Pharmacokinetic and Pharmacodynamic Studies of a Human Serum Albumin-Interferon- Fusion Protein in Cynomolgus Monkeys

Interferon(IFN) is indicated for the treatment of certain viral infections including hepatitis B and C, and cancers such as melanoma. The short circulating half-life of unmodified IFNmakes frequent dosing (daily or three times weekly) over an extended period (6–12 months or more) necessary. To improve the pharmacokinetics of IFNand decrease dosing frequency, IFNwas fused to human serum albumin producing a new protein, Albuferon. In vitro comparisons of Albuferon and IFNshowed similar antiviral and antiproliferative activities, although Albuferon was less potent on a molar basis than IFN. Pharmacokinetic and pharmacodynamic properties of the fusion protein were enhanced in monkeys. After a single intravenous injection (30 g/kg,) clearance was 0.9 ml/h/kg, and the terminal half-life was 68 h. After 30 g/kg subcutaneous injection, apparent clearance (clearance divided by bioavailability) was 1.4 ml/h/kg, the terminal half-life was 93 h, and bioavailability was 64%. The rate of clearance of Albuferon was approximately 140-fold slower, and the half-life 18-fold longer, than for IFNgiven by the subcutaneous route in other monkey studies. Sera from Albuferon-treated monkeys demonstrated doserelated antiviral activity for 8 days based on an in vitro bioassay, whereas antiviral activity from IFN-treated animals was only slightly elevated relative to vehicle on day 0. Significant increases in 2 ,5 -oligoadenylate synthetase mRNA relative to IFNor vehicle-treated animals were maintained for 10 days after subcutaneous dosing. The improved pharmacokinetics of Albuferon are accompanied by an improved pharmacodynamic response suggesting that Albuferon may offer the benefits of less frequent dosing and a potentially improved efficacy profile compared with IFN. Interferons (IFNs) are a class of cytokines that play a key role in the regulation of cell growth and differentiation via activation of a cascade of intracellular pathways. Interferons possess antiviral, immunomodulatory, and antiproliferative effects. There are two types of IFNs. Type I includes IFN, IFN, IFN1 , and IFN; type II IFN includes IFN(Foster, 1997; Maeyer and Maeyer-Guignard, 1998; LaFleur et al., 2001). Most cells in the human body produce type I IFNs within hours of viral infection, with the IFNfamily having greater antiviral activity against hepatitis C infection than the others (Hu et al., 2001). Treatment with unmodified IFNfor chronic hepatitis C requires frequent injections (e.g., once daily or three times weekly) over the course of therapy due to the molecule’s short circulating half-life of 2 to 3 h in humans (Intron A package insert). Although studies have demonstrated the benefit of IFNin the treatment of chronic hepatitis C, the regimen of 3 million international units three times per week for 6 to 12 months is effective in less than half of all patients (Thevenot et al., 2001). Among those who do respond to therapy, 50 to 80% relapse within 6 months after dose reduction or treatment discontinuation (Davis et al., 1989; Tine et al., 1991; Fried and Hoofnagle, 1995). Although prolonged therapy (up to 36 months) may improve the sustained response rate (Ahmed and Keeffe, 1999; Damen et al., 2001), it may also lead to an increase in the frequency and severity of adverse events (Medical Letter, 1997). Patient compliance in these long-term dosing regimens is difficult to maintain. The development of more slowly cleared pegylated IFNs has led to reduced dosing frequency and improved responses. The conFinancial support was provided by Human Genome Sciences, Inc., Rockville, MD. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. DOI: 10.1124/jpet.102.037002. ABBREVIATIONS: IFN, interferon; PEG, polyethylene glycol; OAS, oligoadenylate synthetase; EMCV, encephalomyocardititis virus; VSV, vesicular stomatitis virus; ELISA, enzyme-linked immunosorbent assay; AUC, area under the curve; CL/F, clearance adjusted for fraction (F) of drug absorbed (bioavailability); Cmax, maximal plasma concentration; EC50, concentration to achieve 50% maximal effect; (bioavailability); PBMC, peripheral blood mononuclear cell; Tmax, time to maximal plasma concentration; Vz, terminal volume of distribution; Vz/F, terminal volume of distribution adjusted for bioavailability. 0022-3565/02/3032-540–548$7.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 303, No. 2 Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics 37002/1014527 JPET 303:540–548, 2002 Printed in U.S.A. 540 at A PE T Jornals on O cber 2, 2017 jpet.asjournals.org D ow nladed from jugation of polyethylene glycol (PEG) to IFNsignificantly decreases plasma drug clearance, so that treatment using a once-weekly injection schedule is possible. Two versions of pegylated IFNare commercially available. PEG Intron (Schering Plough, Kenilworth, NJ), with a linear 12-kDa PEG molecule, has a t1/2 in humans of approximately 35 h (Glue et al., 2000). A larger molecule, approved only in Europe at this time, contains a branched 40-kDa PEG molecule and has a t1/2 of 77 h in humans (Perry and Jarvis, 2001). In this paper we present an alternative strategy to pegylation, which also permits less frequent dosing. Allometric scaling of pharmacokinetic data from monkeys to humans predicts a longer half-life than that resulting from pegylation of IFN. This might reduce the occurrence of adverse events and increase patient compliance, which has the potential to further increase efficacy. In an attempt to improve the pharmacokinetic and pharmacodynamic profile of IFN, a novel genetic fusion product composed of recombinant human serum albumin and recombinant IFN, Albuferon fusion protein (Human Genome Sciences Inc.), has been developed. Albumin is an ideal candidate for conjugated IFN sustained-activity agents as it is the most prevalent naturally occurring blood protein in the human circulatory system, and it has a long half-life of 19 days (Peters, 1996). Albumin has little enzymatic or immunological function and is widely distributed in vivo (Peters, 1996). More importantly, research has shown that therapeutic proteins genetically fused with albumin have longer circulating half-lives and improved stability characteristics (Yeh et al., 1992; Syed et al., 1997). Albuferon was designed to combine the activity of IFNwith the pharmacokinetic advantages of a protein such as albumin. The objective is to reduce the dosing frequency while potentially improving efficacy and reducing side effects of conventional IFN therapies. To evaluate the in vitro properties, the antiviral and antiproliferative activities of Albuferon and IFNwere compared in a number of cell lines. To evaluate the in vivo properties of such a fusion protein, a pharmacokinetic and pharmacodynamic study of Albuferon was conducted in nonhuman primates. The objectives were: 1) to describe the pharmacokinetic behavior of Albuferon after a single intravenous or subcutaneous dose, 2) to evaluate the ability of Albuferon and IFNin serum sampled at various time points after drug administration to stimulate antiviral activity in an in vitro bioassay, 3) to evaluate whole blood samples for activation of 2 ,5 -oligoadenylate synthetase (2 ,5 -OAS) genes after Albuferon and IFNtreatment as a biomarker of antiviral activity, and 4) to evaluate the immunogenicity of Albuferon. Materials and Methods