MECHANISM OF ACTION AND PHARMACOKINETICS OF INTERFERON WITH AND WITHOUT PEGYLATION

Overview

Interferons (IFNs) are naturally occurring proteins produced by a wide variety of cells. They have antiviral, antiproliferative, and immunomodulatory properties and are classified into 2 main groups based on antigenic and structural differences. Type 1 includes IFN-alpha and IFN-beta, and type 2 includes IFN-gamma. The first part of this article focuses on IFN-alpha in relationship to hepatitis C, and the second part summarizes the landmark clinical trials of pegylated IFN (PEG-IFN) alfa-2a monotherapy and combination therapy with ribavirin for chronic hepatitis C virus (HCV) infection.

IFN-ALPHA

Mechanism of action

At least 13 different subtypes of IFN-alpha exist, with molecular weights ranging from 17.5-23 kilodaltons (kd). All exhibit approximately 80% sequence homology and are produced by activated B lymphocytes, null lymphocytes, and macrophages in response to infections or tumors. An important mechanism through which IFN-alpha induces its effects is through a signaling cascade called the Janus-activated kinase/signal transducer and activator of transcription (JAK-STAT) pathway. This was the first signaling pathway demonstrated to be activated by IFNs and remains the most extensively studied. IFN-alpha binds to a multichained receptor at the cell surface called the type 1 IFN receptor. This is composed of at least 2 distinct subunits: IFN-alpha receptor 1 (IFNAR1) and IFN-alpha receptor 2 (IFNAR2). Both subunits are associated with the JAK family; IFNAR1 is associated with tyrosine kinase 2 (TYK2), and IFNAR2 is associated with JAK1. These kinases phosphorylate the tyrosine residues of STAT1 and STAT2, leading to formation of the STAT1-STAT1 homodimer complex and the STAT1-STAT2-IFN-regulatory 9 (IRF9) complex, also known as the IFN-stimulated gene factor 3 (ISGF3) complex. The STAT1-STAT2 complex translocates to the nucleus and binds to IFN-gamma–activated sites (GAS) present in the promoter region of certain interferon-stimulated genes (ISGs), while the ISGF3 complex, after nuclear translocation, binds to IFN-stimulated response elements (ISRE) in DNA to initiate transcription. Since the discovery of the JAK-STAT signaling pathway in 1992, several other signaling pathways have been identified. These pathways frequently need to work synergistically to generate the biological effects of IFN and include, but are not limited to, the following: the Crk family of adaptor proteins, mitogen-activated protein kinases (MAPKs), and phosphatidylinositol 3-kinase (PI3K).

Although IFN-alpha’s mechanism of action in the treatment of chronic hepatitis C remains to be fully elucidated, its immunomodulatory action appears to play a major role. IFN-alpha can inhibit viral replication indirectly by altered cytokine synthesis, which in turn can amplify the cytotoxic T cell (specific) and natural killer cell (nonspecific) response to virally infected cells. Mechanisms of direct inhibition of viral replication by IFN include prevention of virus attachment to host cells and uncoating of the virus; increase of viral antigen expression by infected cells; induction of cellular enzymes, such as 2’5’ oligoadenylate synthetase, that impair RNA synthesis; and downregulation of cellular proteins, such as protein kinases and the Mx protein, which are involved in cellular synthesis and division, respectively. However, virus kinetic modeling studies suggest IFN-alpha works primarily through prevention of de novo infection of susceptible cells rather than intracellular inhibition of replication.

Pharmacokinetics

IFN-alfa is commonly administered subcutaneously in order to maximize absorption because intravenous administration results in rapid renal clearance and because oral administration of such a large protein is unlikely. The subcutaneous route results in protracted absorption, which leads to maximum plasma concentrations occurring after 1-8 hours and measurable levels following for an additional 4-24 hours. Initial IFN concentrations are known to drop several orders of magnitude following subcutaneous administration, with peak levels after 7-12 hours, an elimination half-life of 4-16 hours, and virtually undetectable levels after 24 hours. The volume of distribution ranges from 12-40 L, which approximates to 20-60% of body weight. Although a paucity of research has been conducted in humans, no evidence yet indicates that IFN penetrates the blood-brain barrier. Similarly, most data on the catabolism of IFN are from animal research, which has demonstrated similarities in the natural handling of proteins across most species. In general, IFN-alfa is filtered through the renal glomerules and reabsorbed in the proximal renal tubules where proteolytic degradation by lysosomal enzymes occurs. This degradation results in negligible amounts of intact IFN excreted in the urine, unlike in nephrectomized animals, in which the clearance of IFN is greatly diminished. The liver, in turn, has a minor role in IFN-alfa catabolism, although it appears to be the predominant catabolic site for IFN-beta and IFN-gamma.

Pegylation

Pegylation is a process in which a polyethylene glycol (PEG) polymer is attached to a protein to produce a product with a larger molecular weight and bulk. This results in a decrease in renal clearance and an increase in the elimination half-life; this allows the dosing interval to be lengthened. Pegylation also decreases peak-trough fluctuations in IFN plasma concentrations, which have previously been associated with rebound and drug resistance because of the short half-life and rapid replication rate of HCV. Pegylation can also create a “water-cloud” effect, which decreases antigenicity, immunogenicity, and proteolytic degradation of IFN. A PEG polymer can be attached as either a small or large PEG polymer at a single site; as a branched (2 or more chains) PEG polymer at a single site; or as multiple, small chains at multiple sites.

PEG-IFN alfa-2a consists of 2 branched, 20-kd PEG chains attached via hydrolytically stable amide bonds to a lysine residue of IFN alfa-2a. This results in pharmacokinetic properties that are distinctly different from conventional, nonpegylated IFN. After a single subcutaneous dose of 180 mcg of PEG-IFN alfa-2a, plasma drug concentrations are detectable within 3-8 hours. In patients with chronic HCV, 50% of the administered dose is absorbed after 60 hours and peak plasma levels are attained 72-96 hours after administration. The larger size of the PEG-protein conjugate results in a more restricted volume of distribution of 4-16 L because of its predominant distribution in the intravascular compartment and its reduced renal clearance with a mean elimination half-life of approximately 80 hours.

CLINICAL STUDIES OF PEGINTERFERON ALFA-2A MONOTHERAPY

PEG-IFN alfa-2a monotherapy

In 2000, Zeuzem reported the results of a multicenter, randomized prospective study of 531 patients with chronic HCV infection, comparing PEG-IFN alfa-2a weekly versus IFN alfa-2a 3 times a week. Patients in the PEG-IFN alfa-2a group received 180 mcg weekly. Patients in the IFN alfa-2a group received 6 million units 3 times a week for the first 12 weeks followed by 3 million units 3 times a week for 36 weeks. Both groups were similar with respect to duration of treatment, frequency of treatment, and severity of side effects. However, patients randomized to the PEG-IFN group achieved an eradication rate significantly greater than the nonpegylated group at both week 48 (69% vs 28%, P = .001) and at week 72 (39% vs 19%, P = .001). Normalization of aminotransferases at week 72 was also more common in the PEG-IFN group than the IFN group (45% vs 25%, P = .001). The researchers concluded that, for treatment of chronic HCV, PEG-IFN alfa-2a administered once a week is more effective than IFN alfa-2a 3 times a week.

In the same journal, Heathcote evaluated the effectiveness of PEG-IFN alfa-2a versus IFN alfa-2a in patients with HCV who had bridging fibrosis or cirrhosis. In this study, 271 patients were randomly assigned to receive 3 million units of IFN alfa-2a 3 times a week (n = 88), 90 mcg of PEG-IFN alfa-2a once a week (n = 96), or 180 mcg of PEG-IFN alfa-2a once a week (n = 87).Treatment duration was for 48 weeks, and patients were monitored for an additional 24 weeks when liver biopsies were repeated. In an intention-to-treat analysis, a sustained HCV eradication (undetectable HCV RNA at 72 weeks or a sustained viral response [SVR]) occurred in 8%, 15%, and 30%, respectively, of patients treated with 3 million units of IFN alfa-2a, 90 mcg PEG-IFN alfa-2a, and 180 mcg PEG-IFN alfa-2a (P = .001 for the comparison between 180 mcg PEG-IFN alfa-2a and IFN alfa-2a). Histologic improvement at week 72 was present in 31%, 44%, and 54%, respectively (P = .02 for the comparison between 180 mcg PEG-IFN alfa-2a and IFN alfa-2a). The researchers concluded that 180 mcg of PEG-IFN alfa-2a administered weekly is the most effective of the 3 regimens for eradicating HCV in patients with bridging fibrosis or cirrhosis.

PEG-IFN alfa-2a plus ribavirin therapy

A landmark randomized study of 1121 patients with chronic HCV compared the efficacy of 180 mcg/wk of PEG-IFN alfa-2a plus 1000-1200 mg/d of ribavirin, 3 million units 3 times a week of IFN alfa-2b plus 1000-1200 mg/d of ribavirin, and 180 mcg/wk of PEG-IFN alfa-2a plus a placebo (Fried, 2002). This study treated patients for 48 weeks. The SVR was significantly higher in patients who received PEG-IFN alfa-2a plus ribavirin than in patients who received IFN alfa-2b plus ribavirin (56% vs 44 %, P < .001) or PEG-IFN with a placebo (56% vs 29%, P < .001). Among patients with genotype 1 and high pretreatment viral loads, an SVR occurred in 41%, 33%, and 13% of patients, respectively, with overall safety profiles similar in all 3 groups. In conclusion, the investigators reported that, for HCV, PEG-IFN alfa-2a plus ribavirin produces significant improvements in SVR compared to the other regimens.

In a subsequent study designed to determine the optimum dose and duration of treatment, Hadziyannis randomized 1311 patients with HCV to 180 mcg/wk of PEG-IFN alfa-2a for 24-48 weeks plus a low dose (800 mg/d) or a standard dose (1000-1200 mg/d) of ribavirin. The researchers concluded that patients with genotype 1 require treatment for 48 weeks with a standard dose of ribavirin while patients with genotypes 2 or 3 can be adequately treated for 24 weeks with a low dose of ribavirin.

To evaluate the effectiveness of PEG-IFN and ribavirin in prior nonresponders to IFN alfa with and without ribavirin, Shiffman re-treated 604 patients with 180 mcg/wk of PEG-IFN alfa-2a and 1000-1200mg/d of ribavirin. Treatment duration was 48 weeks with an additional 24 weeks of follow-up. An SVR occurred in 18% of patients, and factors associated with the SVR were prior treatment with IFN monotherapy, genotypes 2 and 3, a lower aspartate aminotransferase:alanine aminotransferase (AST:ALT) ratio, and absence of cirrhosis. The researchers concluded that selected nonresponders to IFN-based therapy may achieve an SVR following re-treatment with PEG-IFN alfa-2a and ribavirin.

Because blacks have a high prevalence of HCV but are also underrepresented in many HCV studies, Jeffers studied the effectiveness of 180 mcg/wk of PEG-IFN alfa-2a plus 1000-1200 mg/d of ribavirin in a group of 78 black patients and 28 white patients in a prospective, multicenter trial. Pretreatment and posttreatment biopsies were compared to determine the impact of treatment on necroinflammation and fibrosis. An SVR occurred in 26% of black patients compared to 39% in the white group; however, interestingly, an improvement in fibrosis occurred in 25% of black patients. Jeffers concluded that PEG-IFN alfa-2a and ribavirin can be safely administered to black patients with HCV, particularly because the SVRs were greater than in prior studies.

In a multinational study to evaluate the impact of HCV treatment in patients with normal aminotransferases, Zeuzem randomized 491 patients (3:3:1) to 180 mcg/wk of PEG-IFN alfa-2a plus 800 mg/d of ribavirin for 24 weeks (n = 212) or 48 weeks (n = 210) or a placebo (n = 69). All 3 groups were monitored for a total of 72 weeks: 24 weeks of treatment with 48 weeks of follow-up, 48 weeks of treatment with 24 weeks of follow-up, and 72 weeks of untreated follow-up, respectively. Not surprisingly, an SVR did not occur in the placebo group but did occur in 30% of patients treated for 24 weeks and 52% of patients treated for 48 weeks. An SVR in patients with genotype 1 occurred in 13% of patients after 24 weeks of treatment and in 40% of patients after 48 weeks of treatment (P < .001). An SVR in patients with genotypes 2 and 3 was achieved in 72% and 78% with 24 and 48 weeks of treatment, respectively (P = .452). This study supports the treatment of patients with chronic HCV who have normal aminotransferases.

References

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Hadziyannis SJ, Sette H Jr, Morgan TR, et al. Peginterferon alpha-2a and ribavirin combination therapy in chronic hepatitis C: a randomized study of treatment duration and ribavirin dose. Ann Int Med. 2004;140: 346-55.

Heathcote EJ, Shiffman ML, Cooksley WG, et al. Peginterferon alpha-2a in patients with chronic hepatitis C and cirrhosis. N Eng J Med. 2000;343: 1673-80.

Jeffers LJ, Cassidy W, Howell CD, et al. Peginterferon alpha-2a (40kd) and ribavirin for black American patients with chronic hepatitis C genotype 1. Hepatology. 2004;39: 1702-8.

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Shiffman ML, DiBisceglie AM, Lindsay KL, et al. Peginterferon alpha-2a and ribavirin in patients with chronic hepatitis C who have failed prior treatment. Gastroenterology. 2004;126: 1015-23.

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Zeuzem S, Diago M, Gane E, et al. Peginterferon alpha-2a (40kilodaltons) and ribavirin in patients with chronic hepatitis C and normal aminotransferase levels. Gastroenterology. 2004;127: 1724-32.

Zeuzem S, Feinman SV, Rasenack J, et al. Peginterferon alpha-2a in patients with chronic hepatitis C. N Eng J Med. 2000;343: 1666-72.