BIOPHARMACEUTICS & DRUG DISPOSITIONBiopharm. Drug Dispos. 29: 481–484 (2008)

Published online 4 November 2008 in Wiley InterScience

(www.interscience.wiley.com) DOI: 10.1002/bdd.630

SHORT COMMUNICATION

Pharmacokinetic Properties of a 40 kDa BranchedPolyethylene Glycol-modified Form of Consensus Interferon-a (PEG-CIFN) in Rhesus Monkeys

Yu Du, Hong Tian, Xiang-Dong Gao and Wen-Bing Yao�

School of Life Science and Technology, China Pharmaceutical University, Nanjing, China

ABSTRACT: The pharmacokinetic properties of a branched 40 kDa polyethylene glycol (PEG)conjugate formulation of consensus interferon-a (CIFN) was evaluated in rhesus monkeysfollowing subcutaneous administration. Four groups of rhesus monkeys (n 5 6 per group) received1250, 300 and 150 mg=kg of PEG-CIFN and 150 mg=kg CIFN, respectively. Serum concentrations ofthe interferons were measured with an antiviral activity assay at various time points afteradministration. The PK profiles of the pooled data were described by a noncompartmental method.Peak concentration of PEG-CIFN was observed at 27–31 h, followed by a prolonged decay incomparison with the unmodified CIFN, the PEG-CIFN had a 4–5-fold longer terminal half-life. Theapparent clearance (dosesc=AUC) decreased from 150 mL=h=kg for CIFN to 19.0–45.5 mL=h=kg forPEG-CIFNs. The AUC was lower for PEG-CIFN than that for CIFN at the 150 mg=kg. Copyright r2008 John Wiley & Sons, Ltd.

Key words: consensus interferon-a; pharmacokinetics; polyethylene glycol; bioactivity; antiviralactivity assay

Introduction

Consensus interferon-a (CIFN) is a synthetic typeI interferon, derived by scanning several inter-feron-a nonallelic subtypes and assigning themost frequently observed amino acid in eachposition [1,2]. CIFN has demonstrated a highin vitro antiviral activity that is equivalent to thatof IFN-a-2a or a-2b, suggesting that CIFN may bemore effective at lower concentrations than otherIFNs clinically [3]. Studies have shown that CIFNis a safe and effective therapy for patients withchronic hepatitis C virus (HCV) who have notresponded to previous interferon therapy or

relapsed after discontinuation of interferon ther-apy [4,5], and offers a potential advantage in thetreatment [6,7].

Interferons are metabolized rapidly; the half-life is in the order of a few hours. Thus,PEGylated forms are developed to improve thepoor pharmacokinetic profiles [8] and enhancethe pharmacological properties by reducingclearance [9,10] and potentially reducing proteinimmunogenicity [11,12]. Bailon et al. reportedthat PEGinterferon a-2a (40 kDa) displays aprolonged elimination half-life of 77 h comparedwith 5 h for the unmodified IFN [13]. Anotherstudy confirmed that systemic clearance and theelimination half-life of PEGylated IFN-a-2b(12 kDa) are 10-fold lower and 7-fold greater,respectively, than those of unmodified IFN-a [14].Here, the pharmacokinetic behavior of PEG-CIFN after a single subcutaneous dose in rhesus

*Correspondence to: School of Life Science and Technology,China Pharmaceutical University, Nanjing, 210009, China.E-mail: wbyao@cpu.edu.cn

Received 17 January 2008Revised 10 July 2008

Accepted 15 August 2008Copyright r 2008 John Wiley & Sons, Ltd.

monkeys is described to highlight the effects ofPEGylation on CIFN.

Materials and Methods

Materials

CIFN was purified from E. coli with twoconsecutive procedures of Q-Sepharose and CMSepharose chromatographies (Amersham Phar-macia). The purity of the CIFN verified byreverse-phase HPLC was 98%, and there wasno evidence of aggregated material by sizeexclusion HPLC.

PEGylation was conducted in sodium boratebuffer with CIFN and 40 kDa mPEG-2-N-hydro-xysuccinimide (NHS), and then the product waspurified by a Q-Sepharose column. The unmodi-fied protein or multiple modified forms withadditional PEG groups were not detected in thepurified PEG-CIFN by SDS-PAGE. Size exclusionHPLC resolved the mono-PEGylated proteinfrom oligo-PEG-CIFN forms to result in a singlemodified PEG-CIFN of 95% purity. The molecu-lar weights of CIFN and PEG-CIFN from MAL-DI-TOF-MS were 19.6 kDa and 62.6 kDa,respectively (data not shown).

Animals

This study was conducted in adherence to the‘Principles of Laboratory Animal Care’ (NIHPublication No. 85-23, revised in 1985) andapproved by the Jiangsu Administration Officeof Laboratory Animals. Twenty four rhesusmonkeys (Macaca mulatta), weighing 2.2–4 kg, ingood health were used for the study.

Pharmacokinetics

Four groups of rhesus monkeys (six per group)received 1250 mg=kg, 300 mg=kg, 150 mg=kg PEG-CIFN, and 150 mg=kg CIFN subcutaneously un-der the skin on the back. Blood samples weredrawn at the specified time points: PEG-CIFN at0 (pre-dose), 4, 8, 12, 24, 28, 32, 48, 96, 120, 144,168, 192, 216 and 336 h; CIFN at 0, 1, 2, 3, 4, 6, 12,24, 48, 72, 96, 120 and 140 h. Samples wereallowed to clot and centrifuged at 14000� g for10 min to obtain serum, then stored at �701C

until analysed. Levels of interferons were deter-mined using the antiviral activity assay describedby Rubinstein et al. [15]. Pharmacokinetic para-meters were calculated using the Drug andStatistics software version 1.0 (manufactured bythe Mathematical Pharmacology ProfessionalCommittee of China). The AUC(infinity) wasestimated as the sum of the AUC(0–last) bytrapezoidal rule, and the extrapolated area(Clast=elimination rate constant).

Statistical analysis

All values are expressed as mean7SD. Thestatistical significance was tested with an un-paired Student’s t-test. A value of po0.05 wasdeemed to be statistically significant.

Results and Discussion

The quantity of CIFN and PEG-CIFN wasdetermined relative to a reference preparationof interferon. Compared with the CIFN, the PEG-CIFN retained approximately 12% of antiviral

activity (8.1�107 U=mg). This observation iscomparable to the marketed PEGylated IFN-adrug, PegasysTM, which carries a 40 kDabranched mPEG and is about 7% of the un-modified protein in vitro antiviral activity [13]. Itis clear that conjugates with 40 kDa mPEG have

Figure 1. Mean serum antiviral activity versus time of PEG-CIFN and CIFN after single-dose subcutaneous injection inmonkeys. Blood from these animals was collected at the timepoints indicated and assayed for levels of consensusinterferon in the antiviral assay. Bars represent standarddeviation

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Copyright r 2008 John Wiley & Sons, Ltd. Biopharm. Drug Dispos. 29: 481–484 (2008)DOI: 10.1002/bdd

lower antiviral activity in vitro, presumablybecause the large sized PEG interfered withbinding with the Type I interferon.

The pooled mean serum curves for CIFN andPEG-CIFN that define the PK parameters ofdifferent doses are shown in Figure 1. The areaunder the concentration-time curve from zero toinfinity (AUC), apparent clearance (CL=F), elim-ination half-life (t1=2), and other pharmacokineticparameters are shown in Table 1. CIFN peaked at6 h post-dose and was cleared rapidly. Bycontrast, serum PEG-CIFN was maintained at arelatively high level over a period of 70 h beforeslowly declining and exhibited similar absorp-tion and elimination profiles for all doses. All thethree doses of PEG-CIFN demonstrated pro-longed terminal half-lives of similar values(34–38 h) and increased Tmax (�5-fold) relativeto those for the CIFN. The apparent clearance forPEG-CIFN via the subcutaneous route wasreduced �5-fold compared with the CIFN. Themaximal observed concentration (Cmax) for theCIFN was higher than that for the PEGylatedprotein at 150 mg=kg as PEGylation reduced theantiviral activity of the interferon. PEGylationalso yields a lower AUC compared with the samedose of CIFN (150 mg=kg), suggesting a lowerexposure of the PEGylated form. Monkeysreceiving 1250 mg=kg and 300 mg=kg of PEG-CIFNdisplayed disproportionately higher AUCs about1.6-fold and 2.4-fold, relative to that for the150 mg=kg dose. This finding suggests some formof nonlinearity within the PEG-CIFN doses.

This study quantified the impact of PEGylationon CIFN pharmacokinetics. With a single 40 kDamPEG attached, a relatively constant exposure ofthe PEG-CIFN was observed for 70 h post-peak.Moreover, PEGylation resulted in increased t1=2

and decreased the apparent clearance compared

with CIFN. The lower relative bioavailabilitymay suggest the reduced antiviral activity result-ing from PEGylation and possible bindingoccurred at the site of injection when adminis-tered subcutaneously. The pharmacokinetic be-havior of PEG-CIFN in more detail deservesfurther study.

Acknowledgements

This work is supported by the High Technologiesof China (2004AA2Z3380, 2007AA02Z101), Pro-gram for New Century Excellent Talents inUniversity (NCET-04-0506) and the ‘Six TalentPeaks’ of Jiangsu Province (06-C-027).

References

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Table 1. Pharmacokinetic parameters for CIFN and PEG-CIFN in Rhesus monkeys following subcutaneous administration

Treatment Dose(mg=kg)

Cmax

(U=mL � 103)Tmax (h) AUC

(U �h=mL � 105)AUC=dose(U �h=mL � 103)=(mg=kg)

t1=2 (h) CL=F(mL=h=kg)

CIFN 150 47715a 6.070.1a 7.572.5 5.071.7 8.770.6a 150740a

PEG-CIFN 150 2.7570.06 2773 2.6870.16 1.7870.11 3476 45.572.7300 1473 3179 13.071.6 4.370.5 3675 19.072.41250 26.872.4 28.072.5 35.872.8 2.8670.23 3873 28.572.4

aSignificantly different (po0.05).

Values are expressed as mean7SD.

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6. Poynard T, Bedossa P, Chevallier M, et al. Acomparison of three interferon alfa-2b regimensfor the long-term treatment of chronic non-A, non-B hepatitis. N Engl J Med 1995; 332: 1457–1463.

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Copyright r 2008 John Wiley & Sons, Ltd. Biopharm. Drug Dispos. 29: 481–484 (2008)DOI: 10.1002/bdd