein and overlap with the binding domain for the interferon receptor.

In a one-step immune reaction, the peginterferon- -2b contained in the sample was bound by the peroxidase-conjugated anti-h-peginterferon- (Li-1). This complex binds via the capture-anti-peginterferon- antibody (Li-9) to the multi-protein layer coated surface of the microtitre plate. Unbound complexes were removed by washing. 3,3',5,5'-tetramethylbenzidine (Fluka, CH, Switzerland) substrate solution was than added, which was converted by the peroxidase to a coloured product that was determined photometrically. Data acquisition was performed by means of an ELISA plate reader (SLT Spectra ELISA Reader; Tecan, Durham, NC, USA). A calibration curve was prepared plotting optical density vs the concentration of the standards. For the standard, a vial from the purchased lot of PegIntron^® was used. The peginterferon- -2b concentration in the sample was then calculated from this standard curve. All calibration standards, quality control samples and study samples were analysed in duplicate. The assay employed has a sensitivity as low as 125 pg/mL of peginterferon- -2b and is linear up to a concentration of 2000 pg/mL. The interassay coefficients of variation of standards and quality control samples never exceeded 15% and the interassay recovery rate of standards and quality control samples does not differ more than 20% from nominal concentrations and expected concentrations, respectively.

The method was validated according to the US Food and Drug Administration [] guidelines and the following results were obtained for inter- and intra-assay accuracy and precision.

Statistical analysis

This exploratory study was powered to detect a 70% difference in viral load at days 7 and 14 between the two groups with a type I error defined as 5% (two-sided) and the type II error set to 20%. Viral load and peginterferon- -2b concentration in the two groups were compared by an unpaired two-sided Student's t-test.

Clinical outcome

Twenty interferon-naïve patients infected with chronic hepatitis C, HCV genotype 1 (1a or 1b) were studied. Demographic and baseline characteristics of the patients are shown in . There was no significant difference between the two groups with respect to ALT, viral load, age, sex, weight, body mass index and histologic grade or stage. All patients completed the study phase according to the protocol. One patient received the second dose of peginterferon- -2b on day 8 instead of day 7 because of public holiday. At the time of writing this manuscript, all 20 patients were still on combination therapy.

Peginterferon- -2b concentration

The maximum blood concentration of peginterferon- -2b was observed 24 h after the first dose (day 0). During the subsequent days there was a linear decline in blood concentrations of peginterferon- -2b. In group A (once-weekly dose of peginterferon- -2b), this decline continued throughout the week (see ). On days 6 and 7 (immediately before administration of the next dose), peginterferon- -2b was below the limit of detection in two and nine of the 10 patients, respectively. The same pattern was observed during the next 3 weeks of therapy. In group B, peginterferon- -2b was detectable at any given time point. On days 4, 7, 9, 10, 11, 14 and 21, the difference in peginterferon- -2b levels was significantly higher in Group B (P between 0.01 and <0.0001).

Viral load

All 20 patients responded to the first dose of peginterferon- -2b by a marked drop in the viral load [day 1: group A: 0.96 ± 0.79 log change from day 0 (range: 0.15-2.73); group B: 1.19 ± 0.74 (range: 0.35-2.56); NS; day 2: group A: 1.13 ± 0.86; Group B: 1.57 ± 0.86 log; NS] (see ). In eight patients (from both groups), the 24-h drop was >0.8 log, indicating they were not primarily 'interferon-resistant' []. However, on day 3 values increased again (group A: 0.71 ± 0.64 log change from day 0; group B: 1.09 ± 0.74 log; P vs day 2 <0.01; group A vs group B: NS). In group A, virus concentration increased further until day 7. A similar pattern was observed in the second week. In group B, viral load decreased on day 4 (24 h after the second injection of peginterferon- -2b). Viral load was significantly different (P  <  0.001) between the two groups at each time point until the end of the kinetic study period (day 28). At day 28, four patients (three of them were HCV RNA-negative) in group A and nine patients (five of them were HCV RNA-negative) in group B had a drop in viral load >1 log (P = 0.057).

Platelet count and neutrophil count

As expected, both platelets and neutrophils decreased on treatment. There was no significant difference between the two groups at any given time point. However, like viral load, both parameters decreased continuously during the first 2 weeks in patients receiving peginterferon- -2b twice weekly but increased from day 3 (and day 10 on in the other group) (see ).

Discussion

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The results of this study reconfirm the previously reported description of peginterferon- -2b pharmacokinetics [] and on-treatment viral kinetics [], but offer a different explanation for the findings. Based on the findings of the present study, once-weekly administration of peginterferon- -2b does not provide continuous exposure to interferon over a period of 144 h. This was shown by large differences in interferon-induced viral decline compared with a group of patients receiving peginterferon- -2b twice weekly. This difference cannot be explained by the doubling of the dose of peginterferon- -2b, as patients receiving even higher doses up to 3  g/kg week-1 had a similar rebound in HCV viral load [].

Early virological response to interferon therapy can be described by two different phases. The initial decline of serum virus concentration is most pronounced after the first dose of interferon administered []. This rapid decline may be because of an inhibition of virus production by hepatocytes [] or to the prevention of the infection of noninfected hepatocytes []. A slower second phase of viral decline occurs between 2 and 14 days of treatment. This phase possibly reflects the death rate of infected cells. Ribavirin had no impact on these two initial phases of virus elimination []. Although the biphasic model of HCV decline following the initiation of treatment has held true regardless of the type of interferon product [], a leveling-off or slight increase in viral levels may occur after the rapid initial drop in HCV RNA levels, [] despite daily doses of interferon. Buti et al. [] noted a similar increase in HCV RNA levels after 72 h with peginterferon- -2b. After a single dose of 3.0  g/kg or 0.5  g/kg peginterferon- -2b, they observed a very rapid and dramatic decrease in HCV RNA levels during the first 48 h, and a second, slower decrease in HCV RNA levels in the ensuing days of the first week. However, in the second half of the week, viral concentration increased overall, and some patients (16%), mostly in the high-dose group, even had a rebound in viral load after the first 2 days of therapy. This type of rebound is rarely observed on daily administration of standard interferon [] or on peginterferon- -2a []. These treatment regimens provide continuous interferon in the blood. Thus, the observation of the present study that viral load increases in parallel with the decrease of peginterferon- -2b clearly indicates that this rebound of HCV RNA is because of insufficient interferon levels present in the body. This rebound may be predictive for nonresponse to peginterferon- -2b/ribavirin therapy [].

It should be noted that Buti et al. [] investigated viral kinetics in patients receiving combination therapy. While the precise role of ribavirin in the treatment of chronic hepatitis C is still unknown, ribavirin has no impact on viral kinetics, at least during the first few weeks of therapy []. In an ongoing multicentre trial in Austria, the impact of ribavirin and the dose of peginterferon- -2b on viral kinetics is being explored. Preliminary data in 60 patients showed no effect of ribavirin on viral disappearance. The increase in viral load in the second part of the week was observed both in patients with or without ribavirin administration [].

The clinical importance of the differences in pharmacokinetics of the two available pegylated interferons is unknown at present. However, there are several lines of evidence suggesting that early viral clearance in patients infected with HCV genotype 1 augments the efficacy of interferon and ribavirin combination therapy. Early viral clearance can be achieved by large doses of interferon and/or daily administration of the drug []. HCV kinetics suggest the consideration of more aggressive dosing regimens in particular in patients infected with HCV genotype 1 []. These data imply the potential importance of using higher interferon- doses in combination with ribavirin for patients infected with HCV genotype 1. If pegylated interferons are used instead of daily standard interferon, their efficacy is certainly influenced by their pharmacokinetic properties. In patients with HCV genotype 1 and low viral load, both pegylated interferons (2a and 2b) in combination with ribavirin were more effective than standard therapy, [] but peginterferon- -2b was not different to standard treatment in patients with HCV genotype 1 and high viral load []. This may be because of the lack of continuous exposure to interferon during therapy. From the current study a difference in efficacy of once- or twice-weekly administration cannot be inferred. The study was not designed and powered to detect such a difference.

In conclusion, the results of our study show that in order to achieve constant blood levels of peginterferon- -2b the drug has to be given twice weekly. Whether twice-weekly dosing will improve the results of combination therapy in patients with HCV genotype 1 has yet to be explored.