Pharmacokinetic Consequences of Pegylation

Abstract

Pegylation, generally described as the molecular attachment of polyethylene glycols (PEGs) with different molecular weights to active drug molecules or surface treatment of drug-bearing particles with PEGs, is one of the most promising and extensively studied strategies with the goal of improving the pharmacokinetic behavior of the therapeutic drugs. A variety of PEGs, both linear and branched, with different molecular weights have been exploited successfully for use in this procedure in the form of reactive PEG species. Both reversible and irreversible PEG-drug conjugates have been prepared with relative advantages/disadvantages. The main pharmacokinetic outcomes of pegylation are summarized as changes occurring in overall circulation life-span, tissue distribution pattern, and elimination pathway of the parent drug/particle. Based on these favorable pharmacokinetic consequences leading to desired pharmacodynamic outcomes, a variety of proteins/peptides as well as small molecule drugs have been pegylated and evaluated successfully. Also a number of corresponding products have been approved by the U.S. FDA for specific clinical indications and some others are underway. In this article, the chemistry, rationale, strategies, pharmacokinetic outcomes, and therapeutic possibilities of pegylated drugs are reviewed with pharmacokinetic aspects presented with more details.

Keywords :

• PEG
• Pegylated Drugs
• Pegylation
• Pharmacokinetic
• Systemic Drug Delivery

Pegylation, originally defined as attachment of polyethylene glycol (PEG) to drug molecules, was first introduced by Davis, and colleagues in the 1970s (Davis et al. 1978). At that time, and later on (Abuchowski et al. 1984), the main focus of this procedure was to prepare conjugates of protein/peptide drugs with PEGs having a variety of degrees of polymerization. The main objective of which was modifying the pharmacokinetic profile and in vivo fate of the bound drug in host body upon systemic administration (Harris et al. 1997; Holle 1997). In addition, surface treatment of the particulate drug delivery carriers using PEGs to alter the pattern of particle biodistribution is referred to as pegylation (Gabizon et al. 1996; Iwanaga et al. 1997; Bucke et al. 1998; Reddy 2000; Sharp et al. 2002). A number of pegylated drugs have been approved by the U.S. Food and Drug Administration (FDA), for a variety of clinical situations (Table 1) (Medina et al. 2003; Pedder 2003).

TABLE 1 Pegylated drugs approved by the FDA

Trade name	Generic name	Approval date	Company
Adagen	Pegademase	03/21/1990	Enzon
Oncaspar	Pegaspargase	02/01/1994	Enzon
Peg-Intron	Peginterferon alfa-2b	01/19/2001	Schering
Neulasta	Pegfilgrastim	01/31/2002	Amgen
Pegasys	Peginterferon alfa-2a	10/16/2002	Hoffman-la roche
Somavert	Pegvisomant	03/25/2003	Pharmacia and Upjohn
Peginterferon	Peginterferon alfa-2a	06/04/2004	Hoffman-la roche
Acumen	Pegaptanib sodium	12/17/2004	Eyetech

http://www.accessdata.fda.gov/scripts/cder/drugsatfda

Science has challenged many methods to improve the delivery of pharmaceutical agents. Two classical strategies by which drug delivery can be modified include changing the molecular structure of the active drug substance and development of formulations and/or delivery systems offering desired pharmacokinetic characteristics to the parent drug. Pegylation, as mentioned, is a procedure capable of changing both the basic drug structure and drug-bearing particles properties, that alone or together can result in remarkable alterations in the pharmacokinetic parameters of the drug of interest. These changes, in turn, may result in significant consequences in pharmacodynamic features of the parent drug (Gabizon et al. 1996; Sharp et al. 2002). In fact, the basis for these pharmacokinetic/pharmacodynamic modifications is the changes in size, polarity, structure and surface properties of drug molecule or drug-bearing particles. These changes ultimately lead to altered physicochemical characteristics(e.g., solubility and stability), immunogenicity (Hershfield 1997), cellular uptake, elimination (both biotransformation and excretion) (Yamaoka et al. 1994), and tissue localization profiles of the parent drug (Gabizon et al. 1996; Illum et al. 2001; Ishida et al. 2001; Sharp et al. 2002) while maintaining its original or, even improved, biological activity.

PEG CHEMISTRY

PEGs are amphiphilic and relatively chemically inert polymers consisting of repetitive units of ethylene oxide. PEGs are named based on the number of ethylene oxide units in the polymer chain; a large variety of PEG molecules with different molecular weights are commercially available. PEGs can be presented with various configurations categorized in two main classes of linear or branched polymers (Figure 1) (Monfardini et al. 1995). In addition, PEGs can be divided into two groups: PEGs with free hydroxyl (─;OH) groups at both ends and PEGs with one or two methoxylated end group(s) (i.e., ─;OH replaced by ─;OCH₃). This latter property is of remarkable implications in pegylation procedure (Monfardini et al. 1995) as discussed later in this article.

FIG. 1 Typical molecular structures of poly-ethylene glycols (PEGs).

PEGYLATION PROCEDURE

PEG Conjugation (Molecular Pegylation)

The majority of studies on the molecular conjugation with PEGs involve the attachment of PEGs to protein/peptide drugs (Fujita et al. 1994; Holle 1997; Kozlowski et al. 2001). Therefore, it is obvious that some serious limitations in reaction conditions as well as the choice of reacting PEG-derived functional molecules should be considered. In this context, the formation of some stable (i.e., covalent) bonds between PEGs and drug molecules is a prerequisite for formation of desired conjugates. These associations are from two possible types,—reversible and irreversible PEG-conjugates—each having its own relative advantages and drawbacks (Fujita et al. 1994). As reversible PEG-conjugate forming derivatives, PEG-succinimidyl succinate (Zalipsky 1995; Bullock et al. 1996) PEG-dimethyl maleic anhydride (Garman and Kalindjian 1987), and PEG-ortho-pyridyl-disulfide (Woghiren, Sharma, and Stein 1993) have been studied. In addition, PEG succinimidyl esters have been developed with various ester linkages in the PEG structure to control the degradation rate at physiological pH (Zhao et al. 1997). As the representative irreversible PEG derivatives, first generation compounds (e.g., PEG-dichlorotriazine, PEG-tresylate, PEG-succinimidyl carbonate, PEG-benzotriazole carbonate, PEG-p-nitrophenyl carbonate, PEG-trichlorophenyl carbonate, and PEG-carbonyl imidazole) and second generation compounds (e.g., mPEG-propion aldehyde, PEG-maleimide, PEG-vinyl solfon, and PEG-orthopyridyl disulfide) have been developed (Roberts et al. 2002).

In Figure 2 and Figure 3 the chemical structures of some representative reactive PEG-derivatives are shown. Both pegylation modes (reversible and irreversible) can be carried out using linear as well as branched PEGs (Algranati, Sy, and Modi 1999; Bailon et al. 1999; O'Brien et al. 1999; Shiffman et al. 1999; Bailon et al. 2001). Compared with linear molecules, conjugates bearing branched chain PEGs show increased pH and thermal stability and higher resistance to proteolytic digestion (Monfardini et al. 1995). The first step in molecular pegylation, in all forms, is “activation” of the PEG molecule and is achieved by attachment of some electrophilic “reactive groups” to hydroxyl end of the PEG molecule (see above). A variety of reactive “ends” have been exploited successfully in pegylation process on different drug molecules (Zalipsky, Gilon, and Zilkha 1985; Greenwald, Pendri, and Bolikal 1995; Greenwald et al. 1996a; Greenwald et al. 1996b; Bedeschi et al. 1997; Conover et al. 1997; Conover et al. 1998; Greenwald et al. 1998; Pendri et al. 1998; Greenwald et al. 1999; Ochoa et al. 2000; Zhao et al. 2000; Dosio et al. 2001). Mostly, it is the monofunctionalized PEG molecule, methoxy-PEG (Monfardini et al. 1995; Algranati et al. 1999) that enters the activation procedure. This is because these PEGs have a single hydroxyl group for activation, with methoxy group being inert to standard chemical reactions (Katre 1993). The reactive functional groups of activated PEGs can then be attached to a specific site on proteins, mainly on an amine, a sulfhydryl or other nucleophilic groups. In most cases, the preferred site of modification is the amino group of lysine and the N-terminus of polypeptide chain (Zalipsky et al. 1992; Kinstler et al. 1996). Although amino groups of lysine and N-terminus of the polypeptide chain are of critical importance in pegylation procedure, low frequency of these binding sites (particularly lysine) decreases the degree of pegylation: the lower degree of pegylation, the lower extent of pharmacokinetic/pharmacodynamic enhancements. Recently, protein carboxyl groups have been exploited as binding sites to overcome such problems (Li et al. 2004).

FIG. 2 First-generation irreversible PEG reactive forms (Roberts et al. 2002).

FIG. 3 Second-generation irreversible PEG reactive forms (Roberts et al. 2002).

Alternatively, site-directed mutations in amino acid sequence of proteins has been studied as a way to have more lysines in the peptide chain (Hershfield et al. 1991; He, Shaw, and Tam 1995), which ultimately results in increased degree of pegylation.

Particle Pegylation

A variety of particulate drug delivery carriers have been coated with PEGs to alter their surface properties. The choice of PEG-coating procedure in each case should be based on the physiochemical nature of the particle intended to be surface-treated. PEG-treated liposomes (Gabizon et al. 1996; Alberts et al. 1997; Bedeschi et al. 1997; Iwanaga et al. 1997; Bucke et al. 1998; Maruyama et al. 1999; Ishida et al. 2001; Laverman et al. 2001; Bakker-Woudenberg et al. 2002; Levchenko et al. 2002; Sharp et al. 2002;), nanoparticles (Peracchia et al. 1999; Brigger et al. 2001; Calvo et al. 2001; Fresta et al. 2001; Illum et al. 2001; Otsuka et al. 2003), and microparticles (Diwan et al. 2001; Chandy et al. 2002; Kim et al. 2002) have been studied extensively with more or less success in modification of particle bio-fate.

Pegylated nanoparticles (Peracchia et al. 1999; Brigger et al. 2001; Calvo et al. 2001) and polymeric micelles (Otsuka et al. 2003) have been prepared using copolymers of PEG and main encapsulating polymer. Pegylated liposomes have been produced as commercially available matrices for encapsulation of different drugs and other bioactive agents using simply blending the liposome-constituting phospholipids with a (PEG-PE) copolymer (Levchenko et al. 2002) or alternatively using PEG-derivatives of phospholipids in the preparation step of liposomes (Yuan et al. 1994; Laverman et al. 2001; Bakker-Woudenberg et al. 2002). In addition, the stabilization of protein/peptide agents against denaturation as a result of encapsulation procedure conditions, using pegylated form of protein/peptide, has been of interest for some studies (Diwan et al. 2001; Kim et al. 2002).

In addition to using PEG-polymer copolymers in preparation of pegylated particles, blends of main polymer with PEG have been used to prepare pegylated particles (Couvreur et al. 1983; Illum et al. 1983; Illum et al. 1984; Manil et al. 1986; Kubiak et al. 1988). However, the relative success of PEG-copolymers is much higher than these blends thanks to the structural attachment of PEG to particle surface and, homogeneity of PEG distribution throughout the particle structure.

PEGYLATION STRATEGIES

Molecular Pegylation

Molecular pegylation, as a discussed, is a chemical attachment of a PEG to a drug molecule. In the majority of cases, molecular pegylation takes place primarily on therapeutic proteins and enzymes, and there is a limited data published, far on other small-molecule drug classes. Antigens (e.g., venoms and plant pollens), antibodies and their fragments (Lee et al. 1999; Cheng et al. 2000; Hurwitz et al. 2000; Koumenis et al. 2000), growth factors (e.g., Gh-Rf) and colony stimulating factor (Kinstler et al. 1996; Eliason 2001; Morstyn et al. 2001), cardiovascular agents (e.g., streptokinase, catalase, tissue plasminogen activator) (Collen et al. 2000), blood constituents (e.g., albumin and hemoglobin) (Fujita et al. 1994), immunologically active agents (e.g., cytokines, interleukins) (Yang et al. 1995; Francis et al. 1998; Eliason 2001), free radical scavengers (e.g., superoxide dismutase and catalase) (Somack et al. 1991), antineoplastic agents (e.g., Uri case and interferon), biological receptors (e.g., tumor necrosis factor receptor) (Edwards 1999), drugs for hepatitis C (e.g., interferons) (Burnham 1994; Algranati et al. 1999; Bailon et al. 1999; Glue et al. 1999a; Glue et al. 1999b; O'Brien et al. 1999; Shiffman et al. 1999; Wang et al. 2000; Bailon et al. 2001; Grace et al. 2001; Zeuzem et al. 2001; Gupta et al. 2002;, Medina et al. 2003; Pedder et al. 2003), and finally enzymes (e.g., asparaginase, adenosine deaminase) (Abuchowski et al. 1984; Hershfield 1995; Holle 1997; Aguayo et al. 1999; Sun et al. 2003; Li et al. 2004) are among the proteins pegylated with more or less successes in corresponding therapeutic outcomes (O'Brien et al. 1999).

Attachment of a PEG moiety to a protein generally results in dramatic changes in drug elimination and distribution profiles owing to the structural change(s) in the basic drug molecule (discussed later in this article). Furthermore, PEGs also have been attached to oligodeoxynucleotides (ODNs). The delivery of ODNs has been of great research interest in the past decade (Chirila et al. 2002). The pegylation process on the parent ODN, mainly carried out at the 3′ and 5′ terminus of the molecule, has resulted in desirable pharmacological consequences (Jaschke et al. 1993; Jaschke et al. 1994; Kawaguchi et al. 1995; Bonora et al. 1997; Seelig et al. 1997; Burcovich et al. 1998; Vorobjev et al. 1999).

Pegylated Particles

Particulate drug carriers, with liposomes the most extensively studied cases, have been coated with PEGs to improve their circulation life span as well as the biological fate of the encapsulated drugs. Classically, liposomes are useful in delivering drugs to specific targets in the body. Hydrophilic nature of PEGs incorporated into the bilayer of a liposome brings a shield around the liposome that in turn protects these particles from being phagocyted by natural particle eliminating mechanisms, mainly organs of reticuloendothelial system (RES). This surface modification has been reported to result in as much as 8-fold increase in plasma half-life of liposome over the parent particle (Burnham 1994) that in turn decreases the drug leakage in circulation (Gabizon et al. 1997).The most extensively exploited drug-loaded pegylated liposomes are doxorubicin-containing particles with resulting profound improvements in pharmacokinetic parameters of this antineoplasm drug (Alberts et al. 1997; Gabizon et al. 1996; Gabizon et al. 1997; Sharp et al. 2002). In addition to the incorporation of PEG molecules into the phospholipid bilayers of drug-loaded liposomes, another approach, attachment of the pegylated drugs to the external surface of the liposomes has been attempted to prolong the drug life span in circulation (Ishida et al. 2001).

Nanoparticles also have been coated with PEGs to modify their biological fate, and the findings of these studies have been promising (Peracchia et al. 1999; Brigger et al. 2001; Calvo et al. 2001; Fresta et al. 2001; Illum et al. 2001; Otsuka et al. 2003). For example, pegylated polycyanoacrylate nanoparticles have proved to be potential drug carrier systems for brain delivery (Calvo et al. 2001). Pegylated nanoparticles also can be used as a carrier for diagnostic agents to localize them to specific regional organs/tissues (e.g., regional lymph nodes) (Illum et al. 2001). Pegylated nanoparticles are discussed further in a later section of this article.

PHARMACOKINETIC OUTCOMES

The main purpose of pegylation is defined as achievement of a favorable pharmacokinetic profile for the drug of interest which ultimately results in more desirable therapeutic properties. The pharmacokinetic outcomes of pegylation can be classified within the following areas: life span in circulation, drug elimination, and time distribution.

Life Span in Circulation

Pegylation may result in physicochemical alterations in the parent drug compound, which may collectively lead to decreased efficiency of drug eliminating processes such as renal excretion, proteolysis, and opsonization (Brenner and Rector 1996). Therapeutic proteins, as a general rule, have very limited life span in circulation which results from the effective eliminating mechanisms in place for these agents in the body. Among these mechanisms, proteolysis, specific cell-mediated protein degradation routes, and being captured by the RES are noteworthy. The dependence of protein clearance on the protein net ionic charge in the physiologic pH, its molecular weight, and the presence of protein-specific receptors on the cells responsible for protein uptake is of importance in this context (Burnham 1994).

The attachment of a PEG moiety, in particular branched PEGs, increases the overall size of the parent drug (Bailon et al. 1999). Yamaoka et al. (1994), have shown that renal clearance of intravenously (IV) injected PEGs in mice is conversely related to the molecular weight of the PEG molecule used, with the most profound changes occurring at molecular weights ∼30KDa. The circulation half-life (t_1/2) of PEGs showed a concomitant increase with the increases in molecular weight (e.g., t_1/2 rises from 18 min to 16.5 hr as the molecular weight increases from 6 to 50KDa). In the same study, PEG molecules with molecular weights less than 20KDa were cleared mainly in the urine, whereas higher molecular weight PEGs were cleared more slowly both in urine and feces (Yamaoka et al. 1994).

It Glomerular filtration of small molecules can be retarded by attachment to PEGs with molecular weights in the order of 40 to 50 KDa (Jaschke et al. 1994; Seelig et al. 1997). As a typical protein drug, the systemic clearance of unmodified interferon-alpha (IFN-α) after an IV dose was reported to be 6.6–29.2 lit/hr (Wills et al. 1984; Chatelut et al. 1999), decreasing remarkably to 2.5–5 lit/hr upon conjugation to linear PEG of 5KDa (Nieforth et al. 1996), 0.725 lit/hr when attached to a linear PEG of 12 KDa (Glue et al. 1999), and 0.06–0.10 lit/hr after pegylation with a branched PEG of 40 KDa (Xu et al. 1998; Algranati et al. 1999; F. Hoffmann-La Roche, Ltd.). In another study, mean total body clearance of IFN-α -2b showed a significant (∼10-fold) decrease (from 231.2 ml/hr kg to 22.0 ml/hr kg) as a result of conjugation to PEG (Bukowski et al. 2002). With respect to other pegylated proteins, systemic clearance of pegylated forms of interleukin(16)–2, asparaginase, and brain-derived neurotrophic factor reduced up to 10-fold (i.e., from 1.15 to 0.11 ml/min) (Knauf et al. 1988), 17-fold (i.e., from 2196 to 128 ml/m²/day) (Holle 1997), and 2.6-fold (i.e., from 5.5 to 1.9 ml/min/kg) (Pardridge et al. 1998), respectively, compared with their corresponding free proteins.

In a study to determine the influence of pegylation on protein targeting and systemic clearance of three derivatives of bovine serum albumine (BSA), i.e., lactosylated BSA, mannosylated BSA, and cationized BSA, 7-fold, 45-fold, and 130–fold decrease was observed on the systemic clearance of these compounds, respectively, as a result of PEG conjugation. At the same time, the liver accumulation of all variants tested remained unchanged (Fujita et al. 1994). In fact, PEG moieties prevented the parent protein /peptide from being metabolized enzymatically and/or recognized by the immune defense mechanisms (Hershfield 1997), mainly phagocytosis in circulation by RES organs. This in turn, lead to considerably longer residence time in systemic circulation and therefore in whole body.

Pegylation influences the biological half-lives (t_1/2) of many pharmaceuticals. Many studies indicated of dramatic enhancements in t_1/2 of particular drug molecules as a result of pegylation. For instance, the serum half-life of F (ab′)₂ segment of a humanized anti-IL-8 antibody increased from 8.5 to 48 hr, upon pegylation (Koumenis et al. 2000). In another study, the result of PEG attachment to a polypeptide agent, hirudin (a naturally occurring anticoagulant polypeptide found in a leech), was indicative of the significant increment in the relatively short terminal half-life of the original molecule (Esslinger et al. 1997). In addition, t_1/2 of IFN-α -2a has been reported to increase from 2.3 to 50 hr with corresponding terminal elimination phase t_1/2 changed from 3.8 to 65 hr after pegylation (Harris et al. 2001). Furthermore, the pegylated forms of IL-6, IL-2, tumor necrosis factor (TNF), and brain–derived neurotrophic factor (BDNF) have shown terminal elimination t_1/2 increased from 2.1 to 206 min (∼ 100-fold) (Tsutsumi et al. 1997), from 44 to 57–256 min (up to 6-fold) (Knauf et al. 1988), from 3 to 45–136 min (14 to 43-fold) (Tsutsumi et al. 1995), and 10 to 50 min (5-fold), respectively, compared with corresponding free drugs (Pardridge et al. 1998).

In the same way, terminal elimination t_1/2 of megakaryocyte growth and development factor increased by 10-fold upon pegylation (Hokom et al. 1995). There are several similar reports in literature indicating that PEG conjugation is beneficial in elevating the terminal half-lives of protein drugs with asparaginase (from 20 to 357 hr) (Holle 1997), superoxide dismutase (from 3.5 to 540–990 min) (Beauchamp et al. 1983) streptokinase (from 4 to 7–20 min) (Rajagopalan et al. 1985), and lactoferrin (3 to 15–60 min) (Beauchamp et al. 1983) being more pronounced among others.

It is obvious that the circulation life span of drug-bearing particles exerts profound effects on the pharmacokinetic profile of the encapsulated drug(s). As an effective method for prolongation of the particle residence time in circulation, pegylation of the particle surface has been exploited extensively with favorable pharmacokinetic outcomes in all cases. In a study with PEG-liposomal doxorubicin, an antineoplasm agent, circulation time of the particle increased significantly (Sharp et al. 2002). In addition, transferrin-PEG-liposomes showed a prolonged residence time in the circulation compared with the unmodified drug (Ishida et al. 2001). The same liposomes proved to have a lower RES uptake in colon 26 tumor-bearing mice resulting in enhanced extravasation of liposomes into the solid tumor tissue.

Like any colloidal drug carriers not specially designed to avoid being uptaken by the RES, polymeric nanoparticles are rapidly removed from the blood stream after vascular administration and preferentially accumulate in liver and spleen (Bazile et al. 1992; Stolnik et al. 1994). Blood half-lives of these particles are generally around 2-3 min (Bazile et al. 1992; Verrecchia et al. 1995; Li et al. 2001). After IV administration, the first step of the process that leads to the nanoparticle uptake by the RES is the opsonization phenomenon. Opsonins, including complement proteins, apolipoproteins, fibronectin, and immunoglobulins (Allemann et al. 1997), interact with specific membrane receptors of monocytes and tissue macrophages, resulting in recognition and phagocytosis. It is generally admitted that hydrophobic surfaces promote protein adsorption and that negative surfaces are activators of the complement system (Moghimi et al. 2001). Fallowing the rule, hydrophobic and negative polylactide (PLA) or polylacticlgycolic acid (PLGA) nanoparticle surfaces (Riley et al. 1999; Chognot et al. 2003) activate the complement system (Gref et al. 2000) and coagulation factors (Sahli et al. 1997) in vitro.

In contrast, hydrophilic coating with PEG sterically stabilizes PLA or PLGA nanoparticles and reduces opsonization and phagocytosis in vitro (Gref et al. 2000) or ex vivo (Bazile et al. 1995) and uptake by neutrophilic granulocytes in vivo (Zambaux et al. 2000). Compared with nonpegylated nanoparticles, pegylated nanoparticle surfaces have lower zeta (ζ) potential values, due to the surface shielding by the PEG corona (Gref et al. 1995; Riley et al. 1999; Chognot et al. 2003). Gref et al. (1995) showed a maximum antiopsonic effect with PEG molecular weights of 5000 and above. Covalent linkage of the PEG coating and sufficient PLA block molecular weight is essential to ensure sufficient stability and to avoid loss of the coating benefit by desorption and/or displacement in vivo (Stolnik et al. 1994; Verrecchia et al. 1995; Mosqueira et al. 2001; Chognot et al. 2003). In mice, blood circulation times of ¹¹¹In-labeled mPEG-PLGA nanoparticles (140 ± 10 nm diameter) increased compared with PLGA alone with an advantage to higher PEG molecular weights (Gref et al. 1994). Within 5 min, however, ∼50% (PEG ₂₀₀₀₀) to 75% (PEG ₅₀₀₀) of injected nanoparticles (estimated from the blood clearance curves) had been cleared from the blood compartment (compared with 95% with control PLGA nanoparticles). In another study performed in rats, the blood half-lives of [C¹⁴] PLA-labeled mPEG-PLA₃₀₀₀₀ nanoparticles with PEG molecular weight of 2000 (Verrecchia et al. 1995) (205 nm diameter) or 5000 (Bazile et al. 1995) (140 ± 60 nm) were markedly higher (6hr) and independent of the PEG molecular weights. Less prolonged blood circulation times were observed with PLGA coated with PLA₃₀₀₀-PEG₄₀₀₀ (147 ± 3.6 nm) or PLA₃₀₀₀-PEG₅₀₀₀ (161 ± 3.7 nm) (i.e., t_1/2 = 15 min and t_1/2 = 1hr, respectively) (Stolnik et al. 1994). With nanoparticles made of mPEG₅₀₀₀-PLA₇₀₀₀−¹²⁵I (150 ± 2 nm diameter) or of mPEG₁₄₀₀₀-PLA₆₀₀₀−¹²⁵I (35.8 ± 0.5nm diameter) blood half-lives determined in rats were 29.9 ± 12.4 and 42.3 ± 16.2 min, respectively (no statistical difference) (Novakova et al. 2003). In rats, a blood half-life of 270.9 min was determined for ¹²⁵I-BSA loaded in mPEG₅₀₀₀-PLGA₄₅₀₀₀ nanoparticles (∼200 nm diameter), compared with 13.6 min when formulated in PLGA nanoparticles (Li et al. 2001).

The large variability in blood half-lives determined in these works, even with the same PEG block molecular weight of 5000, may be ascribed to the density-related PEG conformation in the coating layer. The polydispersity of the PLA block molecular weights also should be considered, which renders the pegylated nanoparticles system more complex than liposomes and could lead to a surface heterogenecity pointed out by Gbadamosi et al. (2002). Such a surface heterogeneity may explain the rapid clearance of a significant fraction of IV injected long-circulating nanoparticles by the RES (Gref et al. 1994; Stolnik et al. 1994; Moghimi 2002). Because of this polydispersity, space available for PEG block expansion is likely to be variable on nanoparticle surface. Mushroom-like and brush-like conformations may coexist within a single nanoparticle or among a population of polydispersed nanoparticles, thus explaining variability observed in blood half-lives. Therefore, molecular weights of PEG and PLA block, as well as polydispersity of copolymers, should be carefully selected in designing long-circulating pegylated nanoparticles.

Drug Elimination Pathway

In addition to extending the drug presentation time in circulation, pegylation seems to be capable of changing elimination patterns, via modifying physicochemical characteristics influencing different elimination routes of a given compound, as well as the relative contribution of these routes in overall drug elimination. With the increase in molecular weight of the PEG moiety attached to the drug, the dominant elimination route shifts from renal to hepatic pathway (Yamaoka et al. 1994; Harris et al. 2001). This is probably due to the decreasing possibility of glomerular filtration in response to size increment and the decreasing role of hepatic enzymatic activities producing more polar metabolite(s) capable of being excreted more readily in urine.

Tissue Distribution Profile

Pegylation has the potential to alter the tissue/organ distribution profile (so-called biodistribution) of the conjugated/encapsulated drug. This effect is due to its role in modification of physicochemical properties of the parent drug: a feature central to all possible effects of this process on the biological fate of a drug. The relatively preferred distribution of a pegylated drug to some tissue sites can be regarded as a basis for drug targeting. PEG molecular weight is of major importance in determining targeting characteristics. Researches have documented that macromolecules with extended circulation periods will show a substantial tumor accumulation (Noguchi et al. 1998). For PEG molecules of 10KDa or greater, relative uptake by tumor tissues is higher than by the normal tissues irrespective of the tumor site (Murakami et al. 1997). In a series of studies (Gabizon et al. 1996; Alberts et al. 1997; Gabizon et al. 1997; Sharp et al. 2002), tumor localization of PEG-coated liposomes loaded by doxorubicin have increased remarkably according to the particular tumor sites. Furthermore, pegylated nanospheres utilized in a diagnostic procedure for imaging regional lymph nodes have shown substantial modifications in the distribiution profile and enhanced localization to sites of interest compared with nonpegylated control (Illum et al. 2001).

Regarding surface-treated liposomes, administration of drug-PEG-liposome association significantly modified the biodistribution of the parent drug. Transferrin-PEG-liposomes have shown the capability of specific binding and receptor-mediated endocytosis to target tumor cells in vivo (Ishida et al. 2001). In addition, in a study on PEG-conjugation of camptothecin (CPT), the PEG-CPT conjugation resulted in higher tumor accumulation (30-fold) of the drug in mice bearing subcutaneous tumors compared with the free CPT (Conover et al. 1998). The volume of distribution (V_d) of PEG conjugates of INF-α -2a decreased from 31–73 lit to 8–12 lit (Wills et al. 1984; Algranati et al. 1999; Chatelut et al. 1999; Xu et al. 1990; F. Hoffmann-La Roche, Ltd.) and that of pegylated BDNF increased from 80 to 135 ml /kg (Pardridge et al. 1998). In the case of asparaginase and IL-2, V_d values have remained relatively unchanged (Knauf et al. 1988; Holle 1997).

THERAPEUTIC POSSIBILITIES

Protein/peptide drugs

Protein and peptide drugs are progressively attractive therapeutic tools used in treatment, diagnosis, and monitoring of several disease states. Innovative developments occurred in recombinant DNA technology, and the identification of therapeutic targets for protein drugs in the body has evoked research activities on novel delivery systems for these drugs. Pegylated IFNs, particularly IFN-α -2a, and to a lesser extent IFN-α -2b, are the first and the most extensively studied pegylated protein drugs approved for application in hepatitis C and tumors. Many studies on these products are indicative of the superiority of these compounds over their conventional nonpegylated analogues (Harris et al. 1997; Algranati et al. 1999; Bailon et al. 1999; Glue et al. 1999a; Glue et al. 1999b; Heathco et al. 1999; O'Brien et al. 1999; Shiffman et al. 1999; Sulkowski et al. 1999; Wang et al. 2000; Zeuzem et al. 2000; Bailon et al. 2001; Grace et al. 2001; Harris, Martin, and Modi 2001; Zeuzem et al. 2001; Medina et al. 2003; Pedder 2003). Currently, there are two variants of pegylated IFN-α on the market: one with PEG of 12KDa (Wang et al. 2000; Grace et al. 2001) (i.e., PEG-Interon®, Table 1), and the other conjugate with a single, branched PEG with higher molecular weight (i.e., Pegasys®, Table 1) (Monfardini et al. 1995; Martinez et al. 1997; Zeuzem et al. 2001).

In recent years, extensive studies to gather more information regarding PEG-IFN pharmacokinetic/pharmacodynamic features have been carried out (Gupta et al. 2002; Imaz 2002), and this topic has been reviewed (O'Brien et al. 1999; Imaz 2002). PEG-conjugated L-asparaginase (Pegaspargase®, Table 1) is a registered therapeutic agent used successfully for the treatment of acute lymphocytic leukemia (Abuchowski et al. 1984; Holle 1997). Also, Pegaspargase® has been coadministered successfully in a combination therapy regimen with methotrexate, vincristin, and prednisone for a patient with refractory or recurrent acute lymphoblastic leukemia (Aguayo et al. 1999). Adenosine deaminase pegylated product (Pegademase®, Table 1) has been approved as replacement therapy in patients with adenosine deaminase deficiency (Hershfield 1995). Recombinant methioninase (rMETase), a plasma methionin-depleting agent (tumors have abnormally higher requirements for methionin), has been studied in its pegylated form as a new anticancer agent (Sun et al. 2003). In a novel approach to treat chronic inflammatory diseases, recombinant human soluble TNF receptor type I (rHu-STNF-RI) was pegylated and showed promising results in treatment of rheumatoid arthritis (Edwards 1999). Granulocyte colony stimulating factor conjugated to PEG (known as Neulasta® or PEG- filgrastim®) has been applied to reduce the duration and severity of neutropenia that occurs commonly after cytotoxic cancer therapy (Morstyn et al. 2001; Holmes et al. 2002; Molineux 2003). In a study to determine its pharmacodynamic and safety outcomes, recombinant hirudin was conjugated to PEG; as a result, antithrombotic activity was enhanced significantly with no observed immunogenicity (Esslinger et al. 1997). Salmon calcitonin (sCT) (a bone-reabsorption inhibitor polypeptide hormone) also was pegylated, and the researchers concluded that pegylated sCTs may have therapeutic advantages over the free drug (Yoo et al. 2000).

To benefit the pharmacokinetic improvements just mentioned, antibodies and their fragments also have been involved in the pegylation procedure and met expected enhanced therapeutic characteristics (Lee et al. 1999; Cheng et al. 2000; Hurwitz et al. 2000; Koumenis et al. 2000). Finally, clinical studies on successful applicability of other PEG-conjugated agents including IL-2 (Yang et al. 1995), growth hormone antagonists (Trainer et al. 2000), hemoglobin (Nucci et al. 1996; Lusting et al. 1999), and staphylokinase muteine (Collen et al. 2000) have been published.

Small Molecule Drugs

The main focus of using the pegylation process as a clinical advantage for small molecule (nonprotein) drugs has been on coating the particles loaded by these compounds with PEGs of different molecular weights. As representative small molecule conjugates with PEGs, PEG-derivatives of doxorubicin and amphetamine have been prepared, with only in vitro data presented (Zalipsky et al. 1985). In addition, attachment of PEG to paclitaxel, an antineoplasm agent, was studied by Greenwald et al. (1995) to of improve solubility and tissue targeting of this highly potent drug. Since then, to improve the therapeutic approaches of cancer therapy, many researchers have dedicated their efforts to the potent anticancer characteristics of such virtually water-insoluble drugs as paclitaxel) Greenwald et al. 1995; Greenwald et al. 1996; Pendri et al. 1998; Dosio et al. 2001), camptothecin (Prothecan®) (Greenwald et al. 1996; Bedeschi et al. 1997; Conover et al. 1997; Conover et al. 1998; Greenwald et al. 1998; Conover et al. 1999; Ochoa et al. 2000; Zhao et al. 2000), and podophylotoxin (Greenwald et al. 1999) in corresponding conjugated forms with PEGs.

The authors thank Dr. A. H. Zarrin (Pharm. D.) and Dr. N. Zarei (Pharm.D.) for their valuable comments and assistance in the preparation of this article.