Antiviral Chemistry & Chemotherapy 15:287–297

Review Pegylating IFNs at His-34 improves the in vitro antiviral activity through the JAK/STAT pathway Michael J Grace1* and David Cutler2 1

Schering-Plough Research Institute, Biotechnology Development, Union, NJ, USA Schering-Plough Research Institute, Early Clinical Research & Experimental Medicine, Kenilworth, NJ, USA

2

*Corresponding author: Tel: +1 908 820 6816; Fax: +1 908 820 6829; E-mail: [email protected]

Pegylated interferon alpha-2, alone or in combination with ribavirin, has become the standard therapy for patients with chronic hepatitis C infection. Pegylation of interferon alpha-2 results in a substantially extended half-life that permits once-weekly dosing, because of reduced clearance and more sustained absorption. The size of PEG moiety appears to influence the relative antiviral activities of peginterferon alpha-2. Increasing the size of the polyethylene glycol (PEG) moiety results in a reduction of the specific antiviral activity of the pegylated protein. For example, peginterferon alpha-2b (12 kDa) has an in vitro antiviral specific activity 25–35-fold higher than peginterferon alpha-2a (40 kDa). The antiviral activity of pegylated interferon alpha-2 is also governed by the site of pegylation of the interferon alpha core proteins. Interferon alpha2a is monopegylated at four major positional lysine (Lys) residues. The major site of interferon

alpha-2b monopegylation is histidine (His34), with additional pegylation sites at lysine and cysteine residues. The 12 kDa pegylated His34 positional isomer of peginterferon alpha-2b has the highest antiviral and antiproliferative in vitro specific activity compared with both the 12 kDa Lys positional isomers and the 40 kDa Lys positional isomers. The correlative effects of size and site of pegylation on the JAK/STAT signalling pathway, as evidenced by differences in the formation of the Stat1 homodimer complex, are suggestive of a receptor-mediated mechanism that governs the antiviral activity of pegylated interferons. The elucidation of the in vitro effects of pegylation is important and this will ultimately have a positive impact on the in vivo efficacy of treatment for patients with hepatitis C. Keywords: pegylation, histidine, peginterferon alpha, antiviral activity, PEG

Introduction Hepatitis C virus (HCV), a small RNA virus identified in 1989, is the leading cause of chronic viral liver disease and the principal indication for liver transplantation in the USA and Europe (Choo et al., 1989). It is estimated that HCV currently infects 170 million people, 3% of the world’s population, with 3–4 million people newly infected each year (Lauer & Walker, 2001). Approximately 80% of patients with acute hepatitis C develop chronic infection, defined as persistently detectable HCV ribonucleic acid for at least 6 months after exposure, with about 20% of patients progressing to cirrhosis (Poynard et al., 1997; Hoofnagle & Di Bisceglie, 1997; Leung, 2002). Chronic infection with HCV is also associated with a 1–4% increased risk of hepatocellular carcinoma (Caselmann & Alt, 1996; Fattovich et al., 1997). Transmission of HCV is primarily by blood-toblood contact and, in the Western world, infection is common in those with a history of drug abuse or who

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received contaminated blood products before the introduction of screening. There are six major HCV genotypes (designated 1–6) and multiple subtypes (designated a, b, c etc.) and their global distribution varies widely (Zein, 2000). Knowledge of the genotype is predictive of antiviral response and genotypes 1 and 4 are more resistant to therapy than genotypes 2 and 3 (Zylberberg et al., 2000; National Institutes of Health, 2002; Leung, 2002). Until recently, the most effective treatment for chronic hepatitis C (CHC) was subcutaneous injection of either recombinant interferon alpha-2b or interferon alpha-2a combined with oral ribavirin, a broad-spectrum antiviral nucleoside analogue whose exact mechanism of action is unclear. Ribavirin may interfere with viral transcription and inhibit ribonucleoprotein synthesis and also may have an immunomodulatory effect (Heagy et al., 1991; European Association for the Study of Liver Disease, 1999; Crotty et

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al., 2000; Maag et al., 2001; Hepatitis Interventional Therapy Group et al., 1998; International Hepatitis International Therapy Group et al., 1998). However, both interferon drugs have short serum half-lives (50%)

PEG-IFN alpha-2a Lys31, 121, 131, 134

O C

O O

R1

R2

O

C

NH (CH2)4

O

O

HC

C

C

NH

N IFN alpha-2b N R2 R1 = CH3(OCH2CH2)n where n=239–318

biologically active mono-pegylated positional isomers (Grace et al., 2001; Wang et al., 2002). The primary site of conjugation is on the histidine (His)34 amino acid residue (≥50% of all the positional isomers) (Grace et al., 2001; Wang et al., 2002) (Figure 3).The remaining positional isomer sites that make up the other 50% are distributed among the various lysine (Lys) residues, another histidine residue, cysteine and serine residues (Table 1). In terms of antiviral activity, the His34 positional isomer has been shown to be the most active, possessing approximately 37% of the antiviral activity of interferon alpha-2b. The antiviral activity of peginterferon alpha-2b is about 28% by weight of the interferon alpha-2b core protein compared with interferon alpha-2b (Grace et al., 2001). The specific activity for peginterferon alpha-2b is 7.3×107 IU/mg, whereas the average specific activity for interferon alpha-2b is 2.6×108 IU/mg (Table 2). Peginterferon alpha-2a has a 40 kDa branched PEG molecule attached by a covalent bond to the interferon alpha-2a core protein. The N-hydroxysuccinimid (NHS) pegylation chemistry and manufacturing process leads to a mixture of four major positional isomers at Lys31, Lys121, Lys131 and Lys134 and several minor positional isomers (Bailon et al., 2001) (Figure 3). The antiviral activity of peginterferon alpha-2a is approximately 1% of the antiviral activity of interferon alpha-2a (Foser et al., 2003). Interestingly, the Lys31 and Lys134 positional isomers of peginterferon alpha-2a have been shown to be more active than the mixture, possessing approximately 2% of the antiviral activity of interferon alpha-2b (Foser et al., 2003).

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O

H N

IFN alpha-2a

R2 = CH3(OCH2CH2)n where n=420–510

Table 1. The positional isomers of peginterferon alpha-2b and peginterferon alpha-2a* Peginterferon alpha-2b

Peginterferon alpha-2a

Major positional isomers

His34 (>50%) Cys1 Lys31 Lys121 Lys49

Lys31 Lys121 Lys131 Lys134

Minor positional isomers

Lys83 Lys112 Lys161 Lys133 His7 Tyr129 Ser163

Lys70 Lys83 Lys49 Lys112 Lys164 Lys23 Lys133

*Grace et al., 2001; Foser et al., 2003. Cys, cysteine; His, histidine; Lys, lysine; Ser, serine; Tyr, tyrosine.

The specific activity for peginterferon alpha-2a has been reported to be about 1.1×106 IU/mg, while the average specific activity for interferon alpha-2a is 2.4×108 IU/mg (Table 2). The differences in the size of the PEG molecules and the distribution of the monopegylated isomers are believed to be the contributing factors in the difference in antiviral activity observed between the two peginterferons. In particular,

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Figure 3. Positional assignment map of PEG molecules for PEG isomers of interferon alpha-2b and interferon alpha-2a proteins for peginterferon alpha-2b and peginterferon alpha-2a, respectively

B

A

(A) Pearl diagrammatic representation of interferon alpha-2b. (B) Pearl diagrammatic representation of interferon alpha-2a.

Table 2. Specific activity of the interferon alphas Interferon

Specific activity

CPE and anti-viral assay

Peginterferon alpha-2a Peginterferon alpha-2b Interferon alpha-2a Interferon alpha-2b

1.1×106 7.3×107 2.4×108 2.6×108

MDBK cells/VSV virus infection (Foser et al., 2003) FS-71 cells/EMC virus infection (Grace et al., 2001) FS-71 cells/EMC virus infection (Schering-Plough data on file) FS-71 cells/EMC virus infection (Shering-Plough data on file)

IU/mg IU/mg IU/mg IU/mg

peginterferon alpha-2a does not contain the highly active His34 isomer.

Impact of size and site of pegylation on the pharmacology of pegylated interferon alphas Both the position and size of the PEG group are important to the physiological and pharmacological characteristics of pegylated interferons. For instance, there is a direct corre-

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lation between the molecular size of the attached PEG group and the in vivo half-life of a pegylated protein (Yamaoka et al., 1994). Pegylated interferon alpha-2b has a prolonged serum half-life of 40 h compared with 7–9 h for interferon alpha-2b (Glue et al., 2000a). The larger PEG size of interferon alpha-2a acts to further reduce glomerular filtration of the molecule, thus increasing its half-life to 72–96 h relative to interferon alpha-2a (6–9 h) (Reddy et al., 2001). Conversely, pegylation can reduce the specific activity of the conjugated protein (Bailon &

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Influence of pegylation on the antiviral activity of interferons

Berthold, 1998). As discussed earlier, pegylation of the interferon alpha-2a and alpha-2b core proteins results in reduced antiviral activity. For both peginterferon alpha-2a and alpha-2b, the loss in activity does not appear to be due to a structural perturbation of the core interferon alpha-2b protein (Grace et al., 2001). However, there is a significant difference between the antiviral activities that have been reported for peginterferon alpha-2a and for peginterferon alpha-2b. The mechanism of action of peginterferon alpha-2b against HCV is presumed to be the same as that of nonpegylated recombinant human interferon alpha-2b. Interferons induce their antiviral activity via extracellular receptor binding. This receptor interaction is cogent either for the induction of a direct antiviral or antiproliferative signalling activity on an infected hepatocyte, or for the induction of an indirect immunomodulatory antiviral activity via on the immune system. The interferon alpha signalling pathway involves the activation of Janus kinase ( Jak)1 and Tyk2 tyrosine kinase, initiated by the binding of interferon alpha-2 or peginterferon alpha-2 to the interferon heterodimeric receptor complex (IFNAR1/ IFNAR2), which leads to activation of their downstream substrates, signal transducers and activators of transcription (Stat)1 and Stat2. Activated Stat1 and Stat2 then assemble as a multimeric complex and translocate into the nucleus where they bind to interferon alpha-2-stimulated response elements in the promoters of interferon alpha-2-stimulated genes (Figure 4) (Schindler & Darnell, 1995; Ihle, 1996). Several laboratories have reported that the antiviral and antiproliferative activities of interferon alpha-2 are dependent upon JAK/STAT signalling through a potentially rate-limited interaction with the IFNAR1/IFNAR2 heterodimeric receptor complex (Lee et al., 1997; Pestka, 1997; Piehler et al., 2000; Kozlowski et al., 2001). Recently, in vitro Stat1 translocation studies investigated the effect of pegylation size and site on JAK/STAT interferon alpha signal transduction (Grace et al., 2003a). The advantage of using Stat1 translocation assays for signalling assessment is that the translocation event is linked to both the receptor–ligand interaction and the ultimate expression of interferon alpha gene transcription, which is necessary for antiviral and antiproliferative activity. Because antiviral assay results can vary between laboratories due to differences in assay methodology and reference standards, in vitro studies comparing the antiviral and antiproliferative activities of peginterferon alpha-2b and peginterferon alpha-2a have been performed in one laboratory. These assays have shown that both the antiviral and antiproliferative activities of peginterferon alpha-2b are 25–35-fold higher than those of peginterferon alpha-2a (Grace et al., 2003a; Cox et al., 2002; Leaman et al., 2002; Brassard et al., 2002). The higher activity associated with

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peginterferon alpha-2b was shown to occur early in the signalling cascade, during the formation of the STAT homodimer and subsequent nuclear translocation. Nuclear translocation is a requisite for a STAT-mediated, interferon-induced response gene mRNA transcription, which is needed for the antiviral and antiproliferation response. The formation of the Stat1 homodimer complex, an event upstream of nuclear translocation, was significantly diminished with peginterferon alpha-2a as compared with peginterferon alpha-2b. It is particularly interesting to note the disparate specific activities observed between the major pegylated isomer for peginterferon alpha-2b (12 kDa His34) and that for peginterferon alpha-2a (40 kDa Lys31). The difference in activity for these two pegylated isomers may seem surprising since they are only separated by two amino acids and are located in close proximity on the AB1 loop (amino acids 22–51) of interferon alpha-2, which appears to be involved in one of two putative binding domains for IFNAR2 (Radhakrishnan et al., 1996; Walter, 1997; Roisman et al., 2001). Thus, the size of the PEG moiety appears to influence the relative antiviral activities of the 12 kDa peginterferon alpha-2b and the 40 kDa peginterferon alpha-2a; in particular, increasing the size of the PEG moiety results in a reduction of the specific antiviral activity of the pegylated protein. The different distribution of site-pegylated isomers, differences in the core interferon alpha proteins and/or differences in the bonds linking the PEG molecules to their respective core protein may also result in the differences observed in the relative antiviral and antiproliferative activities of the two peginterferon alphas (Kozlowski et al., 2001; Luxon et al., 2002). Biological characterization studies of the positional isomers of peginterferon alpha-2b have revealed that these have significantly different relative specific antiviral activities. In particular, the His34 positional isomer of peginterferon alpha-2b displays the highest specific antiviral activity (37% of that of interferon alpha-2b), whereas the lysine and N-terminal-pegylated positional isomers retain approximately 11% of the native interferon alpha-2b activity (Grace et al., 2001). Furthermore, NMR studies on peginteferon alpha-2b have revealed that the His34 positional isomer is mainly pegylated with a urethane, or urethane-like, linkage at the Nδ1 position of the imidazole side chain (Figure 5) (Wang et al., 2000). Similar urethane-like bonds are created with the εN on the respective pegylated lysine R-groups. On the other hand, the NHS-chemistry process used to manufacture peginterferon alpha-2a results in the creation of an amide bond with the ε-N on the pegylated lysine R-groups (Bailon & Berthold, 1998; Foser et al., 2003). Additional studies will be required to elucidate the respective roles of PEG size, site of pegylation and the bonds linking the

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Figure 4. Interferon alpha signalling pathway

Type I IFN IFN-α/β

Type II IFN IFN-γ IFNAR1

IFNAR1

IFNGR1 IFNGR2

IFN-α/β IFNAR2

IFNAR2 Jak2 Jak1 Jak1 Tyk2

Jak1 Tyk2

Jak2 Jak1

Jak1 Tyk2

P

Stat2 Stat1

P

Stat2

P P

Stat1 Stat2

Stat1

P

P

P P

P

P

P

P

P

Stat1

Stat1

Stat1 Stat1

IRF-9

P P

IRF9

P P

IRF9

ISGF3 Stat1-p48

P P

GAF/AAF

Nucleus IRF-1

ISRE

IRF-E

GAS

IRF-1 gene IRF-1

IRF-2

Activation of Jak1 and Tyk2 tyrosine kinases is initiated by the binding of interferon alpha-2 to the interferon heterodimeric receptor complex (IFNAR1/IFNAR2), which leads to activation of their downstream substrates, signal transducers and activators of transcription Stat1 and Stat2. Activated Stat1 and Stat2 then assemble as a multimeric complex and translocate into the nucleus where they bind to interferon alpha-2-stimulated response elements in the promoters of interferon alpha-2-stimulated genes.

PEG molecules to their respective core protein on the direct impact to antiviral activity. Most likely, all three have a role in the mechanism that attenuates the interferon alpha core protein activity associated with pegylation. One potential explanation for the higher activity of His34 is that the site of pegylation may reduce the impact of steric hindrance from the PEG molecule (Figure 6). A model of interferon alpha-2b binding to IFNAR1/ IFNAR2, based on X-ray crystallography, suggests that His34 is not located at the receptor interface, even though it is on the AB1 loop, but rather it is located at the interferon alpha-2b dimer interface (Nagabhushan et al., 2002). Whether the pegylation at the dimer interface is responsible for the reduction in activity associated with the His34 pegylated isomer is an interesting hypothesis for further

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Figure 5. The His34 positional isomer is mainly pegylated at the Nδ1 position of the imidazole side chain

His 34

Hδ2

O H3C-(OCH3CH2)n-O-C-Nδ1

Nε1 Hε1

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Influence of pegylation on the antiviral activity of interferons

Figure 6. Steric hindrance of peginterferon alpha-2b

A

B

(A) Interferon alpha-2b. (B) Peginterferon alpha-2b.

study. The Lys31 site is, however, located within, or subsequent pegylation may influence, the thermodynamically important IFNAR2 cognate interaction site on interferon alpha-2 (Roisman et al., 2001). In agreement with these data, the 12 kDa Lys31 isomer for peginterferon alpha-2b retains only 11% antiviral activity compared with the 12 kDa His34 isomer’s 37% antiviral activity (Grace et al., 2001). The additional impact of increasing the PEG size to 40 kDa might be the cause of the marked 98% decrease in the bioactivity reported for the 40 kDa Lys31 isomer for peginterferon alpha-2a (Foser et al., 2003). The impact of the change in PEG size on potential steric hinderance cannot be understated. The binding surface area of interaction between interferon alpha-2 and IFNAR2 resides at two domains and is estimated to be about 2450 Å2 (Roisman et al., 2001). The Stokes radius of peginterferon alpha-2b is estimated to be about 21 Å; a single, linear 12 kDa PEG molecule attached to interferon alpha-2b increases the resulting Stokes radius to about 46 Å (Schering-Plough data on file). Thus, pegylation with 12 kDa essentially fills the available binding domain area

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for interferon alpha-2 and IFNAR2. Obviously, a 40 kDa PEG molecule will have a concomitantly greater Stokes radius, creating greater potential disruption between the two binding domains. Therefore, a larger PEG molecule may significantly encroach into the binding domains, leading to the decreased antiviral activity observed with increased PEG molecule size. Alternatively, increased steric hindrance at the dimer binding surface may also contribute to the decreased activity observed with increased PEG molecule size. A second possible explanation for diminished antiviral activity based on the site of pegylation is a charge-based differential that influences the interferon alpha-2 core protein and IFNAR1/IFNAR2 interaction kinetics (Schreiber & Fersht, 1996; Piehler et al., 2000). It may be possible to postulate that the loss in positive charge on the imidazole ring associated with His34 pegylation may perturb the electrostatic potential at or near the receptor binding site less than the loss in positive charge that occurs with pegylation on the ε-amine of lysine (Wylie et al., 2001). However, it has also been observed that the

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interferon alpha binding site on IFNAR2 is not a highly negatively charged area on the protein surface, and this potentially limits the role for electrostatic forces in determining the rate of association (Piehler & Schreiber, 1999). Nevertheless, a cooperative interaction, first through electrostatic steering, then followed by docking of the interferon alpha-2b and IFNAR1 and IFNAR2, might be impacted by the site of pegylation. In both cases, the His34 site of pegylation appears to be favoured relative to Lys31 on the core interferon alpha-2 protein.

Stability of pegylation It has been suggested that the urethane-like bond formed by the SC conjugation chemistry with the imidazole ring of His34 is hydrolytically labile (Kozlowski & Harris, 2001). In such an instance, one can hypothesise that the resultant hydrolytic depegylation would lead to the binding surface of the interferon alpha-2b becoming more available for receptor interaction. This may result in the increased specific activity of this isomer. However, this hypothesis becomes problematic when one attempts to interpret simple organic chemistry principles in the context of complex macromolecular protein chemistry. For example, NMR studies have shown that the urethane-like bond for 12 kDa His34 is 18-fold more stable in the context of the tertiary structure of the interferon alpha-2 protein than when it is found in a simple peptide (Wang et al., 2000). Furthermore, these studies determined a pseudo-first order rate dissociation constant for peginterferon alpha-2b of around 0.0016/h (Table 3). Follow-up studies investigated peginterferon alpha-2b stability in human serum at 37oC. These studies used HPLC recovery of intact peginterferon alpha-2b and free interferon alpha-2b followed by antiviral bioassay. The results demonstrated that the His34 isomer had a pseudofirst order dissociation constant of 0.0042/h. Interestingly, the pooled pegylated lysine isomers also exhibited depegylation. It is not clear whether the depegylation observed was hydrolytic or proteolytic (see Table 3). Additional in vitro studies have also confirmed that the higher STAT translocation, antiviral and antiproliferative activities for the 12 kDa His34 positional isomer occur with the intact pegylated molecule, and are not due to depegylation and liberation of free interferon alpha-2b (Grace et al., 2003b; Cox et al., 2002; Leaman et al., 2002; Brassard et al., 2002; Brassard et al., 2004). Thus, pegylation at the His34 site does not result in reduced stability of peginterferon alpha-2b. Therefore, the higher activity observed with the His34 pegylated isomer cannot readily be explained by the hydrolytic release of free interferon alpha-2b and must be due to other mechanisms. It should also be noted that the relatively slow depeglyation

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of peginterferon alpha-2b in human serum occurs at a rate that does not theoretically limit the antiviral activity of peginterferon alpha-2b relative to the once-weekly dosing schedule of the drug (Poynard et al., 2002; Grace et al., 2001; Lindsay et al., 2001).

Future perspectives Pegylated interferon alpha-2 in combination with ribavirin is now the recommended treatment of choice for CHC. Compared to their native interferon alphas, peginterferon alpha-2b and peginterferon alpha-2a have both demonstrated substantially extended half-lives that permit onceweekly dosing. Also, both peginterferons have produced sustained viral response rates superior to those achieved with their respective unpegylated formulations. Pegylation of the interferon alpha-2 core protein leads to the loss of in vitro activity that does not appear to be a result of structural perturbation of the core protein. Rather, the overall in vitro activity of peginterferon alpha-2 is governed by the size of the attached PEG molecule and by the specific siteof-pegylation. The 12 kDa peginterferon alpha-2b demonstrates a 25–35-fold higher in vitro antiviral specific activity than 40 kDa peginterferon alpha-2a.

Peginterferon alpha-2b and peginterferon alpha-2a differ not only in the size of their PEG group but also in the distribution of pegylated isomers. Peginterferon alpha2a contains only lysine isomers while peginterferon alpha2b contains >50% His34 pegylated isomer. The 12 kDa pegylated-His34 positional isomer has a significantly greater antiviral and antiproliferative in vitro specific activity compared with both the 12 kDa Lys positional isomers and the 40 kDa Lys positional isomers reported for peginterferon alpha-2a. Taken together, these data suggest that the His34 positional isomer is responsible for the higher specific activity observed for peginterferon alpha-2b compared with peginterferon alpha-2a. Furthermore, stability and dissociation kinetics studies have shown that pegylation at His34 site does not reduce the stability of peginterferon alpha-2b. The correlative effects of size and site of pegylation observed so far, and the connection to the STAT/JAK signalling pathway, allude to a receptor-mediated mechanism. This suggests that reduced antiviral activity in vitro may result from a pegylation chemistry that interferes with the interaction and binding of interferon alpha to the IFNAR1/IFNAR2 heterodimeric receptor. The choice of the size of PEG molecule and the site of pegylation are important in producing the molecule with the highest in vitro activity that also retains superior in vivo pharmacokinetics. The precise mechanisms of action that govern the diminution of activity at the receptor are still speculative

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Influence of pegylation on the antiviral activity of interferons

Table 3. Stability of peginterferon alpha-2b and its major positional isomers Pseudo first order Rate association (h-1) Peginterferon alpha-2b His34 isomer of peginterferon alpha-2b Pooled lysine isomers of peginterferon alpha-2b

0.0016 0.0042 0.0012

Method

Conditions

NMR (Wang et al., 2000) HPLC x bioassay (Schering-Plough data on file) HPLC x bioassay (Schering-Plough) data on file)

25˚C at pH 5.5 isolated 37˚C at pH 7.0 in 10% nhs 37˚C at pH 7.0 in 10% nhs

His, histidine; nhs, normal human serum.

and further investigation is needed. However, the elucidation of the in vitro effects of pegylation is very important for understanding the best balance between high receptor activity and prolonged serum half-life. This understanding will ultimately have a positive impact on the in vivo efficacy for the treatment of patients with hepatitis C.

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