Current Topics
Current Status and Prospects of Polyethyleneglycol-Modified Medicines
2013 Volume 36 Issue 6 Pages 883-888
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2013 Volume 36 Issue 6 Pages 883-888
Polyethyleneglycol (PEG) compounds have a large hydrogen bonding capability and possess various functions that depend on the length of the chain and conformational diversity. Modification of a drug with PEG is a well-known technology for improving the physicochemical properties and biological responses of a drug. There are many reports about the modification of small molecules with PEG, however, there are no modified small molecule products currently on the market. Several protein products for medical use are commercially available. In this review, the effects of modification with PEG on biopharmaceuticals are described through the comparison of two interferon-α products modified with PEG, one with 12 kDa linear PEG and the other 40 kDa branched PEG. There is one original drug delivery system product, Doxil®/Caelyx®, on the market in which liposomes modified with PEG and encapsulating doxorubicin are stabilized sterically and invisible to the reticuloendothelial system. The benefits of modification with PEG are described here with examples of modified products on the market and used in clinical trials.
Polyethyleneglycol (PEG) is a polymer of ethylene oxide, also called polyethylene oxide (PEO) and polyoxyethylene (POE). The polymers have been graded with respect to their range of molecular weight and demonstrate unique properties (e.g., freezing/melting point, solubility) based on their molecular weight and structure (e.g., linear or branched). PEGs are widely used as pharmaceutical ingredients in ointments, suppository formulations, creams, lotions, lubricants, coating material of tablets, and solubilizers for injection. This polymer has low toxicity because it is eliminated through renal and hepatic pathways.1) Also, PEG3350 is an active pharmaceutical ingredient in the medical treatment of chronic constipation and mixtures of PEG3350 and electrolytes are used as mild cathartics and laxatives as pretreatments for surgery and colonoscopy.2) Several polymeric surfactants, for example, Pluronic®, a tri-block polymeric surfactant made from PEG and polypropylene oxide, are used to stabilize suspensions and emulsions.3) Various non-ionic detergents, for example, a polyoxyethylene derivative of sorbitan monolaurate (Tween® 20),4) are used in many liquid formulations.
PEGs have large hydrogen bonding capability, and as a result the chemical modification of materials with PEG (“PEGylation”) yields significant changes to their physicochemical properties.5) Several activated PEG derivatives for the chemical modification of drug substance have been reported previously6–8) and PEGylation of various drug substances and pharmaceutical ingredients, such as proteins,6) RNAs,9) small organic compounds,10) polymers11) and lipids,12) can be performed in a laboratory. PEGylation can improve the solubility in aqueous and organic media, stability after administration, bio-compatibility and lack of antigenicity, and consequently, enhancement of the pharmacological effect and safety are achieved.7,9,13) In recent exploratory research conducted by pharmaceutical companies, the solubility of drug substance is a major issue in the structural optimization and further development.14) Prodrug strategies, more specifically the introduction of ionizable and/or hydrophilic moieties,15) are effective for improving solubility. Furthermore, PEGylation improves not only the physicochemical properties of a compound, but also its pharmacokinetics and biodistribution.16) The molecular weight increases with PEGylation, depending on the molecular size of the PEG moiety, and a change in molecular size affects the glomerular filtration rate and hepatic clearance.17)
In this paper, the application of PEGylation as a strategy to improve the properties of drug substance and pharmaceutical ingredients is discussed.
Among small molecules, some PEGylated compounds are in the clinical development stage (Table 1). Paclitaxel is a potent inhibitor of cell replication used in the treatment of various cancers.18) Because of its poor water solubility, paclitaxel is currently formulated in taxol and a mixture of polyoxyethyleneglycerol triricinoleate 35 (Cremophor EL®) and dehydrated ethanol.19) Cremophor EL® is toxic when administered intravenously and produces vasodilatation, labored breathing, lethargy and hypotension.19) Greenwald et al. reported PEG conjugation for solubilizing and delivering paclitaxel.20) In a water-soluble ester of paclitaxel with linear PEGs having several different molecular weights, in vivo experiments clearly established that the molecular weight of PEG must be larger than 30 kDa in order to prevent rapid elimination of the PEGylated species from the kidney. PEG-SN38 (EZN-2208) has a multi-arm PEG backbone (40 kDa) linked to four SN38 molecules and has been successfully prepared with high drug loading and significantly improved water solubility (400- to 1000-fold of SN38).21) This conjugate has non-permanently bounded PEG, functionalized as a prodrug system which releases SN38 with 12 min of half-life in human plasma. The drug release is quite fast in treated mice, and EZN-2208 showed a 207-fold higher exposure to SN38 compared to irinotecan, with the tumor to plasma drug concentration ratio being increased over time during the four day long pharmacokinetic and biodistribution studies. Sapra et al. reported that the passive accumulation of SN38 in solid tumors after intravenous injection of EZN-2208 results from an “enhanced permeation and retention (EPR)” effect.22) The EPR effect exists because of the discontinuous endothelial lining and other capillary anomalies in the tumor vasculature and these leaky vessels facilitate the extravasation of supramolecular structures, such as polymers and liposomes, into the interstitial space in solid tumors.23) Additionally, multi-arm PEG can introduce drug substances to the terminal of each outshoot and seems to be a promising system for delivering insoluble antitumor agents to tumor tissues.24) In contrast, several programs for the clinical development of small molecules conjugated with linear PEG have been recently suspended or discontinued.
| Conjugate | Original drug | PEG | Status of development |
|---|---|---|---|
| PEG-SN38 (EZN-2208) | SN38 | 40 kDa, 4 arm-branched PEG | Metastatic breast cancer (Phase 2), Metastatic colorectal cancer (Phase 2), Pediatric solid tumor (Phase 1), Solid tumors (Phase 1) |
| Etirinotecan pegol (NKTR-102) | Irinotecan | 40 kDa, 4 arm-branched PEG | Metastatic breast cancer (Phase 3), Platinum-resistant ovarian cancer (Phase 2), Second-line colorectal cancer (Phase 2), GI and solid tumor in combination with 5-FU (Phase 1) |
| Naloxegol (NKTR-118), oral | Naloxol | Opioid-induced constipation (Phase 3) |
Many therapeutic proteins must be administered at a high dosage and high frequency because of their short half-life in the systemic circulation and low stability after intravenous injection. PEGylation of these proteins significantly increases the half-life in the systemic circulation and stability against proteolytic degradation,25) and PEGylated conjugates also exhibit reduced immunogenicity and antigenicity.5) Several PEGylated protein products are currently commercially available for therapeutic use (Table 2). There are two PEGylated interferon-alpha (IFN-α) products, PegIntron® (PEGylated INF-α2b) from Schering-Plough Corp.26) and Pegasys® (PEGylated INF-α2a) from Hoffmann La Roche Inc.27) Clarifying the differences between the two products should lead to an understanding of typical approaches using PEGylation for solving issues related to the medical use of proteins. Recombinant human IFN-α was developed as a drug for the treatment of viral hepatitis.28) However, this protein having poor solubility, short half-life and high immunogenicity, is eliminated from the bloodstream in only 4–8 h, and subcutaneous administration three times a week is necessary. In addition, IFN-α monotherapy for chronic hepatitis C leads to a sustained virological response rate in only 10–15% of patients. PegIntron® is a conjugated protein with a 12 kDa linear PEG through a releasable urethane bond. In the case of Pegasys®, a 40 kDa branched PEG2 chain (a molecule having two dendrons of 20 kDa PEG) is induced to one lysine residue of the protein with a stable amide bond. Both PegIntron® and Pegasys® are a mixture of more than ten isomers, depending on the position of the PEGylation. The molecular weights of standard IFN-α, PegIntron® and Pegasys® are 19 kDa, 31 kDa and 60 kDa, respectively, and the difference between the two PEGylated IFN-α is due to the difference in molecular weight of induced PEG. The smaller PegIntron® retains 28% of the native protein and the larger Pegasys® retains 7% because the large and branched mPEG2 moiety hinders the specific binding of the modified protein to the receptor on the surface of target cells to a wider degree than small and linear PEG moieties.27) The reduction of specific activity by the modification entails the increase of dosage amount because it is important that the delivery of a known amount of activity (IU) will provide an equivalent effect to secure the therapeutic efficacy. However, the decrease of affinity to a specific receptor is often counterbalanced by prolongation of the half-life in the systemic circulation or by a higher total exposure. In other words, large and branched PEG allows the masking and protection of protein surface due to an “umbrella-like effect,”29) and the glomerular filtration rate of the modified protein is smaller than that of protein modified with small and linear PEG. In fact, the higher PEG size prevents the free diffusion of the molecule to tissue and organs, as confirmed by the lower distribution volume (Vd) of Pegasys® (8–12 L, the approximate volume of the plasma and extracellular water)30) in comparison to the larger Vd of PegIntron® (69 L, comparable to Vd of native IFN-α2b).31) Additionally, the size of the PEG moiety affects indirectly the possibility to rapidly reduce the effects of adverse events by a dose-reduction of treatment discontinuation, since a larger PEG derivative (with a more prolonged half-life) will require a more prolonged period of time to reduce the plasma concentration of the active molecule after a dose reduction. From the viewpoint of physicochemical stability, the different stability of the two PEG-IFNs also leads to a difference in the formulation of the marketed derivatives. The releasable, less stable, derivative PegIntron® is provided as a lyophilized powder that should be administered immediately after reconstitution, whereas the stable derivative Pegasys® is a ready-to-use solution, with the advantage of easier use and less waste of product. Therefore, both of the PEGylated INF-α, which were developed in each strategy, fulfill the requirements of a long-acting INF-α while providing a significant clinical benefit to the patients with an improved elimination half-life, and furthermore, co-administration of a PEGylated INF-α and ribavirin has been shown to further improve anti-hepatitis C virus activity without placing any additional burden on the patients.32,33) This example illustrates the complexity of PEGylation technology caused by differences in the molecular species of PEG and structure of the linker in the PEGylation of a protein, and the flexibility to select a strategy makes it possible to apply this technology to other proteins.
| Conjugate | Original protein | PEG | Indication |
|---|---|---|---|
| Adagen® (pegademase) | Bovine adenosine deaminase | 5 kDa linear PEGs | Severe combined immunodeficiency disease |
| Oncaspar® (pegaspargase) | l-Asparaginase | 5 kDa linear PEGs | Acute lymphoblastic leukemia |
| PegIntron® (peginterferon-α2b) | Interferon-α2b | 12 kDa linear PEG | Hepatitis C |
| Pegasys® (peginterferon-α2a) | Interferon-α2a | 40 kDa branched PEG | Hepatitis C |
| Neulasta® (Pegfilgrastim) | Granulocyte colony-stimulating factor | 20 kDa linear PEG | Neutropenia during chemotherapy |
| Somvert® (pegvisonmant) | Growth hormone-receptor antagonist | 5 kDa linear PEGs | Acromegaly |
| Mircera® (methoxyPEG-epotin-β) | Epoetin-β | 30 kDa linear PEG | Symptomatic anaemia associated with chronic kidney disease |
| Cimiza® (certolizumab pegol) | Fab′ fragment of a humanized monoclonal antibody against tumor necrosis factor-α | 40 kDa branched PEG | Crohn’s disease and rheumatoid arthritis |
| Krystexxa® (pegloticase) | Urate oxidase | 10 kDa PEG | Severe debilitating chronic tophaceous gout |
PEGylated bovine adenosine deaminase (Adagen®)34) is used to treat patients afflicted with a type of severe combined immunodeficiency disease (SCID), which is caused by a chronic deficiency of adenosine deaminase.35) Injections of unmodified adenosine deaminase are not effective because of its short half-life in the systemic circulation (less than 30 min) and the potential for immunogenic reactions to the bovine-sourced enzyme.35) The multiple PEGylation of this non-human bovine enzyme (amide bond, 5 kDa linear PEG) allows it to achieve its full therapeutic effect by maintaining the plasma concentration in the bloodstream and masking the exogenous protein to avoid immunogenic reactions. Clinical pharmacokinetic profiles and pharmacodynamic results (depletion of adenosine metabolites) support once weekly subcutaneous dosing in patients.36) Also, PEG-l-asparaginase (Oncaspar®) is a multiple PEGylated enzyme37) like Adagen®. Native l-asparaginase from Escherichia coli (E. coli) and Erwinia chrysanthemi are available for clinic use, but these proteins are not devoid of limitations that often are responsible for treatment failures, such as clinical hypersensitivity, acute allergic reactions, silent hypersensitivities, or easy development of antibodies.38) The half-life of this enzyme in the systemic circulation showed a marked increase after PEGylation, it increased from about 15 d compared to 24 h for the non-modified enzyme.39) The PEGylated enzyme allows less frequent administrations with an improved patient compliance. The PEG-l-asparaginase is less immunogenic than either of the two native enzymes and can be administered safely to most patients with allergenic reactions to E. coli or Erwinia asparaginase, even though a certain degree of immunological cross-reaction has been highlighted between the formulations of E. coli asparaginase and conjugate.40) The conjugated protein is less prone to elicit an immunological response but, at the same time, if antibodies have been previously raised against the E. coli native enzyme, they may be able to recognize and eliminate the PEGylated enzyme. In a recent study, the presence of an antibody against PEG in the serum of some patients with undetectable asparaginase activity after receiving PEG-l-asparaginase was reported.41) This drawback has been highlighted in the case of other non-human enzymes, such as PEG-methioninase (PEG-METase). Methioninase (methionine-α-deamino-γ-mercaptomethanelyase, METase), isolated from Pseudomonas putida, is a pyriodoxal-l-phosphate (PLP)-dependent enzyme that transforms l-methionine into α-ketobutyrate, methanethiol and ammonia and is able to induce methionine depletion. The recombinant enzyme produced from E. coli showed high immunogenicity in a phase 1 clinical trial. PEGylation using methoxy-PEG of 5 kDa decreased the immunogenicity of the unmodified recombinant enzyme without loss of activity and showed longer-lasting serum methionine depletion, in accordance with an increased number of induced PEG molecules.42) Some antibodies against PEG-METase were induced upon repeated challenges in a primate model. However, the level of such antibodies was 100–1000 fold less than those elicited by the unmodified enzyme, and each challenging dose was effective in depleting the serum methionine level. This induction of antibodies against the non-human enzyme with PEGylation can be prevented by establishing a dosing-regime that depends on the unique situation of each individual patient, such as the minimum amount and appropriate interval of administration based on the concentration of substance and titer of antibodies in the bloodstream of the patient.
2.4. Other SubstancesCertolizumab pegol (CIMZIA®) is the only PEGylated derivative of antibody on the market.43) It is a PEGylated Fab′ fragment of a humanized monoclonal antibody against tumor necrosis factor (TNF) alpha for the treatment of Crohn’s disease and rheumatoid arthritis. In terms of PEGylated oligonucleotides, the PEGylated anti-vascular endothelial growth factor (VEGF) aptamer Pegaptanib (MACUGEN®) is a single strand 28mer oligonucleotide and is currently being marketed for the treatment of neovascular (wet) age-related macular degeneration, an eye disease associated with aging that destroys central vision.44)
In 2012, the European Medicines Agency released a scientific guideline that includes recommendations on clinical trials for PEGylated drug product in the pediatric population, based on reports about ependymal cell vacuolation.45) There are currently many bio-medicine candidates, including monoclonal antibodies and their conjugates, in clinical stages and PEGylation technology is very useful for improving the physicochemical and biological properties of proteins. However, the clinical development of PEGylated molecules should be undertaken carefully because the risks and benefits of PEGylation technology are still not entirely clear.
Unfortunately, the direct PEGylation of compounds very often leads to significant loss of biological activities. However, if the encapsulation of compounds into drug carriers, e.g., liposomes, without any chemical modification is available, there is a possibility to improve their pharmacokinetics and pharmacodynamics without loss of activity. The modification of the liposomal surface with PEG was a very important event in the history of drug delivery systems.46) Liposomes without PEGylation are rapidly cleared from the systemic circulation by interaction with opsonins and entrapment by the reticuloendothelial system (RES).47) PEGylation of liposomes increases the hydrophilicity of the liposomal surface and prevents interactions with proteins, cells and tissues, and therefore, the liposomes are stabilized sterically and invisible to the RES.47,48) PEGylation of liposomes not only bestows stability and increases circulating time, but also results in passive targeting to tumor tissues by the ERP effect.23,49) PEGylated liposomes circulate in the bloodstream without loss of encapsulated drug and are small enough to extravasate through the highly permeable discontinuous endothelium of tumor vessels and passively accumulate in the interstitial fluid compartment due to the lack of functional lymphatic drainage.
At present, approved PEGylated liposome products include Doxil®50,51) and Lipodox®. Doxil® was approved by the U.S. Food and Drug Administration (FDA) in 1995 as a doxorubicin hydrochloride (DXR) injection for second line treatment of AIDS-related Kaposi’s sarcoma. It is marketed as Caelyx® in Europe and Canada. Lipodox® is the first generic version of Doxil® in the United States. Doxil® is supplied as a ready-to-use solution and contains liposomes that consist of hydrogenated soy phosphatidylcholine, cholesterol and mPEG2000-DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000]). The liposomes have a mean diameter of 80 nm, 2 kDa linear PEGs grafted to the liposomal surface, and stability for retaining DXR in its interior phase while circulating in the bloodstream, so that they have all the functionality that the stealth liposomes52) provide. DXR is one of effective cytotoxic drugs for cancer therapy and an adverse effect that has been reported is cumulative, dose-related, progressive myocardial damage that may lead to congestive heart failure.53) The reason for this disadvantage is that DXR is a small molecule (molecular weight (MW) 580) and its non-specific distribution to both tumor and normal tissues, including the myocardium, occurs rapidly after intravenous administration. In order to overcome this drawback to DXR therapy, non-PEGylated liposomes with encapsulating DXR (Myocet®) were developed and reported to increase the half-life and total exposure while reducing Vd and clearance.54) Additionally, Doxil® is able to enhance the capability for improving the pharmacokinetics profile of a non-PEGylated liposomal formulation and shows longer circulating time in blood (half-life as 2–3 d for clearance).50) The Vd of Doxil® is slightly larger than the total volume of plasma in humans and this data indicates that the distribution of Doxil® to healthy tissues is only very slight. The PEGylated liposomes decreased the distribution of DXR to the myocardium and improved the safety significantly. Additionally, a ten-fold higher concentration of DXR in tumor cells after administration of Doxil® in comparison with free DXR was observed, as was a consequent 20-fold higher concentration of DXR in tumor cells compared to non-tumor cells. The current indications of Doxil® include recurrent ovarian cancer, metastatic breast cancer, and multiple myeloma in combination with bortezomib. Furthermore, there are some PEGylated liposomal products currently in clinical trials. ThermoDox® is PEGylated liposomes with encapsulated DXR and is currently in phase III study for the treatment of hepatocellular carcinoma. ThermoDox®, which contains low temperature-sensitive liposomes, is administered intravenously in combination with radio frequency ablation (RFA). Localized mild hyperthermia (39–42°C) created by the RFA releases the entrapped doxorubicin from the liposomes.55) S-CKD602 is a PEGylated liposome formulation of the topoisomerase inhibitor CKD602 (Belotecan®), which is a semi-synthetic analog of camptothesin.56) Finally, the PEGylated liposome formulation of cisplatin, SPI-077, is being used clinically for the treatment of non-small-cell lung cancer.57)
There are many small molecular compounds with high biological activity and poor “drugability” in the compound libraries of pharmaceutical companies. Chemical modification, including prodrugs, is one of effective approaches with which to improve various properties and extract the potential of compounds. Additionally, the use of drug carriers like liposomes is one approach for masking their pharmacokinetic and safety drawbacks without chemical modification. Several cytotoxic antitumor agents for anti-cancer therapy are commercially available, however, only one original PEGylated liposomal formulation product, Doxil®, is currently on the market. Some liposomal products have been approved, indicating that manufacturing processes for liposomal products have been established by some pharmaceutical companies. However, there are not many liposomal products on the market. It may be difficult to encapsulate drug substances into liposomes with a high encapsulation efficiency for both small molecules and biopharmaceuticals because their physicochemical properties are not suitable for encapsulation into the drug carriers. Encapsulation efficiency is one of the important factors that has an effect on the cost of manufacturing a liposomal product, and the balance between cost and benefit is always a focus in the life cycle management of products. In other words, if the physicochemical properties of discarded compounds can be changed by chemical modification (like PEGylation) and the optimization of encapsulation into liposomes achieved, we can create opportunities to develop them as liposomal products.
