Double-blind, randomized controlled trial of interleukin-2 treatment of chronic hepatitis B.

Pilot studies have demonstrated that recombinant interleukin 2 (rIL-2) has an indirect antiviral activity against hepatitis B virus, but the minimal dose of rIL-2 for induction of this effect was not defined. The aim of the study was to ascertain the most efficient dose of rIL-2 for induction of the loss of detectable serum HBV-DNA or a 50% or greater decrease in its level. Thirty-one patients with chronic hepatitis B, hepatitis B e antigen and serum HBV-DNA positive were enrolled in this double-blind randomized controlled trial. Patients were divided: Group I (n = 8) placebo; Group II (n = 7) treated with 0.9 MU of rIL-2 subcutaneously administered daily for 8 weeks; Group III (n = 8) treated with 1.8 MU of rIL-2 under the same schedule; Group IV (n = 8) which received 3.6 MU of rIL-2 under the same conditions. At the end of treatment 25% of the patients in the placebo group, and 13% and 25% in rIL-2 groups III and IV, respectively, had a decrease in HBV-DNA higher than 50% of the basal value. None of the patients lost serum HBV-DNA. Only three patients (one from group II and two from group IV) normalized the ALT levels. Overall, during treatment, ALT levels decreased in the treated groups. This decrease occurred simultaneously with an increase in serum HBV-DNA concentration. Since the response rate in the treated groups was similar to that of the placebo group, rIL-2 is not useful as monotherapy for the treatment of chronic hepatitis B at the doses and schedules used in this study.

Assays for antibodies to human interferon-alpha: the need for standardization.

Since the first reported occurrence of anti-interferon (IFN) antibodies in 1981, the reported incidence of antibody production has differed enormously. In some clinical trials of human IFN preparations, no patients developed antibodies, whereas other studies reported an incidence of more than 80%. In patients with hepatitis C, the reported incidence varies from 7% to 61%. One of the factors contributing to the variability of the results is the lack of a standard assay system to measure antibodies to IFNs. In 1994, a Concerted Action funded by the European Commission started to coordinate studies into the immunogenicity of recombinant DNA-derived pharmaceuticals. These studies aimed to examine whether antibodies could interfere with the efficacy of treatment and also studied the long-term effects on cytokines produced by the patients themselves. Only when a well-calibrated and standardized assay is available, however, will it be possible to define the biologically relevant titer of antibody. Assays for both binding and neutralizing antibodies are discussed here.

Clinical investigation of the immunogenicity of interferon-alpha 2a.

The following confounding array of variables can affect the reporting of antibodies to interferon (IFN)-alpha or any other protein injected into humans for prophylactic or therapeutic purposes: route, dose, frequency, and duration of administration; timing and frequency of blood sampling; methods used to determine antibody presence (sensitivity); calculation of the units used to report the results; intrinsic immunogenicity of the protein product; major histocompatibility complex genotype of the challenged individual; associated diseases and medication. Without specification of all these factors, it is difficult to put forward a valid statement from the published literature regarding the comparative incidence of antibodies to various forms of IFN. Furthermore, because units are not standardized and rarely reported, it becomes impossible to make any comparisons of antibody titers. This evaluation confirms, by clinical trial results, the decrease in immunogenicity observed in vitro and in vivo of different IFN-alpha 2a products manufactured between 1989 and 1993 following incremental improvements in process specifications and expanded quality control. Serial serum samples were taken, prepared, stored, and shipped according to identical protocols in five different clinical trials performed with IFN-alpha 2a between 1989 and 1995 in comparable patient populations with chronic hepatitis C. The coded samples were screened using sensitive enzyme immunoassay (EIA). Positive samples were further analyzed and quantified by bioassay [antiviral inhibition assay (AVIA)]. The data from these clinical trials confirm the results from extensive preclinical testing. Refrigerated lyophilisate and a new human serum albumin (HSA)-free formulation of IFN-alpha 2a, produced according to the latest process specification, are less immunogenic than earlier products.

Treatment of chronic hepatitis C with interferon: how long should a lasting response last if a lasting response could last?

In chronic hepatitis C infection to document spontaneous remission or response to treatment, more than one parameter is required. Both the activity of liver disease and of viral replication need to be recorded. The former is most accurately done by liver biopsy. The number of such biopsies which a patient or physician are willing to have performed is necessarily limited, such that serum-ALT has traditionally been used as surrogate marker. Viral replication can be quantified using a standardized and validated polymerase chain reaction (PCR) test system. Drawing on a database of over 2300 patients with documented chronic hepatitis C for whom data were recorded longitudinally for up to 5 years, a proposal is presented on how to document more precisely quality of response and its duration. Using both biochemical and virological parameters quantified repeatedly at regular intervals, three response patterns can be described, and less frequent histological assessments are used for confirmation: non-response, where no relevant changes are seen in either parameter; transient response that consists of a response induction, usually biochemical in nature, accompanied by a transient decrease in viral replication only. This response is subsequently lost either during (breakthrough) or after the end of treatment (relapse); finally, a durable response is defined by a rapid return to normal or fall to undetectable levels, as a rule within 2 weeks to 3 months, of serum ALT and hepatitis C virus (HCV) RNA that is sustained until the end of the follow-up period (up to 5 years).

Usefulness of interferon for treatment of hepatitis C.

The integrated results of a worldwide clinical research program studying interferon-alpha 2a (IFN-alpha 2a) (Roferon-A, F. Hoffmann-La Roche Ltd, Basel, Switzerland) for the treatment of chronic hepatitis C in 1831 patients are reviewed in this paper. According to a multivariate analysis of the data available from this extensive program, which studied fixed, escalating, and deescalating dose regimens of 1, 3, 4.5, and 6 million international units (MIU) for 3, 6 and 12 months in 10 clinical trials, the best response rates were obtained with an induction dose of 6 MIU given thrice weekly for 3 months, followed, in responding patients, by a maintenance regimen of 3 MIU thrice weekly for an additional 3 months. Before the decision to treat is taken, a careful assessment of individual patients' clinical history and prognosis based on stage and activity of their chronic hepatitis and associated other diseases must be made and weighed against the statistical odds of treatment success. Liver histology and repeated measurements of biochemical markers of liver disease such as alanine transpeptidase (ALT) are classical indicators. More recently, quantitative assessment of viremia has become possible with the polymerase chain reaction (PCR) to measure the number of viral genomes per milliliter of serum. After the decision to treat, monthly monitoring of those markers gives a dynamic picture of the therapeutic effect. Depending on the course observed, the indication for the treatment must be reevaluated after induction therapy. Responding patients should receive an additional 3 months of maintenance therapy. If a patient shows no satisfactory evidence of response, treatment must be discontinued.

The fluctuations of hepatitis C virus RNA and IgM anti-HCV (core) serum levels correlate with those of alanine aminotransferases during the hepatitis relapses of patients treated with interferon.

Variations in the serum levels of hepatitis C virus (HCV) RNA. IgM antibody against the HCV 'core' structural protein (c22) and alanine amino-transferase (ALT) were measured in 23 patients with chronic hepatitis C who underwent therapy with interferon-alpha 2a (IFN alpha 2a). Low pretreatment levels of viraemia and undetectable IgM anti-core were significantly associated with a long-term response to treatment. In patients with hepatitis relapses after the end of treatment, HCV RNA levels increased before or at the same time as ALT in 29 out of 34 cases (85%). ALT flares occurred before or simultaneously with IgM anti-core elevations in 18 out of 20 cases (90%). Therefore, post-treatment hepatitis C exacerbations show the same sequence of events seen as in hepatitis B exacerbations (increases of viraemia followed by those of ALT and IgM anti-'core'). These findings underscore the diagnostic and prognostic usefulness of monitoring anti-HCV-positive patients with quantitative assays for HCV markers.

Comparative study of three doses of interferon-alpha 2a in chronic active hepatitis B. The International Hepatitis Trial Group.

To determine the efficacy of interferon-alpha 2a in chronic active hepatitis B, 238 patients were randomly divided, into four groups: three groups received either 2.5 MIU m-2, 5.0 MIU m-2 or 10.0 MIU m-2, three times weekly by intramuscular injection for 12-24 weeks; and a control group received no treatment. Patients were followed for up to 12 months after treatment was discontinued. There was a statistically significant difference in response [clearance of hepatitis B e antigen (HBeAg) and hepatitis B viral DNA (HBV-DNA)] between treated and untreated patients (37 vs 13%) but no statistically significant difference was seen between treatment groups (33%, 34% and 43% for the 2.5, 5.0 and 10.0 MIU m-2 groups, respectively). A transient rise in transaminases (seroconversion hepatitis) was seen in responders, but levels returned to within the normal range after response to treatment. In patients responding to interferon therapy there was a significant reduction in the severity of the hepatitis. Interferon-alpha 2a was generally well tolerated with respect to vital signs and laboratory parameters.

To treat or not to treat? The judicious use of interferon-alpha-2a for the treatment of chronic hepatitis B.

Based on results from extensive clinical research, interferon-alpha-2a (IFN-alpha-2a, Roferon-A, F. Hoffmann-LaRoche Ltd., Switzerland) and other interferons have been registered for the treatment of chronic active hepatitis B. The officially recommended dose regimen is 4.5 MIU (or 2.5 MIU/m2) thrice weekly for 6 months. To present guidelines for the optimization of treatment for individual patients, 3 major controlled trials from our worldwide research program with a total of 416 patients were reviewed in a meta-analysis. Before deciding whether to treat or not, the history, prognosis and chances of treatment success for a given patient must be carefully assessed. Liver histology and repeated quantitative measurements of markers for viral replication (HBV-DNA, HBeAg) and biochemical markers for liver disease such as ALT are valuable indicators. After the decision to treat, monthly quantitative measurements of these markers make it possible to monitor therapeutic success. Depending on the course they run, treatment can continue unchanged, be adjusted in dose or duration until a full response is achieved, or be terminated early in case of evidence of non-response.

Treatment of chronic hepatitis C by continuous subcutaneous infusion of interferon-alpha.

The effectiveness of a daily continuous infusion of interferon-alpha was evaluated in 12 patients (10 males, 2 females; mean age of 33 years, range 19-62) with biopsy-proven chronic active hepatitis C. Nine million units (MU) of recombinant interferon-alpha 2A (rIFN-alpha 2A) were administered by continuous subcutaneous infusion with a portable syringe pump, Graseby model MS 16A, for 24 h over 28 days. A significant decrease (P less than 0.01) in median serum alanine aminotransferase (ALT) levels was observed after the first week of treatment (96 IU/L, range 58-263) with respect to the pre-treatment values (188 IU/L, range 119-670). ALT became normal in four patients only by the fourth week. When IFN was interrupted, an increase in ALT was observed in all patients (1.5 to 5 times the pre-treatment values). The maximum decrease in ALT coincided with a significant increase in serum levels of the enzyme 2',5'-oligoadenylate (2-5A) synthetase (two to fourteen times the pretreatment values) and these parameters were inverse-correlated (r = -0.598, P less than 0.05). 2-5A synthetase levels returned to pre-treatment values after discontinuing IFN administration. Hepatitis C virus (HCV) RNA (as detected by the polymerase chain reaction using oligonucleotide primers of the NS5 region) was positive in all cases, remaining so during the treatment period. IgM antibody to HCV (as tested by ELISA) was present in 10/12 cases at baseline without changes throughout the study. No irreversible side effects were noted during therapy, which needed to modify the schedule.(ABSTRACT TRUNCATED AT 250 WORDS)

Limited protective effect of rough mutant antisera in murine Escherichia coli bacteremia.

Previous studies in mice have demonstrated differing immunoprophylactic activity of antisera against rough mutants of Enterobacteriaceae in the prevention of lethal gram-negative bacteremia. In this study, in which CF1 mice were made bacteremic with a serum-resistant Escherichia coli 06:K2:H1, the composite survival was significantly (p less than 0.001) enhanced by i. v. pre-treatment one to two hours before injection with either normal rabbit sera or antisera to the J5 mutant of E. coli 0111. The protective efficacy of these preimmune and hyperimmune sera did not differ significantly. Since considerable variability in the mortality of control mice occurred in the 25 separate experiments, the results of individual experiments were grouped retrospectively according to survival in the individual control groups and compared for evidence of possible differences in the efficacy of these two sera. With the exception of a statistically significant difference in the efficacy in one group receiving an LD75-95 inoculum, no such differences were noted. Thus, the variable effects of a rough mutant antiserum were not explained by differences in the relative virulence in the inoculum. This study confirms earlier observations by others that the protective efficacy of the anti-J5 antisera in infected mice does not differ appreciably from that of normal rabbit sera, provided the same donor rabbits are the source of both preimmune and hyperimmune sera.

In vitro and in vivo studies of three antibiotic combinations against gram-negative bacteria and Staphylococcus aureus.

The activities of azlocillin, cefotaxime, and amikacin alone and in combination were evaluated in in vitro checkerboard studies, in infected neutropenic mice, and in human volunteers. The combination of cefotaxime plus amikacin was more synergistic in vitro than the others against the Enterobacteriaceae tested, and the combination of azlocillin plus amikacin was more synergistic against Pseudomonas aeruginosa and Staphylococcus aureus. Survival of neutropenic mice infected with Escherichia coli and Klebsiella pneumoniae, respectively, was greater with azlocillin plus amikacin (24 of 40 and 11 of 40) and with cefotaxime plus amikacin (21 of 40 and 17 of 40) than with azlocillin plus cefotaxime (22 of 40 and 3 of 40; P less than 0.05). Median serum bactericidal activity in volunteers receiving these antibiotics alone and in combination was greater than or equal to 1:8 with most agents and with all combinations tested against 10 strains each of E. coli, K. pneumoniae, P. aeruginosa, and S. aureus. These data suggest that clinical trials with combinations of azlocillin or cefotaxime plus amikacin deserve further study in febrile neutropenic patients.