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Pegylated interferon (PEG-IFN) has become standard therapy for hepatitis C virus (HCV) infection. We evaluated whether PEG-IFN pharmacodynamics and pharmacokinetics account for differences in treatment outcome and whether these parameters might be predictors of therapeutic outcome. Twenty-four IFN-naïve, HCV/human immunodeficiency virus-coinfected patients received PEG-IFN ␣-2b (1.5 g/kg) once weekly plus daily ribavirin (1,000 H epatitis C virus (HCV) and human immunodeficiency virus (HIV) are both parenterally transmitted viruses that coinfect more than 250,000 individuals in the United States. 1 In HCV monoinfected patients, viral eradication occurs in approximately 55% of those treated with current standard therapy: weekly pegylated interferon (PEG-IFN) in combination with daily ribavirin (RBV). 2,3 In HIV/HCV-coinfected patients, successful therapeutic outcomes are substantially lower, ranging from 27% to 40% overall and from 14% to 29% for difficult-to-treat genotype 1 patients. [4][5][6] Consequently, understanding why HIV/HCV-coinfected patients respond to current treatment and how to improve responses is of clinical importance. A fundamental question is whether increased serum IFN ␣ concentrations result in improved therapeutic outcomes.One method of assessing the effectiveness of anti-HCV treatment is the analysis of HCV RNA decay using mathematical models. 7-11 These models have generally not incorporated the effects of time-varying IFN concentrations. PEG-IFN ␣-2b concentration, however, wanes to a Abbreviations: PEG-IFN, pegylated interferon; HCV, hepatitis C virus; SVR, sustained virological responder; NR, nonresponder; HIV, human immunodeficiency virus; RBV, ribavirin; ETR, end-of-treatment responder. From the

The type I interferon-alpha (IFN-alpha) family is a family of natural small proteins that have clinically important anti-infective and antitumor activity. We have developed a semisynthetic protein-polymer conjugate of IFN-alpha2b (Intron A) by attaching a 12,000-Da monomethoxypolyethylene glycol (PEG-12000) polymer to the protein. PEG conjugation is thought to increase the serum half-life and thereby prolong patient exposure to IFN-alpha2b without altering the biologic potency to the protein. Matrix-assisted laser desorption ionization/mass spectrometry (MALDI-MS), high-performance size exclusion chromatography (HPSEC), circular dichroism (CD) analysis and tryptic digestion peptide analysis of PEG Intron demonstrated that the IFN-alpha2b protein was approximately 95% monopegylated and that the primary, the secondary, and the tertiary structures were unaltered. Pegylation did not affect the epitope recognition of antibodies used for Intron A quantitation. An extensive analysis of the pegylated positional isomers revealed that approximately 50% of PEG Intron was monopegylated on the His(34) residue of the IFN-alpha2b protein. The highest antiviral activity of the pegylated positional isomers for PEG Intron was associated with the His(34) pegylated isomer. The specific activity for PEG Intron in an antiviral cytopathic protection assay was 28%, relative to Intron A. However, the potency of PEG Intron, defined as bioactivity independent of protein concentration, was comparable to Intron A at both the molecular and cellular levels in a battery of in vitro assays. Equivalent units of PEG Intron and Intron A were indistinguishable for the induction of several key IFN-induced genes, including 2',5'-oligoadenylate synthetase (2',5'-OAS) and protein kinase R (PKR), in Molt 4 cells. The antiviral dose-response curves revealed that there were no significant differences between PEG Intron and Intron A. This demonstrated that the introduction of more IFN-alpha2b protein associated with equivalent unit dosing of PEG Intron did not create any antagonism or agonism in the antiviral assay. In assays for the immune response, PEG Intron and Intron A displayed comparable potency for both natural-killer (NK) and lymphokine-activated killer (LAK) cell cytolytic activity and for the induction of class I major histocompatibility protein. These results demonstrate that PEG Intron maintains an in vitro biologic potency profile for both antiviral and immunotherapeutic activity that is highly comparable to that of Intron A.

Therapeutic pegylated interferon-␣s (IFN-␣) are mixtures of positional isomers that have been monopegylated at specific sites on the core IFN-␣ molecule. The pegylation results in lower in vitro specific activity associated with the core IFN-␣ molecule that is related to the site of pegylation and size of polyethylene glycol (PEG) attached. We prepared purified, homogeneous, positional pegylation isomers of IFN-␣2b that were monopegylated using 5-30-kDa linear PEG molecules attached at 7 primary reactive amino acid residues: Chronic hepatitis C is considered one of the major causes of chronic liver disease, cirrhosis, and hepatocellular carcinoma and is the most common reason for liver transplantation in the United States (1). It is estimated that there are 3 million chronically infected individuals in the United States and over 170 million worldwide (1). Treatment of hepatitis C has evolved from the use of interferon-␣ (IFN-␣), 1 either alone or in combination with ribavirin, to the newer pegylated interferons (PEGIFNs), which have provided a dramatic increase in virological response, especially in combination with ribavirin. Standard IFN-␣ therapy has a short (Ͻ12-h) half-life that requires subcutaneous injection three times weekly to maintain effective levels in the blood (2). The short half-life of IFN-␣ has led to the development of longer lasting preparations achieved by the attachment of a large polyethylene glycol (PEG) molecule directly to IFN-␣. Two different commercial preparations of PEG-IFN-␣ have been developed for clinical use, PEG-IFN-␣2b (PEG-INTRON®) and PEG-IFN-␣2a (Pegasys®); both have long half-lives (40 and 80 h, respectively) that permit once weekly administration (3). Both of these preparations have been demonstrated to be effective for the treatment of patients with hepatitis C (4), and clinical trial results have shown further that both of the pegylated molecules produce sustained viral response rates superior to those achieved with their respective standard IFN-␣s (5-7).Whereas pegylation has proven to be highly effective for slowing the clearance of biological molecules, including IFN-␣, and thus increasing serum half-life, it has been shown to also modify in vitro biological activity (8). For instance, we have reported that pegylation of IFN-␣2b with a 12-kDa linear PEG molecule results in a preparation that has a specific activity of 28% relative to IFN-␣2b; the loss in activity was not due to structural perturbation of the core IFN-␣2b core protein (9). Other groups have reported that pegylation of IFN-␣2a with a 40-kDa branched PEG molecule results in a preparation that contains from 1 to 7% relative specific activity compared with IFN-␣2a (10, 11). These two pegylated interferon-␣s (PEG-IFN␣s) differ substantially in their postpegylation constituent properties. PEG-IFN-␣2b has a 12-kDa linear PEG molecule attached using succinimidyl carbonate polyethylene glycol (SC-PEG) chemistry via a covalent urethane-like bond to the IFN␣2b protein (12). The pegylation linkage process results...

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