Pegylated IFNs for chronic hepatitis C: an update.

For over a decade, IFN-alpha(2) has been the standard treatment for chronic hepatitis C. However, the drug's rapid clearance and short half-life have led to low rates of sustained virological response. Pegylation is a well-established method of modifying the pharmacological properties of IFNs, causing significant improvements in pharmacokinetics, which in turn lead to improved efficacy. Two pegylated forms of IFN-alpha(2) have been developed: PEG-IFN-alpha(2b) and PEG-IFN-alpha(2a), and their efficacy has been established in randomised, controlled trials. However, the two differ significantly in structure, in vitro activity and pharmacological properties, and this may translate into -differences in clinical efficacy. Comparative trials have been initiated that will provide insight into relative importance of pharmacokinetics, bioactivity and dosing regimen.

Interferon-alpha as an immunotherapeutic protein.

Interferon-alpha (IFN-alpha) has proven to be a clinically effective antiviral and antineoplastic therapeutic drug for more than 16 years. During this time, evidence from in vitro laboratory studies and the clinical arena has supported the concept that IFN-alpha is an immunotherapeutic drug. By regulating a diverse set of cytokines and their receptors, IFN-alpha is uniquely positioned to prime the host immune response and provide an effective antineoplastic- and antiviral-immune response. IFN-alpha stimulates the innate cell-mediated response and then participates in the transition of the initial host innate response into an effective adaptive-immune response. IFN-alpha also drives the adaptive cell-mediated CD8+ T-cell response and helps to maintain a CD4+ Th1-cell population balance for an effective antineoplastic and antiviral host defense. This review will describe the current state of knowledge of IFN-alpha as an immunoregulatory protein and address specific issues of IFN-alpha as an immunotherapeutic for antineoplastic and antiviral diseases.

The use of field emission scanning electron microscopy to assess recombinant adenovirus stability.

A field emission scanning electron microscopy (FESEM) method was developed to assess the stability of a recombinant adenovirus (rAd). This method was designed to simultaneously sort, count, and size the total number of rAd viral species observed within an image field. To test the method, a preparation of p53 transgene-expressing recombinant adenovirus (rAd/p53) was incubated at 37 degrees C and the viral particles were evaluated by number, structure, and degree of aggregation as a function of time. Transmission electron microscopy (TEM) was also used to obtain ultrastructural detail. In addition, the infectious activity of the incubated rAd/p53 samples was determined using flow cytometry. FESEM image-analysis revealed that incubation at 37 degrees C resulted in a time-dependent decrease in the total number of detectable single rAd/p53 virus particles and an increase in apparent aggregates composed of more than three adenovirus particles. There was also an observed decrease in both the diameter and perimeter of the single rAd/p53 viral particles. TEM further revealed the accumulation of damaged single particles with time at 37 degrees C. The results of this study demonstrate that FESEM, coupled with sophisticated image analysis, may be an important tool in quantifying the distribution of aggregated species and assessing the overall stability of rAd samples.

Structural and biological characterization of pegylated recombinant interferon alpha-2b and its therapeutic implications.

The type I interferon alpha family consists of small proteins that have clinically important anti-infective and anti-tumor activity. Interferon alpha-2b (Intron A) combination therapy with ribavirin is the current standard of care for the treatment of chronic hepatitis C virus infection. A drawback to the therapy however, is the short serum half-life and rapid clearance of the interferon alpha protein. Schering-Plough has developed a semi-synthetic form of Intron A by attaching a 12-kDa mono-methoxy polyethylene glycol to the protein (PEG Intron) which fulfills the requirements of a long-acting interferon alpha protein while providing significant clinical benefits. A detailed physicochemical and biological characterization of PEG Intron revealed its composition of pegylated positional isomers and the specific anti-viral activity associated with each of them. Though pegylation appeared to decrease the specific activity of the interferon alpha-2b protein, the potency of PEG Intron, independent of protein concentration, was comparable to the Intron A standard at both the molecular and cellular level. Importantly, PEG Intron has demonstrated an enhanced pharmacokinetic profile in both animal and human studies. Recently, PEG Intron in combination with ribavirin has been shown to be very effective in reducing hepatitis C viral load and maintaining effective sustained viral suppression in patients. Because of the improved clinical benefits, it is anticipated that the PEG Intron plus ribavirin combination therapy will become the new standard of care for the treatment of chronic hepatitis C.

Regulation of gene expression by pegylated IFN-alpha2b and IFN-alpha2b in human peripheral blood mononuclear cells.

The pleiotropic biologic effects of interferon (IFN) are mediated through regulation of the expression of numerous IFN-sensitive genes. Peripheral blood mononuclear cells (PBMCs) obtained from healthy donors were analyzed to study the immunoregulatory and antiviral messenger RNAs (mRNAs) and proteins regulated by pegylated IFN-alpha2b (PEG-IFN-alpha2b) and IFN-alpha2b. A dose-dependent and time-dependent response for multiple IFN-regulated genes was observed. IFN-dependent protein production and secretion were correlated with IFN-regulated mRNA induction. Overall regulation of gene expression patterns for PEG-IFN-alpha2b and IFN-alpha2b was comparable, even though the antiviral activity of PEG-IFN-alpha2b demonstrated a longer biologic halflife in vitro compared with IFN-alpha2b. To study the heterogeneity of responses, PBMCs obtained from over 25 healthy donors were analyzed. Within a particular donor dataset, gene-specific and dose-dependent responses to PEG-IFN-alpha2b treatment, demonstrated in both the amplitude of transcriptional upregulation and the duration of sustained mRNA upregulation, were observed. However because of donor heterogeneity, the amplitude of a given transcriptional response could not be predicted for a specific dose of PEG-IFN-alpha2b. Notably, mRNA levels of oligoadenylate synthetase (OAS), double-stranded RNA (dsRNA)-activated protein kinase (PKR), IP-10, IFN-stimulated gene 54 (ISG54), and ISG15 were upregulated after 120 h of continuous PEG-IFN-alpha2b treatment. These results suggest that the use of antiviral and immunoregulatory protein mRNA levels as markers to assess the therapeutic efficacy of IFN-alpha2b and PEG-IFN-alpha2b against viral and neoplastic diseases in clinical trials is promising but will require further analysis using clinical patient samples.

Structure, biology, and therapeutic implications of pegylated interferon alpha-2b.

Derivatization of protein-based therapeutics with polyethylene glycol (pegylation) can often improve pharmacokinetic and pharmacodynamic properties of the proteins and thereby, improve efficacy and minimize dosing frequency. This review will provide an overview of pegylation technology and pegylated protein-based drugs being used or investigated clinically. The novel therapeutic, PEG Intron(R), formed by attaching a 12-kDa mono-methoxy polyethylene glycol (PEG) to the interferon alpha-2b protein, will be discussed in detail in terms of its structure, biological activities, pharmacokinetic properties, and clinical efficacy for the treatment of chronic hepatitis C. Detailed physicochemical and biological characterization studies of PEG Intron revealed its composition of pegylated positional isomers and the specific anti-viral activity associated with each of them. Pegylation of Intron A at pH 6.5 results in a mixture of > or = 95% mono-pegylated isoforms with the predominant species (approximately 50%) derivatized to the His(34) residue with the remaining positional isomers pegylated at various lysines, the N-terminal cysteine, as well as serine, tyrosine, and another histidine residue. The anti-viral activity for each pegylated isomer showed that the highest specific activity (37%) was associated with the His(34)-pegylated isomer. Though pegylation decreases the specific activity of the interferon alpha-2b protein in vitro, the potency of PEG Intron was comparable to the Intron A standard at both the molecular and cellular level. The substituted IFN had an enhanced pharmacokinetic profile in both animal and human studies, and, when combined with ribavirin, was very effective in reducing hepatitis C viral load and maintaining sustained viral suppression in patients.

Pegylated interferons for the treatment of chronic hepatitis C infection.

BACKGROUND: Interferon (IFN) alfa is a clinically effective therapy used in a wide range of viral infections and cell-proliferative disorders. Combination therapy with IFN alfa-2b and ribavirin is the current standard of care for the treatment of chronic hepatitis C (CHC) infection. However, standard IFN alfa has the drawbacks of a short serum half-life and rapid clearance. To overcome this problem, 2 pegylated forms of IFN have been developed and tested clinically. OBJECTIVE: This article reviews the development and properties of pegylated IFN alfa-2b and pegylated IFN alfa-2a, and presents safety and efficacy data from recent clinical trials. METHODS: Relevant clinical studies were identified through a MEDLINE search from 1966 through the present using the key words hepatitis C and interferon. Studies of the pegylated IFNs in humans were then selected. RESULTS: Pegylated IFN alfa-2b is formed by covalent conjugation of a 12-kd mono-methoxy polyethylene glycol (PEG) molecule to IFN alfa-2b, and pegylated IFN alfa-2a by covalent conjugation of a 40-kd branched mono-methoxy PEG molecule to IFN alfa-2a. The 2 pegylated IFNs differ in the mixture of pegylation isomers resulting from their conjugation chemistry. Pegylated IFN alfa-2b has a prolonged serum half-life (40 hours) relative to standard IFN alfa-2b (7-9 hours). The greater polymer size of pegylated IFN alfa-2a acts to reduce glomerular filtration, markedly prolonging its serum half-life (72-96 hours) compared with standard IFN alfa-2a (6-9 hours). In clinical studies, once-weekly dosing of the pegylated IFNs was associated with a sustained virologic response in patients infected with hepatitis C virus (HCV). Once-weekly dosing with either of the pegylated IFNs was more effective than the respective thrice-weekly regimen of IFN alfa, with a comparable safety profile. The combination of once-weekly pegylated IFN and ribavirin effectively reduced HCV viral load and sustained viral suppression. CONCLUSIONS: Once-weekly dosing with either pegylated IFN alfa-2b or pegylated IFN alfa-2a has been shown to produce significantly higher rates of viral eradication than standard thrice-weekly IFN alfa therapy without compromising safety. With respect to the treatment of CHC, the greatest anti-HCV efficacy has been achieved with the combination of once-weekly pegylated IFN and ribavirin.

A randomised trial to compare the pharmacokinetic, pharmacodynamic, and antiviral effects of peginterferon alfa-2b and peginterferon alfa-2a in patients with chronic hepatitis C (COMPARE).

BACKGROUND/AIMS: To compare the pharmacokinetics, pharmacodynamics, and antiviral activity of peginterferon alfa-2b and peginterferon alfa-2a in patients with chronic hepatitis C virus genotype 1. METHODS: Thirty-six patients were randomised to peginterferon alfa-2b (1.5 microg/kg/week) or peginterferon alfa-2a (180 microg/week) for 4 weeks, then in combination with ribavirin (13 mg/kg/day) for a further 4 weeks. The pharmacokinetic profile of both peginterferons, mRNA expression of a selected group of interferon-induced gene transcripts, and serum HCV-RNA levels were assessed. RESULTS: Patients receiving peginterferon alfa-2b had significantly greater up-regulation of interferon-alfa response genes compared with those receiving peginterferon alfa-2a. Correspondingly, patients treated with peginterferon alfa-2b also had a significantly greater log10 maximum and log10 time-weighted average decrease in serum HCV-RNA. A greater proportion of peginterferon alfa-2b patients achieved a > or = 2.0 log10 reduction in serum HCV-RNA levels by week 8 (72% vs 44% of peginterferon alfa-2a patients, P = 0.09). There was an approximately 16-fold greater exposure to peginterferon in the serum of patients treated with peginterferon alfa-2a. CONCLUSIONS: These findings suggest that the biological activity, measured by early interferon-induced gene transcripts and early antiviral responsiveness, may have been greater in patients treated with peginterferon alfa-2b despite their lower exposure to the drug compared with patients treated with peginterferon alfa-2a.

Use of an automated image processing program to quantify recombinant adenovirus particles.

Electron microscopy has a pivotal role as an analytical tool in pharmaceutical research. However, digital image data have proven to be too large for efficient quantitative analysis. We describe here the development and application of an automated image processing (AIP) program that rapidly quantifies shape measurements of recombinant adenovirus (rAd) obtained from digitized field emission scanning electron microscope (FESEM) images. The program was written using the macro-recording features within Image-Pro Plus software. The macro program, which is linked to a Microsoft Excel spreadsheet, consists of a series of subroutines designed to automatically measure rAd vector objects from the FESEM images. The application and utility of this macro program has enabled us to rapidly and efficiently analyze very large data sets of rAd samples while minimizing operator time.

Site of pegylation and polyethylene glycol molecule size attenuate interferon-alpha antiviral and antiproliferative activities through the JAK/STAT signaling pathway.

Therapeutic pegylated interferon-alphas (IFN-alpha) are mixtures of positional isomers that have been monopegylated at specific sites on the core IFN-alpha molecule. The pegylation results in lower in vitro specific activity associated with the core IFN-alpha 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-alpha2b that were monopegylated using 5-30-kDa linear PEG molecules attached at 7 primary reactive amino acid residues: Cys(1), His(34), Lys(31), Lys(83), Lys(121), Lys(131), and Lys(134). The isomers were evaluated for STAT translocation and antiviral and antiproliferative activity. The site of pegylation strongly influenced activity relative to an IFN-alpha2b control. The highest residual activity was observed with the His(34) positional isomers, and the lowest was observed with the Cys(1) positional isomers. The Lys positional isomers demonstrated intermediate activity, with a general order of Lys(134) > Lys(83) approximately Lys(131) approximately Lys(121) > Lys(31). The progressive relationship between decreased activity and increased PEG size suggests that pegylation may interfere with interaction and binding of IFN-alpha to the IFNAR1-IFNAR2 heterodimeric receptor. The higher specific activity associated with the His(34) positional isomer suggests that this site may be favorable for pegylating IFN-alpha2b molecules.