Effects of pharmaceutical PEGylation on drug metabolism and its clinical concerns.

Review

Expert Opin. Drug Metab. Toxicol. Downloaded from informahealthcare.com by Emory University on 11/02/14 For personal use only.

Effects of pharmaceutical PEGylation on drug metabolism and its clinical concerns 1.

Introduction

2.

Pharmaceutical significance of PEGylation

3.

Metabolism and toxicity of PEG

4.

Metabolism of PEGylated proteins and peptides

5.

Metabolism of PEGylated small-molecule drugs

6.

Effects of PEGylated formulations on drug metabolism

7.

Clinical concerns on PEGylated drugs and formulations

8.

Conclusion

9.

Expert opinion

Xingwang Zhang, Huan Wang, Zhiguo Ma & Baojian Wu† †

Jinan University, College of Pharmacy, Division of Pharmaceutics, Guangzhou, China

Introduction: PEGylation refers to covalent conjugation of one or more polyethylene glycol chains to a drug molecule. It also refers to formulation of a drug into PEGylated drug-delivery vehicles in pharmacy. It is well-known that PEGylation can greatly influence the pharmacokinetics and pharmacodynamics of drugs. Areas covered: This article describes the importance of PEGylation in drug development and research. The impact of PEGylation on drug metabolism and the clinical safety of PEGylated drugs (or formulations) are also discussed. Information and data from the literature were collected, analyzed and summarized. Expert opinion: PEGylation is an effective approach to potentiate drugs with undesirable properties. Currently, PEGylation has penetrated into every field of pharmaceutical practice, involving biomacromolecules, small drugs and drug delivery systems. Efficacy enhancement is attained through modification of the pharmacokinetics and toxicity profiles of parent drugs. As a result of PEGylation, the drugs tend to display enhanced solubility, prolonged circulatory time and reduced immunogenicity/antigenicity. The bottleneck of PEGylation is how to break through the limitation of chemical conjugation and to properly preserve the pharmacological activity of the drug. PEGylated formulation is an area deserving more attention in terms of systemic delivery of insoluble small drugs. Keywords: clinical concerns, metabolism, PEGylation, proteins and peptides, small-molecule drugs Expert Opin. Drug Metab. Toxicol. [Early Online]

1.

Introduction

Advances in combinational chemistry and biotechnology have significantly accelerated drug discovery and development. More than 10,000 drugs are undergoing clinical evaluation in 2013. About a third of these drug candidates are proteins and peptides, and a large portion of the rest are small-molecule drugs. Modern pharmaceutical technologies allow new drugs to be produced in such large scale. However, unfavorable physiochemical and pharmacokinetic properties have been limiting their clinical use [1]. These properties include poor water-solubility, high susceptibility to metabolic enzymes, short residence in the body and rapid kidney clearance (CL). Various strategies have been explored with the aim to obtain drugs with improved properties. These strategies include the molecular manipulations to a drug and the utility of more suitable formulations. The former involves the chemical modification of a drug structure, such as salification [2], altering the amino-acid sequence of polypeptides [3], fusing drugs into immunoglobulin or albumin [4,5] and introducing a new group to drugs [6]. The latter is to incorporate drugs into smart delivery vehicles to enhance the clinical efficacy [7]. Therein, PEGylation is an 10.1517/17425255.2014.967679 © 2014 Informa UK, Ltd. ISSN 1742-5255, e-ISSN 1744-7607 All rights reserved: reproduction in whole or in part not permitted

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X. Zhang et al.

Article highlights. .

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PEGylation has been proved to be highly potential in improving the pharmacokinetics and pharmacodynamics of drugs. The pharmaceutical significance of PEGylation was elaborated. PEGylated proteins and peptides as well as their body metabolism were discussed. The PEGylation of small-molecule drugs and its effect on the drug metabolism in vivo were discussed. PEGylated formulations and their effects on pharmacokinetic behavior of encapsulated drugs were summarized and analyzed. The clinical considerations of using PEGylated products were addressed.

This box summarizes key points contained in the article.

effective approach to potentiate the drugs with poor drug-like properties. Compared to non-modified drugs, the PEGylated ones (including PEGylated formulations) display different pharmacokinetic behaviors. For instance, PEGylated drugs tend to possess prolonged body residence, decreased accessibility for metabolic enzymes and reduced renal elimination. The most effective approach to systemic delivery of protein is through structural modification by addition of one or more PEG molecules. For small-molecule drugs, use of PEGylated delivery carriers such as ‘stealth’ liposomes is also highly possible in enhancing drug delivery. PEG is commercially available and has been approved by FDA for use in topical and/ or intravenous (i.v.) routes. Several PEGylated drugs have been successfully marketed such as PEG-IFN-a2a, PEGgrowth hormone receptor antagonist and PEG-anti-VEGF aptamer [8]. However, relatively little is known about the metabolic pattern of PEGylated drug or drug-loaded formulation. The present review aims to elaborate the applications of PEGylation in drug design and metabolic knowledge of PEGylated drugs (and formulations). Additional focus is centered on the clinical safety of PEGylated drugs. 2.

Pharmaceutical significance of PEGylation

Recombinant DNA technique makes the large-scale production of proteins and peptides feasible. However, their clinical uses are significantly limited by the unfavorable properties such as mass molecular weight (MW), low solubility, poor physiological stability, susceptibility to enzymatic degradation and rapid kidney CL. In addition, some native peptides often exhibit obvious immunogenicity and antigenicity, increasing the risk of long-termed and repeated drug administration. These limitations generally result in the failure of i.v. delivery. Further, it is difficult to incorporate protein and peptide drugs into delivery carriers due to strong hydrophilicity. Although in some cases the macromolecules can be encapsulated into carriers, the delivery systems are concerned with low drug load, leakage from the carriers and loss of activity 2

owing to the use of organic solvent. In order to potentiate these biodrugs, scientists have been exploring a more effective means of drug delivery. In the year 1977, Devis and his colleagues discovered that the body residence times of bovine serum albumin and liver catalase were prolonged and the immunogenicity was reduced by covalent attachment of PEG chain [9,10]. Since then, PEGylation has been an important approach to improve the pharmacokinetics and pharmacodynamics of active molecules. PEGylation of drugs can increase the water solubility, lower the enzymatic degradation, reduce the phagocytosis by reticuloendothelial systems (RES), decrease the glomerular filtration and downregulate the immunogenicity or antigenicity [8]. Likewise, modern synthesis technique allows smallmolecule drugs to be produced in a large quantity, even structurally sophisticated compounds. Unfortunately, most of these drug candidates possess serious solubility and systemic toxicity issues, posing a significant barrier to drug administration and to the therapeutic uses [11,12]. Clearly, effective approaches are needed to resolve both the delivery and safety problems. In addition to being miscible with water, PEG can dissolve in the frequently used triglyceride, revealing its lipophilicity. Based on ‘like dissolves like’ principle, PEGylation can enhance the solubility of lipophilic molecules. Therefore, small-molecule drugs can also be reconstructed by PEGylation for the purpose of solubility enhancement. In other words, PEGylated small-molecule drugs share the same rationale of improved properties with PEGylated proteins and peptides. Nevertheless, one should bear in mind that not all drugs can be PEGylated [13]. PEGylation is impossible for the drugs in which the chemical group(s) for PEG conjugation is lacking. For such drugs, PEGylated formulations are the alternative method to enhance the drug-like properties. PEGylated formulations can finely offset the weakness of direct PEGylation. As being encapsulated in PEGylated formulation, the undesirable properties of drugs can be shielded, and the performance in vivo can be enhanced. Thereby, PEGylated formulations achieve the same goal as the PEGylated drugs in a different approach. In summary, PEGylation has been a versatile platform of drug design and drug delivery. Main drawbacks of parent drug and advantages of PEGylated drug (or formulations) are schematized in Figure 1. 3.

Metabolism and toxicity of PEG

PEG is a polymer with repeating units of ether oxygen (-CH2CH2O-) formed by polymerization of ethylene oxide or ethylene glycol under alkaline catalysis. Generally, PEG is a mixture of homologues that can have different MWs and shapes. Each ether oxygen subunit is demonstrated to be firmly associated with two or three water molecules in solution. Liquid PEGs with MW below 600 are miscible with water in any ratio. Certainly, solid PEGs also possess excellent solubility. The solubility and viscosity of PEG

Expert Opin. Drug Metab. Toxicol. (2014) 10(11)

Effects of pharmaceutical PEGylation on drug metabolism and its clinical concerns

Protein and peptide drugs


Small molecule drugs

Low solubility Easy proteolysis Short half-life Protential antigenicity PEGylated or immunogenicity

PEGylation drug or formulation

Low solubility High toxicity Rapid elimination Lack of specificity

Increased solubility owing to hydrophilic PEG Diminished accessibility to enzymes and antibodies Prolonged residence time in body A decrease or lack of immunogenicity Reduced toxicity and side effects Passive accumulation in tumoral or inflammatory tissues

Figure 1. The main advantages of PEGylation: resulting in improvement of pharmacokinetic, pharmacological and toxicological properties.

solutions are not affected by the presence of electrolytes and ionic strength [14]. Another excellence of PEG is the strong solvent power for a great number of substances. These advantages lead to its extensive applications in pharmaceutical, cosmetic and foodstuff fields. The MW of 2000 is regarded the terminal size of PEG that can be absorbed by epithelial membranes through paracellular transport or macropinocytosis [15]. The extent of absorption for PEG 2000 is just 2% by oral gavage and ~ 4% following intranasal administration [16]. PEGs can be eliminated from the body in the form of metabolites or original polymer, which is dependent on their MW and shape. PEGs < 400 in MW could be transformed to toxic metabolites in vivo by alcohol dehydrogenase, whereas larger PEGs used for PEGylation of drugs or formulations undergo less enzymatic degradation [17]. It should be mentioned that large-size PEG polymers can also be metabolized by enzymes, but the rate of biotransformation is significantly slower compared with the systemic elimination. There are clear data demonstrating that the metabolism of PEG does occur on alcohol groups with the production of corresponding acid or di-acid metabolite, because these metabolites have been reported in the plasma and urine of the subjects. The MW exerts an important effect on the Phase I metabolism of PEG. Approximately 25% of dosed PEG 400 is metabolized in vivo, but the metabolism reduces with an increase of the MW. For example, the overall metabolism is < 4% of the dose for PEG 6000 [18]. The Phase I metabolism of PEG was mediated by alcohol dehydrogenase and aldehyde dehydrogenase [19]. Other enzymes like CYPs and sulfotransferase have also been implicated in the metabolism of PEG [20]. The chain length of linear PEG may be shortened slowly in vivo through enzymatic action, and the branched PEG may also miss one chain due to hydrolysis by an anchimeric assistance [21]. PEGs are FDA-approved excipients extensively used in intravascular, topical and oral formulations. The polymers can be completely eliminated either by renal or by

hepatobiliary pathway, depending on their MWs. PEG molecules below 20 kDa are cleared through the renal filtration, whereas larger PEGs are primarily eliminated by liver sequestration followed by the biliary excretion [20,22]. Due to easy elimination, PEG shows insignificant toxicity at a wide range of doses. PEG itself only generates toxicity in the case of parenteral route at a high dose. Kidney is the exclusive target organ subjected to PEG offense when the dose exceeds the maximum renal burden. Based on the recommendation from the World Health Organization, acceptable oral daily intake of PEG is up to 10 mg/kg. The data are unavailable regarding the toxicity assessment of PEG using human subjects. However, the toxicity of PEG has been evaluated in several animal species. The results showed that high dose, over 350 mg/kg/day, can result in acute and chronic toxicities to the rabbits, such as renal tubular swelling, hepatic parenchyma and death, and low dose like 2.276 mg/m2/week caused no acute or chronic toxicity to the trial animals. The LD50 of various PEGs in the mouse and rat following intraperitoneal injection exceeds 3.1 g/kg, demonstrating the low toxicity of PEG [23]. It is noteworthy that the PEG levels used for drug delivery are far below its toxicity level. Figure 2 depicts the in vivo fate of PEG polymer and its toxicity profile.

Metabolism of PEGylated proteins and peptides

4.

In general, protein and peptides cannot be orally administrated as a result of insufficient bioavailability. Gastrointestinal degradation by protease and poor permeability due to lack of lipophilicity limit their oral absorption [24]. The parenteral route is also challenged by many factors, such as frequent administration, potential immunogenicity, fluctuant blood drug levels and poor compliance. Protein and peptide bioactives are rapidly eliminated after injection due to degradation by proteolytic enzymes and liver uptake. Indeed, enzymes


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X. Zhang et al.

Toxicity

2000 is the molecular weight cutoff of oral or nasal absorption

Me ta

O O n

PEGs < 400 can be largely metabolized; higher PEGs (e.g. > 6000) are metabolized less

H

ut

et

tr

ib

io

n

H Dis

Low dose


As 600-fold as the dose of PEGylated products

Ab s

Epithelial mucosa

on pti or

m lis bo

High dose

Phase I

Alcohol dehydrogenase Aldehyde dehydrogenase Cytochrome P450s Sulfotransferase

io n

Ex

cr

Renal route (less 20 kD) Liver-gall route (above 20 kD)

Circulating in the blood stream; less distribution into the tissues

Figure 2. The in vivo characteristics of PEG polymer: absorption, metabolism, distribution and excretion.

either in the plasma or in the cytoplasm do not have the opportunity to act on the substrates until they are freely accessible to the target molecules. PEG molecule can increase the size of parent proteins by 5 -- 10 times [25]. This may result from the attachment of lots of water molecules to the PEG chain and the expansion of PEG molecule itself. PEGylation provides several advantages (as showed in Figure 3) for proteins and peptides, such as protection of susceptible molecules from proteolysis, improved half-life and biodistribution and reduced immunogenicity or antigenicity. For example, PEGylated glucagon-like peptide-1 (PEG/GLP-1) displayed a depot effect on insulin release as well as improved biological activity. The half-life of Lys-modified PEGylated GLP-1 was 40-, 10and 28-fold longer than that of GLP-1 in the plasma, liver and kidney homogenates, respectively. Particularly, the N-terminally modified PEG/GLP-1 exhibited a markedly extended metabolic stability [26]. Likewise, PEGylated uricase (urate oxidase) confer the non-human enzyme a sufficiently reduced immunogenicity and a long half-life in patients, allowing a repeated and convenient administration, for example, once or twice a month [27]. Immunogenicity is the ability of a particular substance, such as an antigen or exogenous species, to provoke an immune response in the body. Induction of immunogenicity is dominated by specific epitopes as well as their exposure to immune system. PEGylation can decrease the immunogenicity of drugs by reducing the exposure of epitope to immunocytes. PEGylation has significant effects on the in vivo fate (particularly on the metabolism) of therapeutical proteins. Also, the nature of PEG used for protein modification plays a role in the body disposition of PEGylated proteins, such as MW 4

and shape. The molecular mass of PEG as PEGylation materials are mostly above 5 kDa, with a maximum of 40 kDa [17]. For example, PEGs used for the PEGylation of anti-IL-17A antibody fragments were 40 kDa [28,29]. There are no reports that PEGs > 40 kDa have already been used to modify the proteins. This may be due to large difficulty in synthesis and serious loss of activity. The molecular mass of PEG determines the hydrophilic conformation and steric hindrance for enzyme accessibility of PEGylated products. As a rule, an increase in molecular size reduces the accessibility of metabolic enzymes, thereby prolonging the circulation half-life of PEGylated molecules [30]. Further, the shape of PEG has an influence on the pharmacokinetic property of PEGylated proteins and peptides. Branched PEG-tailored proteins display a longer half-life and reduced metabolic destruction as compared to linear PEG-tailored ones [31]. On the other hand, the steric effects derived from large size and shape would undermine the pharmacological activity of native proteins or peptides because of inaccessibility of the active molecules to their targets. For instance, the two commercially available PEGylated IFN, PegIntron and Pegasys, distinguish them by a particular pharmacokinetics, pharmacodynamics and biodistribution [32]. Pegasys, an IFN PEGylated with branched PEG, exhibits a weaker specific activity albeit with enhanced pharmacokinetic parameters such as prolonged half-life, smaller volume of distribution (Vd) and reduced body CL, in comparison with the linear PEGmodified form. Nonetheless, the two medications have exhibited equivalent clinical performance. Decreased activity could be compensated by the improved pharmacokinetic properties [33].



ttack

he a

gt ventin

Proteases

m

Circu

Interfering the recognition Bloc


king

Antibodies the

ops

oniz

atio

n

Hydrophilic corona Immunoglobulins

Prolonged blood residence time

Figure 3. PEGylated proteins or peptides and the underlying mechanism of enhanced pharmacokinetics and pharmacodynamics.

Although PEGylation has a prominent advantage of reducing the metabolism of PEGylated bioactives, the conjugates still experience slow degradation by protease or related enzymes in the body [34]. Data on the metabolism of PEGylated proteins in human subjects are not available, although there is clear evidence that degradation occurs in animals, leading to the release of PEG. Mircera is a PEGylated epoetin b marketed by Hoffmann-La Roche. Both uncharged (about 1% of administrated dose) and PEG-like material were found in the urine of rats [35]. Data from animals indicated that once CIMZIA (PEGylated certolizumab, a therapeutic mAb to TNF-a marketed by UCB) cleaved from the Fab’ fragment, the PEG moiety is mainly excreted in urine without further metabolism [36]. Radiolabeled methodology was performed to explore the metabolism of PEGylated recombinant urate oxidase (Pegloticase) following administration of Krystexxa by the i.v. or SC routes. By measuring the amount of radioactivity in the blood and urine, it was found that greater than 89% of radioactivity in the urine was trichloracetic acid-soluble fraction. The results indicated that extensive degradation of 125I radiolabeled protein occurred by vascular proteolytic action and reticuloendothelial processing [37]. Experiment dealt with TNF binding protein has demonstrated the presence of vacuoles in lysosomes after administration of its PEGylated conjugate. However, after a 2-month convalescence, these vacuoles are no longer positive for TNF-binding protein but are associated with PEG alone, demonstrating the metabolism of PEGylated protein [38]. The metabolic process of PEG conjugates in biological fluids could be analyzed if an accurate method is adopted [39]. The ultimate goal of attaching PEG(s) to a protein or peptide is to minimize the enzymatic degradation and prolong their

surviving half-life. As a matter of fact, PEGylation has brought about significant changes in metabolism pattern, particularly in elimination associated with the liver and kidney. The marketed PEGylated proteins and their improved pharmacokinetic properties have been collected and summarized in the literature. Please note that pharmacokinetic properties other than metabolism were not discussed here. The readers can refer to the article by Milla et al. [17].

Metabolism of PEGylated small-molecule drugs

5.

Modern synthetic technique is resulting in numerous drugs or candidates (particularly anticancer agents) that show low solubility and/or high toxicity [40]. Following commercial success of PEGylated proteins, there is an intense and continuous interest in developing PEGylated small-molecule drugs. To date, there are four PEGylated drugs undergoing clinical trials, namely PEG-naloxol (Phase III), PEG-irinotecan (Phase II/III), PEG-SN38 (Phase II) and PEG-docetaxel (Phase I). Still, numerous PEGylated small-molecule drugs are being developed and many of which are ready to clinical investigation [13]. PEGylation of small drugs can not only improve their solubility and biodistribution but also attenuate their metabolism and toxicity by altering drug exposures to enzymes and vital organs. Unfavorable attributes of small-molecule drugs could be addressed using PEGylation strategy by which the properties of drugs are converted to the conjugates. The hydrophilic PEG chains serve as a coating for the drugs, leading to alterations of their in vivo biofate. PEGylation increases the hydrophilicity of drugs, thus reducing the uptake of drugs by


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X. Zhang et al.

A. PEG chain (short) Drug

Active site

Permanent linkage PEG chain (long)

Cell


B.

Releasable linkage

Cell

Figure 4. The PEGylation of small-molecule drugs. A. Permanent conjugate. B. Releasable conjugate.

normal cells. This is because lipophilic compounds are more readily transported through cell membrane. Further, transmembrane transport of drugs is largely limited by MW. PEGylation significantly increases the MW of parent drugs, resulting in accumulation of PEGylated drug conjugates in tumor tissues by enhanced permeability and retention (EPR) effect. Generally, PEGylation can exert an effect on the metabolism of the attached drugs by two mechanisms: shielding plasma enzymes and reducing RES phagocytosis [41]. The shielding effect can be deduced from the prolonged halflife in vivo and decreased pharmacological activity in vitro in the case of PEGylated IFN [42,43]. For some small-molecule drugs, the attachment of PEG chain makes the conjugates amphiphilic owing to the hydrophobicity of drug moiety. The conjugates can be self-assembled into micelle-like nanoparticles and passively target to solid tumor by EPR effect [44]. It is noteworthy that the bonds or spacers linking PEG with drugs also play a role in the metabolism of parent drugs, because they determine the release rate of native drugs. According to the characteristics of the linkages, there are two types of PEGylation of small-molecule drugs, namely ‘permanent’ or ‘releasable’ PEGylation (Figure 4). Permanent style (e.g., NKTR-118, an indecomposable PEGylated naloxol) often results in novel candidates that possess enhanced biomembrane permeability and oral bioavailability [45]. The strategy of permanently attaching a PEG to a small molecule requires using a low MW PEG. Large PEG may cause the loss of activity of parent drug due to increased steric hindrance. For example, Greenwald et al. [46] obtained highly watersoluble paclitaxel derivatives by the attachment of PEG (2 -- 5 kDa) to paclitaxel at the 7-position via a permanent carbamate bond. Although these water-soluble derivatives just showed activity in micromolar range, the activities were much weaker than those of native drugs. Therefore, releasable PEGylation would be a more attractive approach for modification of therapeutic drugs. 6

Releasable PEG-drug conjugates are usually considered as macromolecular prodrugs that have to be chemically or enzymatically transformed into their active forms in the body [47]. For example, the anticancer agent camptothecin has high toxicities and poor stability in vivo. To increase the blood half-life and stabilize the active lactone configuration, PEGylated camptothecin (Pegamotecan) was developed by Enzon Pharmaceuticals, Inc. This prodrug was engineered by attaching two molecules of camptothecin to a diol PEG of 40 kDa via a releasable ester bond [48]. Results from clinical trials showed that the conjugate was highly tolerated with significantly reduced toxicity as compared to the commercial formulation. However, quick hydrolysis in vivo of the linkage caused a parallel toxicity to that of the native drug, leading to the failure of drug development of this conjugate [49,50]. In short, the in vivo metabolism of small-molecule drugs can be manipulated through PEGylation, for example, utilizing a suitable linkage and controlling the release speed of parent drugs.

Effects of PEGylated formulations on drug metabolism

6.

PEGylated formulation is an indirect PEGylation approach for drug potentiation. PEGylated formulations are PEGmodified drug carriers, into which drugs are incorporated. The PEGylated formulation and its featured biofate are illustrated in Figure 5. Compared with the PEGylation of drugs, the PEGylation of formulations possesses several advantages, such as easy production, low cost and good applicability. For those drugs that cannot be PEGylated, it is an advisable alternative to exploit PEGylated formulations. The first commercially available PEGylated formulation is the PEGylated liposomal doxorubicin (Doxil), which was approved in 1995 [51]. Although marketed PEGylated drug-containing formulations are limited to liposomes and the drug of



Toxicity-sensitive organs

Metabolic enzymes

Opsonin proteins

Non-PEGylated carriers

Expo

sure


Reticuloendothelial systems re

posu

d ex

uce Red

Drug

Reduced opsonization PEGylated carriers

Figure 5. Schematic illustration of PEGylated formulation and its advantages relative to non-PEGylated vehicle.

doxorubicin, great endeavors have been made to develop PEGylated carriers to effectively deliver drugs that have a short half-life and/or high toxicity [52-56]. Similar to the PEGylated macromolecules, PEGylated formulations also exert a positive action on the metabolism of encapsulated drugs [57]. The extended circulation half-life is the evidence that drug metabolism is altered by use of PEGylated formulations. Depending on the ‘Stealth’ of PEGylation, PEGylated formulations or carriers can reduce the sequestration of nanoparticle-associated drugs by RES systems, protect them from degradation by metabolic enzymes and increase their accumulation in tumoral or morbid tissues with leaky microvessels. In general, the drugs entrapped into a PEGylated formulation are poorly water-soluble. Phase I and Phase II metabolism reactions are both involved in the disposition of these drugs, because it is necessary to introduce reactive and polar groups into these molecules [58,59]. The liver is the predominant metabolic organ of drugs, which is distinct from the metabolism of proteins and peptides where plasma enzymes play a leading role. Therefore, reducing the uptake of the liver is an effective means to prolong the half-life of delivered drugs. PEGylated formulations have exhibited a significant merit in this regard. Furthermore, reduced exposure of drugs to systemic enzymes and normal cells via PEGylated carriers is beneficial either to prolong the body-residence or to attenuate the drug-associated toxicity. Taking doxorubicin as an example, doxorubicin (Adriamycin) is a strong cytotoxic drug that is widely used in oncology. Short half-life and progressive myocardial damage

are the main defects of doxorubicin. Rapid systemic CL and easy accumulation in toxicity-sensitive tissue such as myocardium limit its clinical applications. To overcome these limitations, two different liposomal formulations (Myocet, non-PEGylated liposomes; Doxil, PEGylated liposomes) were developed. Compared to conventional liposomes, PEGylated formulation showed a much greater potential in altering pharmacokinetic parameters such as half-life (t1/2), systemic drug exposure (AUC), Vd and systemic CL (Table 1). The data clearly demonstrated that PEGylated formulation could markedly modify the in vivo fate of the encapsulated drugs, resulting in enhanced therapeutic efficacy and reduced toxicity. Besides PEGylated liposomes, other PEGylated nanocarriers have been used to deliver cytotoxic drugs for the improvement of adverse events. Table 2 showed the main changes of pharmacokinetic parameters of drugs before and after being encapsulated in PEGylated formulations. It can be concluded that PEGylated formulations have the capability to lowering the metabolic elimination of drugs by reduced enzymatic approachability and improved biodistribution.

Clinical concerns on PEGylated drugs and formulations

7.

PEGylation is shedding light on the improvement of clinical efficacy of therapeuticals [60]. Nevertheless, there are numerous hurdles before PEGylated products can approach the clinical application. For example, it is difficult to precisely characterize PEGylated drugs. In addition to acceptable safety


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X. Zhang et al.

Table 1. Pharmacokinetic parameters in patients of doxorubicin formulations: free doxorubicin, conventional and PEGylated liposomal doxorubicin. Formulation

Tumor type

Dose (mg/m2)

T1/2 (h)

Cmax (mg/l)

AUC (mg/h/l)

CL (ml/h)

Vd (l)

Ref.

Free Dox CL-Dox PL-Dox

Breast Breast Breast

50 75 50

10.4 50.9 75.1

5.9 11.59 22.5

3.5 12.52 2579.5

25,300 6360 19

365 58.08 2.082

[70] [71] [72]

The pharmacokinetic parameters are presented as median values. CL-Dox: Conventional liposomal doxorubicin; Dox: Doxorubicin; PL-Dox: PEGylated liposomal doxorubicin.


Table 2. Pharmacokinetic comparison between various non-PEGylated and PEGylated formulations loading drug. API

PEGylated carriers Control (shadeless)

Dose

Cmax

AUC0!`

Curcumin

PEG-micelles Drug solutions PEG-emulsions FYTOSID injections PEG-polymeric NPs Drug solutions PEG-lipid NPs Drug solutions PEG-dendritic NPs Drug solutions

10 mg/kg 10 mg/kg 10 mg/kg 10 mg/kg 30 mg/kg 30 mg/kg 50 mg/kg 50 mg/kg 2.7 mg/kg 2.7 mg/kg

2,173 3,661 / / 4.56 17.05 / / 2.284 2.578

621.6 474.0 207.1 34.9 5.795 6.263 783.39 61.31 41,731 13,418

Etoposide 5-Fluorouracil Noscapine Aceclofenac

T1/2

Vd

CL

0.057a/1.153b 0.023a/0.254b 0.50a/8.3b 0.22a/3.4b 33.3 0.088 11.94 1.19 12.1 3.97

/ / / / 0.114 0.069 1.03 1.40 / /

16,120 21,120 280 50

60 810 / /

Subjects

Ref.

Mice

[73]

Wistar rats

[74]

Rabbits

[75]

Swiss mice

[76]

SD rats

[77]

The unit of the maximum plasma drug concentration (Cmax), the area under the plasma drug concentration versus time curve (AUC0!¥), the half-life (t1/2), the volume of distribution (Vd) and the total body clearance (CL) is normalized as µg/ml, µg/h/ml, h, l and ml/h, respectively. T1/2a: Distribution half-life. T1/2b: Elimination half-life. NPs: Nanoparticles.

and efficacy, an ideal PEGylated product may embrace the following attributes: . Easy

to manufacture with a good technical reproducibility. . Able to characterize the product and its performance using key parameters. . Good compliance and acceptable payment for patients. . Suitable stability in use, storage and administration. PEGylated products include PEGylated biomacromolecules, small-molecule drugs and drug-loaded formulations. For PEGylated proteins and peptides, the potential immunogenicity and antigenicity, possibly caused by the fragments of native proteins and variable molecular mass of conjugates, should be given close attention [61]. It is possible that PEGylation leads to the formation of new epitopes as a result of partial protein fragmentation or introduction of exogenous species. For example, Phase I clinical trials showed that unusual anti-PEG antibodies was formed in some patients after administration of PEG-uricase [62]. The methoxyl group of PEG chain at the terminus remote from the protein was identified as the presumable cause of antigenicity [27]. Moreover, there was a report on the induction of anti-PEG immune response in the case of repeated administration of 8

PEG-glucuronidase [63]. It is difficult to anticipate the characteristics of PEG-protein conjugates, because they largely depend on the nature of protein, polymer and final product composition. Thus, it is crucial to recognize the key processes of chemical coupling that determine the quality of conjugates and to take steps to surmount them. Generally, small-molecule drugs are chemically more stable than biodrugs and can be PEGylated under a strong reaction condition. It is important to reproduce the drug batches and reduce the side products for stable product quality. Residues control of reactive and catalytic agents employed in PEGylation is another issue that needs to be resolved both for macromolecular and small drugs [64]. As for PEGylated formulations, the biocompatibility of excipients, product stability, particle size and administration integrate the main concerns of clinical applications [65]. To date, PEGylated formulations on the market are limited to PEGylated liposomal doxorubicin, for example, Doxil, Caelyx and Myocet. The materials used in PEGylated liposomes essentially contain phospholipids and cholesterol. They are generally less toxic in physiology. However, the products (e.g., lysophosphatide and peroxide) resulting from hydrolysis and oxidation of phospholipids (particularly those in liquid dosage forms) have negative effects on product quality [66]. The injectable products generally require the particle




size < 0.5 µm and no larger than 5 µm particulates detectable (item in USP 36), so as to avoid embolic syndrome. Unfit infusion rate of PEGylated formulation containing chemotherapeutics would cause extravasation of drug solution and vasculitis [67,68]. The kidney and liver are the main elimination organs of PEG polymers. Special attention should be paid to patients who suffer kidney and/or liver diseases. Acute renal tubular necrosis has been reported after i.v. administration of Lorazepam (Ativan) to a patient who received a cumulative dose of 240 g PEG 400 [69]. Accordingly, therapeutic drug monitoring should be performed when PEGylated drugs are applied to special population, such as pediatrics, geriatrics or patients with kidney failure. The factors above-mentioned have to be considered in developing and use new PEGylated products. 8.

Conclusion

Depending on good biocompatibility and non-toxicity, PEG is being extensively applied to pharmaceutical and medical purposes. PEGylation refers to covalent attachment of PEG chain(s) to another molecule, a drug or biomaterial for fabrication of drug carriers. PEGylation profoundly affects the fate in vivo of the ready-to-refine drugs, prolonging their circulation half-life. The metabolic process of drugs can be regulated with PEGylation by altering the drug exposure to enzymes and biodistribution. Physicochemical and pharmacokinetic properties of macromolecular or small-molecular drugs could be improved by direct PEGylation or use of PEGylated formulations, which rest with the difficulty of chemical synthesis. In conclusion, PEGylation has been shown to be a versatile platform for the elevation of drug performance. 9.

Expert opinion

Attachment of a PEG chain to biomacromolecules (i.e., PEGylation) can change their pharmacokinetic and pharmacodynamic properties. This technique has already been successfully used to modify small-molecule drugs and formulations, resulting in dozens of PEGylated products available on the market. PEGylation confers native proteins or drugs many advantages, including increased solubility, reduced immunogenicity or antigenicity of protein, attenuated toxicity of cytotoxic drugs and prolonged circulation half-life. However, the complexity of manufacture is an obstacle towards development of PEGylated drugs. How to properly preserve the activity of native drugs and obtain a monodispersed product rather than a mixture is the key point of successful PEGylation. Drug-loaded PEGylated formulations are challenged by the scarcity of injectable excipients for carrier preparation, product stability and potential risk of administration. The ultimate goal of PEGylation is to improve the clinical efficacy of therapeuticals. In the last decades, there have been a

number of successful cases on PEGylation. PEGylation technology is involved in an interdisciplinary science that closely concerns pharmaceutics, chemistry and clinical medicine. To perform PEGylation research, one should be very familiar with the physicochemical properties of drugs and materials used for PEGylation. It is also important to design the rational route of PEGylation, characterize the key parameters of products and elucidate the process of drug disposition in vivo. PEGylation is presenting a new opportunity for developing innovative drugs. It can be expected, in the coming decades, there will be more PEGylated products in the market. Although PEGylated proteins and peptides have obtained remarkable successes, there have been limited progresses in developing PEGylated small-molecule drugs. This status may be attributed to the loss of bioactivity of native drug and the difficulty of chemical conjugation and purification. It is of great value to elucidate the structure--activity relationships of small-molecule drug. This information will help to specifically link a PEG chain to the inactive site of drug. It is the authors’ opinion that click chemistry may be the most promising technique for selective modification of small-molecule drugs in the future. PEGylation of formulation does not involve chemical process. However, this type of PEGylation is hardly applied to deliver protein and peptide drugs as a result of low drug load due to strong hydrophilicity. Flexibility in drug loading renders PEGylated formulation suitable for i.v. delivery of poorly water-soluble or insoluble drugs. PEGylated formulation is more suitable for those drugs that cannot be chemically PEGylated. There is a particular interest in using PEGylated formulation to improve the pharmacokinetics of drugs with solubility and/or toxicity issue. PEGylated formulation will not alter the intrinsic characteristics of the drug itself but can actually influence the drug disposition in an indirect manner (e.g., changing the biodistribution and shielding the metabolic enzymes). Therefore, utilization of PEGylated formulation should be an advisable alternative to deliver ‘problem’ drugs, particularly chemotherapeutic agents.

Acknowledgment X Zhang and H Wang contributed equally to this work.

Declaration of interest The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.


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Affiliation Xingwang Zhang1 PhD, Huan Wang2, Zhiguo Ma2 & Baojian Wu†3 PhD † Author for correspondence 1 Postdoc., Jinan University, College of Pharmacy, Division of Pharmaceutics, Guangzhou, China 2 Jinan University, College of Pharmacy, Division of Pharmaceutics, Guangzhou, China 3 Professor, Jinan University, College of Pharmacy, Division of Pharmaceutics, 601 Huangpu Avenue West, Guangzhou, Guangdong 510632, China Tel: +86 20 8522 0482; Fax: +86 20 8522 0482; E-mail: [email protected]

Fetal drug metabolism and its possible clinical implications.

Biochemical effects of the pharmaceutical drug paracetamol on Anguilla anguilla.

Impact of pharmaceutical cocrystals: the effects on drug pharmacokinetics.

Influence of xenobiotics on drug metabolism and its sensitive detection.

Effect of PEGylation on drug entry into lipid bilayer.

Drug reformulations and repositioning in pharmaceutical industry and its impact on market access: reassessment of nomenclature.

Effects of flow intermittency and pharmaceutical exposure on the structure and metabolism of stream biofilms.

Drug repurposing in pharmaceutical industry and its impact on market access: market access implications.

Adipose tissue dysfunction and its effects on tumor metabolism.

Drug chirality: impact on pharmaceutical regulation.

Clinical implications of variable antiarrhythmic drug metabolism.

Comment. Twenty years on: a review of the current practice of drug metabolism and pharmacokinetic studies in the pharmaceutical industry.

Questioning the Use of PEGylation for Drug Delivery.

Presence of the pharmaceutical drug carbamazepine in coastal systems: effects on bivalves.

Effects of insecticide synergists on microsomal oxidation of estradiol and ethynylestradiol and on microsomal drug metabolism.

Drug metabolism by leukocytes and its role in drug-induced lupus and other idiosyncratic drug reactions.

Construction of drug network based on side effects and its application for drug repositioning.

Effects of antibody, drug and linker on the preclinical and clinical toxicities of antibody-drug conjugates.

Inborn 'errors' of drug metabolism. Pharmacokinetic and clinical implications.

Effects of PEGylation on the physicochemical properties and in vivo distribution of organic nanotubes.

Effects of PEGylation on membrane and lipopolysaccharide interactions of host defense peptides.

Fetal carbohydrate metabolism: its clinical importance.

[Practice of drug monitoring based on comprehensive pharmaceutical judgment].

Systematic prediction of drug combinations based on clinical side-effects.