N-Terminally PEGylated Human Interferon-β-1a with Improved Pharmacokinetic Properties and in Vivo Efficacy in a Melanoma Angiogenesis Model

Abstract

PEGylation of IFN-alpha has been used successfully to improve the pharmacokinetic properties and efficacy of the drug. To prepare a PEGylated form of human interferon-beta-1a (IFN-beta-1a) suitable for testing in vivo, we have synthesized 20 kDa mPEG-O-2-methylpropionaldehyde and used it to modify the N-terminal alpha-amino group of the cytokine. The PEGylated protein retained approximately 50% of the activity of the unmodified protein and had significantly improved pharmacokinetic properties following intravenous administration in rats. The clearance and volume of distribution at steady state were reduced approximately 30-fold and approximately 4-fold, respectively, resulting in a significant increase in systemic exposure as determined by the area under the curve. The elimination half-life of the PEGylated protein was approximately 13-fold greater than for the unmodified protein. The unmodified and PEGylated proteins were tested for their ability to inhibit the formation of radially oriented blood vessels entering the periphery of human SK-MEL-1 melanoma tumors in athymic nude homozygous (nu/nu) mice. In a single dose comparison study, administration of 1 x 10(6) units of unmodified IFN-beta-1a resulted in a 29% reduction in vessel number, while 1 x 10(6) units of PEGylated IFN-beta-1a resulted in a 58% reduction. Both treatments resulted in statistically significant reductions in mean vessel number as compared to the vehicle (control)-treated mice, with the PEGylated IFN-beta-1a-treated mice showing a statistically significantly greater reduction in mean vessel number as compared to the unmodified IFN-beta-1a-treated mice. In a multiple versus single dose comparison study, daily administration of 1 x 10(6) units of unmodified IFN-beta-1a for 9 days resulted in a 51% reduction in vessel number, while a single dose of 1 x 10(6) units of the PEGylated protein resulted in a 66% reduction. Both treatments resulted in statistically significant reductions in mean vessel number as compared to the vehicle-treated mice, with the PEGylated IFN-beta-1a-treated mice showing a statistically significantly greater reduction in mean vessel number as compared to the unmodified IFN-beta-1a-treated mice. Therefore, the improved pharmacokinetic properties of the modified protein translated into improved efficacy. Since unmodified IFN-beta is used for the treatment of multiple sclerosis and hepatitis C virus infection, a PEGylated form of the protein such as 20 kDa mPEG-O-2-methylpropionaldehyde-modified IFN-beta-1a may serve as a useful adjunct for the treatment of these diseases. In addition, the antiangiogenic effects of PEGylated IFN-beta-1a may be harnessed for the treatment of certain cancers, either as a sole agent or in combination with other antitumor drugs.

Efficient N‐Terminal Labeling of Proteins by Use of Sortase

“…[3] Each of these methods has its own associated challenges: Selective labeling of a single cysteine residue frequently requires rounds of site-directed mutagenesis to introduce the labeling site and/or remove other cysteine residues, and selective labeling of the N-terminal amino group requires careful control of pH to ensure lysine residues are not also modified. [4] Other modern methods for chemoselective labeling often require the introduction of specific recognition sequences [5] or nonnatural amino acids into the protein to be labeled. [6] In most cases, a substantial excess of the labeling reagent is necessary to ensure complete conversion to the product.…”

In situ growth of a stoichiometric PEG-like conjugate at a protein's N-terminus with significantly improved pharmacokinetics

“…We selected the N-terminus as the site of in situ growth, because the N-terminal amine has a sufficiently different pK a (Ϸ7.8) from the amine group present on lysine side chains (pK a Ϸ 10.5-12) that are typically distributed on the protein surface. As the different pK a of the N-terminal amine allows chemical reactions to be selectively performed at the N-terminus of a protein with no cross reaction at solventaccessible lysine residues (19)(20)(21)(22)(23)(24), we hypothesized that it should be possible to selectively attach a polymerization initiator to the N-terminus and subsequently grow a polymer chain solely from the N-terminus.…”

Natural interferon-beta treatment for patients with chronic hepatitis C in Japan

“…It was reported that recombinant human interferon-beta-1a with or without ribavirin has an excellent safety profile, and after 24-wk-treatment of recombinant human interferon-beta-1a with or without ribavirin, SVR was 21.6% and 27.4% in HCV genotype 1 and genotype 2 patients, respectively [57][58][59][60] . Peginterferonbeta-1a may be beneficial for patients infected with HCV [61,62] .…”