to Modern Drug Discovery home
September 2000
Modern Drug Discovery, 2000, 5(7) 59–60, 62, 65, 67.
© 2000 American Chemical Society.


Improving Hepatitis C Therapy

opening artAttaching a polyethylene glycol “tail” to interferon for better clinical properties

BY STEPHEN A. CHARLES, J. MILTON HARRIS, SIMON PEDDER, AND SARAN KUMAR

The hepatitis C virus (HCV) is one of the major causes of liver disease. Nearly 4 million people in the United States have antibodies to HCV, which indicates at least previous exposure to the disease if not active infection. Acute infections can develop into chronic hepatic diseases, including cirrhosis, liver failure, and liver cancer. About 20% of those infected develop cirrhosis after 10 years or more of infection. Chronic infection leading to liver failure accounts for many of the liver transplants performed in the United States. Many researchers believe that HCV is the most common cause of primary liver cancer in the developed world (1).

Interferon- (IFN-) monotherapy and the combination of IFN- with ribavirin are the only approved treatments for patients with chronic hepatitis C (2). The delivery of IFN-, however, represents a significant clinical challenge. Following intravenous administration, serum concentrations of IFN- decline rapidly. The terminal half-life of IFN- ranges from 4 to 16 h with serum concentrations being measurable between 8 and 24 h after administration (3). Following subcutaneous administration, the absorption of IFN- is rapid, and large fluctuations in serum concentrations occur after each dose. This fluctuation is clinically important, as serum concentrations are largely undetectable for much of the dosing interval despite the drug being administered three times a week. Also, the sharp rises and declines in IFN serum concentrations have been correlated with the severity of the acute flulike symptoms that are seen when IFN is administered (4).

This article traces the development of PEGASYS, a polyethylene glycol (PEG)-modified IFN--2a designed to improve upon the pharmacokinetics of IFN--therapy.

PEGylation
...
Figure 1. PEG (40 kDa)–IFN--2a was modified with activated, branched mPEG-disubstituted lysine with an average molecular weight of 40 kDa.
PEGylation is the process of attaching one or more chains of PEG to another molecule (Figure 1). PEG-conjugated proteins belong to a class of biomolecules that are protein and polymer hybrids (5). PEGylation can alter the characteristics of drugs without affecting the parent molecule’s inherent ability to perform its function (although specific activity can be affected). PEGs of a variety of sizes are nontoxic, nonimmunogenic, highly water soluble, and readily cleared from the body. PEG has many different applications and is commonly used in foods, cosmetics, beverages, and prescription medicines. PEGs are approved for use in the United States by the FDA.

Why attach PEG to drugs? PEGylation of therapeutic proteins and enzymes may provide improved clinical properties, including enhanced solubility, sustained absorption, reduced immunogenicity and proteolysis, reduced renal clearance, decreased dosing due to increased circulation time, optimized biodistribution, and reduced toxicity, all of which contribute to improved efficacy resulting from increased concentration (6). These effects have been shown with a variety of protein drugs, including granulocyte macrophage colony stimulating factor, interleukin-2, tumor necrosis factor, and interleukin-6 (5). Approved drugs that have been PEGylated include

  • PEG-adenosine deaminase (Adagen) for the treatment of adenosine deaminase deficiency and severe combined immune deficiency syndrome, and
  • PEG-asparaginase (Oncospar) for the treatment of acute lymphoblastic leukemia.

Attachment. Pharmacological enhancements from PEGylation can only be expected if the PEG-parent molecule is optimized in terms of stable linkage to the drug molecule, reduction of cross-linking, size of PEG moiety, structure of PEG moiety, and number of attachment sites. Two primary types of linkages are described in the patent literature. The first is protein PEGylation between an amino group in the native protein and an active carbonate, active ester, aldehyde, or tresylate on the PEG moiety. The second is reaction between thiol groups within the protein and vinyl sulfone, maleimide, or orthopyridyl disulfide groups on the PEG moiety. The amide and urethane bonds formed between a lysine in a protein and a PEG active ester or carbonate are stable linkages (7). Similarly stable are the thioether bonds formed between maleimide or vinyl sulfone and protein thiol groups and the secondary amine linkage formed between tresylate or aldehyde and protein amino groups. In contrast, the urethane linkage formed between a histidine in a native protein and a PEG active carbonate is not as stable (8). Instances in which PEGylated proteins are developed using this urethane linkage may result in detachment of the PEG moiety and in rapid clearance and degradation of the native protein.

PEG molecules contain two terminal hydroxyl groups that can be chemically activated. Typically, the hydroxyl groups are substituted with electrophilic functional groups (9); however, to make the PEG molecule monofunctional (no cross-linking potential and therefore more stable), it is common practice to convert one of the two hydroxyl groups to a methoxy or other alkoxy group (5). The methoxy group is chemically inert, while the hydroxyl group is activated through the use of various activating chemistries (9). Typically, however, methoxy-PEG is contaminated with PEG-diol. Thus, it is critical that active PEG derivatives be purified to remove difunctional material (7).

Types of PEG structures. The rate of clearance of native proteins from the body depends on the ionic charge and size of the molecule and the presence of cellular receptors. The size of the PEG moiety attached to the native protein may alter the physical characteristics of the native protein, including its thermal stability, protection against enzymatic degradation, and solubility (9). The structure of the attached PEG moieties is also important in optimizing the PEGylation of proteins. Currently, two different kinds of PEG structures—linear and branched—can be attached to proteins.

Linear moieties are limited in size by the manufacturing process because the amount of PEG-diol increases as PEG molecular weight increases. With linear moieties, increases in PEG–protein molecular size may only be accomplished by increasing the number of PEG attachment sites on the native protein. This has been shown, however, to result in suboptimal pharmacological profiles.

A more useful method is branching the PEG moiety from a single attachment site. Branched moieties increase the size of the PEG molecule without increasing the number of site attachments; they also confer significant enhancements to the pharmacological profile of the native protein.

PEG–protein conjugates can be created in three different ways: A single large PEG moiety can be attached at a single site; a branched PEG moiety (two or more medium PEG chains joined together via a linker) at a single site; or several small chains at multiple sites (5). Theoretically, monosite PEGylated proteins should have higher activity because the PEG attachment is less likely to occur at or near receptor-binding domains. Attaching multiple small-linear-chain PEGs or attaching large PEGs may result in extensive or complete loss of bioactivity.

PEGylated interferon. The drug PEGASYS is the reaction product of IFN--2a with a succinimidyl ester of a branched PEG molecule. The branched PEG moiety consists of two monomethoxy PEG chains, each with an average molecular weight of 20 kDa. The branched PEG moiety is highly purified to be monofunctional, so there is no difunctional material that could potentially lead to cross-linking and a decrease in biological activity. The two monomethoxy PEG chains are branched together through a lysine molecule via urethane bonds, one at the -amino group and the other at the -amino group of the lysine linker. The N-hydroxy succinimidyl ester derivative of the carboxyl group of the lysine linker reacts with primary amino groups of IFN--2a to form stable amide bonds (4).

Pharmacokinetics
As stated earlier, the delivery of IFN-a represented a significant clinical challenge. Modifying the IFN- structure by PEG attachment to increase its effective half-life seemed a logical way to address its pharmacokinetic profile. Early development of a 5-kDa linear PEG molecule attached to native IFN proved unsuccessful, as the increase in circulation half-life was minimal (10). F. Hoffmann-LaRoche, Ltd., however, continued to aggressively pursue its program for the development of a PEGylated IFN--2a. Shearwater Polymers, Inc., with its large array of PEG molecules, supplied F. Hoffmann-LaRoche with various sizes (5–40 kDa) and structures (linear and branched) of PEG molecules that were combined with native IFN. Five PEG–IFN--2a conjugates were evaluated:

  • 5-kDa linear mono-PEG–IFN--2a,
  • 20-kDa linear mono-PEG–IFN--2a,
  • 40-kDa linear di-PEG–IFN--2a,
  • 20-kDa branched mono-PEG–IFN--2a, and
  • 40-kDa branched mono-PEG–IFN--2a.

...
Figure 2. Pharmacokinetic profile of IFN--2a.
...
Figure 3. Pharmacokinetic profile of 5-kDa linear pegylated IFN--2a.
...
Figure 4. Pharmacokinetic profile of PEGASYS.
Conjugation of IFN--2a with PEG moieties of these various sizes, structures, and site-attachment numbers resulted in a variety of pharmacokinetic parameters including half-life, absorption rate, mean residence time, and antiviral activity (11, 12) (Figures 2, 3, 4). The 40-kDa, branched PEG molecule (later named PEGASYS) had the optimal pharmacological profile and was chosen for further clinical development (11). The optimization of PEGASYS confirmed that the size and branching of the IFN-a-2a directly influenced the pharmacokinetic properties and biological activity of the protein drug.

PEGASYS reaches therapeutic concentrations in as little as 3 h after initial subcutaneous injection, peaks at 80 h postdose, is absorbed into the circulation, and lasts for more than 168 h (one-week duration) (13). Because of this change from native IFN-, PEGASYS is injected only once a week (Figure 4). It also reduces the peak-to-trough ratio, which provides for more sustained therapeutic serum levels of IFN—possibly preventing the development of hepatitis C virus quasi-species by eliminating the trough periods between injections—and fewer peak-related side effects.

In addition, results from numerous clinical studies now indicate that PEGASYS has superior efficacy compared with IFN--2a monotherapy and similar efficacy to reported rates for combination IFN and ribavirin therapy (14, 15), while maintaining a similar safety profile to the unmodified IFN--2a in patients with and without cirrhosis (15, 16).

The development of an optimized PEGylated interferon has resulted in a therapeutic molecule that has improved pharmacokinetic and pharmacodynamic parameters. PEGASYS has overcome many of the intrinsic limitations of unmodified IFN, including reducing the administration of the drug from three times weekly to once weekly (which may increase patient compliance) and increasing the clinical efficacy while maintaining a similar side effect profile. The long-term commitment to this PEGylation technology by the professionals at Shearwater Polymers and F. Hoffmann-La Roche has resulted in the development of a novel therapeutic molecule that is poised to significantly affect the treatment of hepatitis C.

References

  1. Chronic Hepatitis C: Current Disease Management. www.niddk.nih.gov/ health/digest/pubs/chrnhepc/chrnhepc.htm (accessed September 11, 2000).
  2. Liang, T. J. et al. Pathogenesis, natural history, treatment, and prevention of hepatitis C. Ann. Intern. Med. 2000, 132 (4), 296–305.
  3. Wills, R. J. Clinical pharmacokinetics of interferons. Clin. Pharmacokinet. 1990, 19, 390–399.
  4. Zhi, J. et al. Influence of human serum albumin content in formulations on the bioequivalency of interferon alfa-2a given by subcutaneous injection in healthy male volunteers. J. Clin. Pharmacol. 1995, 35 (3), 281–284.
  5. Bailon, P.; Berthold, W. Polyethylene glycol-conjugated pharmaceutical proteins. Pharmaceut. Sci. Technol. Today 1998, 1, 352–356.
  6. Fuertges, F.; Abu chowski, A. The clinical efficacy of polyethylene glycol-modified proteins. J. Controlled Release 1990, 11, 139–148.
  7. Harris, N. J. et al. Multiarmed, Monofunctional Polymer for Coupling to Molecules and Surfaces. U.S. Patent 5,932,462, Aug 3, 1999.
  8. Gilbert, C. W.; Park-Cho, M. O. Interferon Polymer Conjugates. U.S. Patent 5,951,974, Sept 14, 1999.
  9. Zaplipsky, S.; Lee, C. Use of functionalized poly(ethylene glycol)s for modification of polypeptides. In Poly(ethylene glycol) chemistry. Biotechnical and biomedical applications; Harris, J., Ed.; Plenum Press: New York, 1992; pp 347–370.
  10. O’Brien, C. et al. A double-blind, multicenter, randomized, parallel dose-comparison study of six regimens of 5kD, linear peginterferon alfa-2a compared with Roferon-A in patients with chronic hepatitis C [abstract]. Presented at the International Conference on Therapies for Viral Hepatitis, Maui, HI, Dec 6, 1999.
  11. Bailon, P. et al. Pharmacological properties of five polyethylene glycol conjugates of interferon alfa-2a [abstract]. Presented at the International Conference on Therapies for Viral Hepatitis, Maui, HI, Dec 6, 1999.
  12. Fung, W-J.; Porter, J.; Bailon, P. Strategies for the Preparation and Characterization of Polyethylene Glycol (PEG) Conjugated Pharmaceutical Protein. Poly. Prepr. (Am. Chem. Soc., Div. of Fuel Chem.) 1997, 38, 565–566.
  13. Algranati, N. E.; Sy, S.; Modi, M. A branched methoxy 40-kDa polyethylene glycol (PEG) moiety optimizes the pharmacokinetics of peginterferon alpha-2a (PEG-IFN) and may explain its enhanced efficacy in chronic hepatitis C. Hepatology 1999, 40 (Suppl), 190A.
  14. McHutchison, J. G. et al. Interferon alfa-2b alone or in combination with ribavirin as initial treatment for chronic hepatitis C. N. Engl. J. Med. 1998, 339, 1485–1492.
  15. Poynard, T. et al. Randomised trial of interferon 2b plus ribavirin for 48 weeks or for 24 weeks versus interferon 2b plus placebo for 48 weeks for treatment of chronic infection with hepatitis C virus. Lancet 1998, 352, 1426–1432.
  16. Heathcote, E. J. et al. Multinational evaluation of the efficacy and safety of once-weekly peginterferon -2a (PEG-IFN) in patients with chronic hepatitis C (CHC) with compensated cirrhosis. Hepatology 1999, 30 (4Pt2), 316A.
  17. Shiffman, M. et al. A controlled, randomized, multicenter, ascending dose phase II trial of Pegylated interferon alfa-2a (PEG) vs standard interferon alfa-2a (IFN) for treatment of chronic hepatitis C [Abstract no. L0418]. Gastroenterology 1999, 116, Part 2, 1275.

    Stephen A. Charles is vice president, corporate development, for Shearwater Polymers, Inc. J. Milton Harris is president of Shearwater Polymers, Inc. Simon Pedder is international project leader for F. Hoffmann-LaRoche, Inc. Saran Kumar is research leader for F. Hoffmann-LaRoche, Inc. Comments and questions for the authors may be addressed to the Editorial Office by e-mail at mdd@acs.org, by fax at 202-776-8166 or by post at 1155 16th Street, NW; Washington, DC 20036.

    Top || Modern Drug Discovery Home Page

    CASChemPortChemCenterPubs Page