Exp,ert
`Opiíríon
`
`1. lntroduction
`2. General aspects of PEGYIation
`3. Polyethylene glycol (PEG), a
`biocor¡jugation polymer
`4. Chemistry of polyethylene
`glycol (PEG) conjugation
`5. PEc-prote¡n coqiugates
`6. PEcylation of drugs
`7. Expert opinion
`
`For reprint orders, please
`contact:
`re pr ¡ nts@ ashl ey - pu b. com
`
`Ashtey Publ¡cat¡ons
`www,ashley-pub.com
`
`Review
`Protein, peptide and non-pePtide
`drug PEGylation for theraPeutic
`application
`G Pasut, A Guiotto & FM Veroneser
`ÍDpartment of Pharmaceutical Sciencæ, [Jnivenily of Padua, vk E lVfarzo]o 5, 3513[-Padoua, Ialy
`
`For many years proteins have been investigated as therapeutic agents, but
`unfortunately the¡r potent¡al advantages could not be completely exploited.
`The main drawbacks are their intrinsic short life in the body, immunological
`adverse reaction and proteolytic digestion. Among all the approaches studied
`for overcoming these problems, PEGylation (the modificat¡on of molecules
`w¡th polyethylene glycol [PEG]) achieved the most interesting results, leading
`to a novel series of products that have already reached the market, and hope-
`fully other promising agents will soon be available. Since the first studies in this
`f¡eld, the cor.¡jugation of PEG to a prote¡n has shown the possibility of improv-
`ing the pharmacokinetic profile of a linked drug. ln the last few years this tech-
`nology, firstly developed for proteins, has been transferred to non-peptide
`drugs, opening a new area of investigation that is now receiving increasing
`¡nterest. This leads to new opportun¡t¡es for many therapeutic treatments as lt
`is possible to use molecules that could not before be exploited due to limita-
`tions such as inadequate water solubility, high nonspecific toxicity and poor
`pharmacokinetic profiles. ln th¡s review the most recent achievements in
`PEGylation of protein, peptide and non-peptide drugs are described concern-
`ing the binding chemistry, and many examples from the literature are
`reported, in the fields of both prote¡n therapeutics and non-peptide drugs.
`
`Keywords: poþethylene glycol (PEG), PEGdrugs, PEG protein, PEGylation, polyrrnr
`therapeutics
`
`Expert Opn. Ther. Patenx (2004) 14(6):855894
`
`t. Introduction
`
`Polymer bioconjugation is receiving inffeasing interest in applied biotechnology,
`and polyethylene glycol (PEG), m particular, has been extensively used in the modi-
`fication of different substrates of therapeutic interest'
`It is usually common to divide therapeutic polymer coniugates into two main cat-
`egoriesl conjugates with drugs having a peptide structure (so far the most studied
`area) and those with a non-peptide structure, in particular low molecular weight
`drugs such as anticancer drugs. The success of this technology is reflected not only
`by the many drug-polymer conjugates already available or under investigation but
`also by the growing number of publications and patents published each year.
`Polypeptides and many low molecular weight molecules usually have shortcom-
`ings that restrict or even prevent clinical use. common limits for many drugs are
`shãrt ft vivo :¡alf-life (due to enzyme degradation or rapid kidney clearance) and
`physicochemical drawbacks, for example low solubility or instability; polypeptides
`may present additional restrictions, such as immunogenicity and antigenicity [1-3ì.
`These problems are often addressed by covalent linking of the pharmaceutically
`active molecule to polymers (Box 1), PEG being the most successful among those
`used [¿-oì. Polymer conjugation, especially for small drugs, is also exploited to
`achieve an active or passive targeting, an improved biodistribution and a selected
`cellular uptake by endocytosis 1z-0¡.
`
`2004 o Ashley Publlcations Ltd ISSN 1 354-3776
`
`859
`
`MPI EXHIBIT 1041 PAGE 1
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`MPI EXHIBIT 1041 PAGE 1
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`

`

`Protein, peptide and non-peptide drug PEGylation for therapeutic apPl¡cat¡on
`
`Box 1. General advantages of bioconjugat¡on ¡n
`therapeutic application.
`
`Stabilisation of labile drugs from chemical degradation
`Protection from proteolytic degradation
`Reduction of immunogenicity
`Decreased antibody ræognition
`lncreased body residence time
`Modif ication of biodistri bution
`Drug penetration by endocytosis
`New strategies lor drug targeting
`lncreased water solubi lity
`Reduced toxicity
`
`All protein and peptide drugs are candidates for alternative
`delivery methods. It is noteworthy that peptide drugs are
`worth > US$10 billion of the world pharmaceutical market
`and represent a rapidly growing area. As drug detivery is
`closely tied with pharmaceutical manufacture, it is anticipated
`that its market will be worth an estimated US$120 billion by
`2007 and bioconjugation appears to be one of the most prom-
`ising approaches to reach this goal.
`Although polymer conjugation to proteins originated in
`the 1950s and 1960s using polysaccharide polymers, the real
`boost in this field was represented by the use of PEG, thanks
`to the pioneering studies conducted in the late 1970s by Pro-
`fessor Frank Davis at Rutgers University [10]. Since then many
`excellent reviews have been dedicated to different aspects of
`this technique, known as PEGylation, whereas this review
`focuses on more recent pharmaceutical applications of PEG
`with a particular attention to the patent literature.
`
`z. General aspects of PEGylation
`
`PEG, approved by the FDA for human use, has a variety of
`interesting properties such as the absence of immunogenicity,
`antigenicity and toxicity, and high solubility in water and in
`many organic solvents. These properties can be transferred to
`the final conjugates by PEGylation, obtaining modification of
`the pharmacokinetic and pharmacodynamic profiles of native
`drugs [ll]. In general, PEGylated drugs, when compared with
`the parent molecules, show: i) reduced kidney excretion and
`altered biodistribution, mainly due to the increased molecular
`weight; ii) reduced degradation by proteolytic enzymes or
`hydrolytic media; iii) enhanced water solubility; iv) reduced
`reticuloendothelial (RES) clearance and v) reduced immuno-
`genicity and antigenicity U2,l3l.
`The evolution of protein PEGylation has been frequently
`described, and is divided into two generations:
`
`. The first generation of conjugates refers to PEGs with low
`molecular weights (< 12 kDa) and with a relevant percent-
`age of PEG Aiot impurities, a potential crosslinking
`agent originating from the synthesis of methoxy-PEG.
`
`Furthermore, the chemistry employed often presented
`side reactions or led to weak and reversible linkages. Despite
`these initial difficulties, important products were created
`and some reached the market, such as Enzon Pharmaceuti-
`cals PEG-adenosine deaminase (Adagen@, Enzon, Inc.)
`tl4,20lì for the treatment of severe combined immunodefi-
`ciency disease (SCID) and PEG-asparaginase (Oncaspar@,
`Enzon, Inc.) [15,202] for the treatment of leukaemia.
`. The second generation of conjugates was an improvement
`on the first, in particular as far as PEG purity is concerned;
`an important reduction in polydispersivity and in diol
`amounts was achieved in industrial production and
`improvements are also rmade in selectivity of protein mod-
`ification and in the range of available activated PEGs.
`Heterobifunctional PEGs were prepared in order to link a
`second molecule with a targeting role. Moreover, spacers
`between the polymer and the drug were studied to allow
`the release of bound drug under specific triggering condi-
`tions. Several products of this second generation reached
`the market, such as PEG-IFN-o¿Zb (PEG-Intron@, Scher-
`ing-Plough) [16,203], branched PEG (40 kDa)-IFN-o2a
`(Pegasys@, Roche Pharmaceuticals) t17,18,204Ì, PEG-growth
`hormone receptor antagonist (Pegvisomant, Somavert@,
`Pfizer) Itg,zosl and PEG-granulocyte-colony-stimulating
`factor (G-CSF) þegfilgrastim, Neulasta@, Amgen) ¡zo,zoo¡
`but many others are presently undergoing clinical trials and
`will hopefully be available in the near future.
`
`s. Polyethylene glycol (PEG), a bioconjugat¡on
`polymer
`
`PEG, as obtained by ethylene oxide polymerisation,
`presents one or two terminal hydroxyl groups (mPEG-OH
`or HO-PEG-OH) depending upon the initiator of polym-
`erisationl methanol or water, respectively. A branched form
`may also be obtained, as well as products with multiple reac-
`tive groups, at one or at both extremes (Figure 1). Whlle the
`monofunctional polymers, linear or branched, are used for
`protein modification, those with multiple groups are indi-
`cated to enhance the loading of low molecular weight drugs.
`The main characteristics of PEG, as compared to other poly-
`mers, are low polydispersivity (fu[*tA'[" spanning from 1.01 for
`PEG < 5 kDa molecular weight and up to I .1 for PEG as high as
`50kDa molecular weight), solubility in both aqueous and
`organic solvents and biocompatibility. The unique solvation
`properties of PEG are due to the ability to coordinate 2 - 3 water
`molecules per ethylene oxide tnit and to the higtrly flexible the
`backbone chain I2l. These characteristics give PEG an apparent
`molecular weight 5 - l0 times higher than that of a globular pro-
`tein of comparable mass, as may be verified by gel permeation
`chromatography (see tztl for a recent discussion) and this explains
`the protein-rejecting property, at the basis of the anti-immuno-
`genicity and antigenicity conveyed to a conjugate tz2l. This also
`explains the prolonged blood residence time and the decreased
`degradation by mammalian cells and enzf/mes [23].
`
`860
`
`Expert Opin. Ther. Patents (2004) 14(61
`
`MPI EXHIBIT 1041 PAGE 2
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`MPI EXHIBIT 1041 PAGE 2
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`

`

`./o
`
`OH
`
`OH
`
`Pasut, Gu¡otto & Veronese
`
`U
`
`OH
`
`a mPEG-OH
`
`b HO-PEG-OH
`
`c mPEG,-COOH
`
`o
`
`o
`
`X
`
`d X=Reactivegroup
`
`"{--^-"']:---"
`
`X
`
`Figure 1. Different PEG structures. a) Linear monometho)ry PEG; b) Linear diol PEG; c) Branched PEG; and d) Multifunctional PEGs.
`PEG: Polyethylene glycol.
`
`In uivo, PEG undergoes limited chemical degradation and
`its clearance depends on its molecular weight, < 20 kDa.
`PEG is easily secreted into urine, whereas for higher molecu-
`lar weights it is eliminated more slowly into urine and faeces,
`up to the threshold of 40 - 60 kDa (a hydrodynamic radius of
`- 45 Ä tzal), which represents the albumin excretion limit.
`Above this limit the polymer remains in circulation and is
`mainly accumulated in the liver. Alcohol dehydrogenase can
`degrade low molecular weight PEGs and chain cleavage can
`also be catalysed by cytochrome P450 microsomal enzymes
`t25ì. Many years of the use of PEG as an excipient in foods,
`cosmetics and pharmaceuticals without toxic reactions are a
`clear proof of its safety 1231.
`Tì"ends for new PEGs are directed towards: i) monodisperse
`polymer batches, not only in the case of low molecular weight
`PEG (< 600 Da), which have recently reached the market, but
`also for an extended range of higher molecular weights, ii)
`PEG polymers with specific functions tailored to provide con-
`jugation with reactive groups on target molecules, also in view
`of a controlled releasei and iii) high loading PEGs to increase
`the payload of active molecules conjugated to the chain,
`through the construction of dendrimeric structures or multi-
`arm forms at the polymer extremes 126,27,2071.
`
`4. Chemistry of polyethylene glycol(PEG)
`coqiugat¡on
`
`The first generation PEGs were mainly designed for amine
`modification, as amines are a widely represented functional
`group in proteins. Among these, of particular not are: i)
`PEG succinimidyl succinate (SS-PEG); iÐ PEG succinimidyl
`carbonate (SC-PEG); iii) PEG ¡rnitrophenyl carbonate
`(pNPC-PEG); iv) PEG benzotriazoþl carbonate (BTC-PEG);
`v) PEG trichlorophenyl carbonate (TCP-PEG);vi) PEG carb-
`onylimidazole (CDI-PEG); vii) PEG tresylate; and viii)
`PEG dichlorotriazine (Figure 2).
`Carbonate PEGs, such as pNPC-PEG, CDI-PEG and
`TCP-PEG, show slower reactivity than other PEG derivatives,
`
`thus allowing a selective conjugation on the basis of reactivity
`of the different amino groups. It is noteworthy that
`PEG dichlorotriazine, PEG tresylate and PEG aldehyde (after
`sodium borohydride reduction) dlow the same total charge of
`the native protein to be maintained because the conjugation,
`based on alkylation, leads to a secondary amine, whereas the
`coupling performed with the other PEGs, based on acylation,
`yields neutral acidic amide or carbamate linkages. Although,
`the primary amino group is the more reactive in proteins,
`PEGs such as SC-PEG, BTC-PEG and PEG dichlorotriazine
`can slowly react with hydroxyl groups (Ser, Tyr) and the histi-
`dine imidazole side chain to give hydrolytically unstable link-
`ages. For example, one histidine residue of a-IFN was
`conjugated to SC-PEG or BTC-PEG under slightly acidic
`conditions t2081. PEG was also linked to hydroxyl groups of
`serine and tyrosine in the decapeptide antide and in the epider-
`mal growth factor (EGF) [28,20eì, respectively.
`The improvement of polymer synthesis and conjugation
`chemistry is now yielding a second generation of PEGs,
`mainly characterised by lower percentages of diol contami-
`nant in polymer batches. This was achieved by the isolation of
`the monocarboxylic acid intermediate of PEG from the bicar-
`boxylic intermediate, coming from the diol by ionic exchange
`chromatography t2r0ì. Higher molecular weight polymers,
`with an improved pharmacokinetic profile and stability for
`non-peptide drugs, were also obtained. Among the new PEGs
`are reportedi
`
`' PEG-propionaldehyde, also in the form of the more stable
`acetal [211]: the reaction with the amino group leads to a
`Schiff base that is reduced by sodium borohydride, giving a
`derivative that maintains the same total ionic charge of the
`parent drug.
`. PEG-succinimidyl derivatives: higtily reactive towards
`amine group. The reaction rate of these derivatives may
`significantly change depending upon the extension and the
`composition of the alkyl chain between PEG and
`succinimidyl moiety Í29,2121.
`
`Expert Op¡n. Ther. Patents(2004\ 14(6)
`
`861
`
`MPI EXHIBIT 1041 PAGE 3
`
`MPI EXHIBIT 1041 PAGE 3
`
`

`

`Protein, pept¡de and nm-peptide drug PEGylation for therapeutic apPlicat¡on
`
`/R
`
`NH
`
`/o
`
`PEG
`
`ô
`
`PEG'
`
`o
`
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`
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`
`/o
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`
`a
`
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`
`b
`
`+
`
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`
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`
`+
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`H2N-R
`
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`
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`d
`
`+ H2N-R
`
`PEG
`
`H
`
`Y-o
`
`R
`
`/o
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`
`cl
`
`+
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`
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`+ H2N-R +
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`-o
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`
`ct
`
`rJY)N1
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`-R
`
`il
`
`Figure 2. Elamples of act¡vated PEG molecules reactive tourards amino groups. a) PEG succinimidyl succinate; b) PEG succinimidyl
`carbonate; c) PEG p-nitrophenyl carbonate; d) PEG benzotriazol carbonate; e) PEG trichlorophenyl carbonate; 0 PEG ærbonylimidazole;
`gf PEG dichlorotriazine; and h) PEG tresylate.
`PEG: Polyethylene glycol.
`
`862
`
`Expett Opin. Ther. Patents(2004) 14(6)
`
`MPI EXHIBIT 1041 PAGE 4
`
`MPI EXHIBIT 1041 PAGE 4
`
`

`

`ì
`
`Linear PEG
`
`Branched PEG
`
`ì
`
`a
`
`Ho'
`
`b
`
`OH
`
`Pasut, Guiotto & Veronese
`
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`
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`
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`
`= Bnanching moiety
`X = Active group
`
`Figure 3. Structure of linear and branched PEG on the
`protein surface. The 'umbrella-like' structure of branched PEG
`explains its higher capacity for rgecting approaching molecules or
`cells compared to linear PEG.
`PEC: Polyethylene glycol.
`
`Figure 5. Different strategies to achieve multifunctional
`high loading PEGs. a) Multiarm PEGs. b) Dendronìsed PEGs, the
`branching moiety may be a bicarboxylic amino acid, lysine or
`other bifunctional molecule.
`PEG: Polyethylene glycol.
`
`Branched PEG
`
`jÉ
`
`,,,
`
`Active site cleft
`
`Linear PEG
`
`Figure 4. Effect of PEG hindrance on the en4me active site'
`The high steric hindrance of branched PEG may be advocated to
`explain the lower inactivation of enrymes compared to linear PEG
`of the same size.
`PEG: Polyethylene glycol.
`
`. 'Y'-shaped branched PEG l¡o,zts,zlal (see Figure lc): for its
`increased surface shielding (Figure 3), this PEG reagent is
`more effective in protecting the coqjugated protein from
`degradative enzymes and antibodies. Moreover, enzymes
`modified with this PEG retain more activity with respect to
`the same en4/me modified by linear PEGs. Tfris effect is
`probably due to the hindrance of the branching polymer
`that prevents the entrance of PEG inside the en4rme active
`site cleft (Figure 4).
`
`. PEGs reactive toward thiol groups: PEG-maleimide
`(MAL-PEG) Í^st, PEG-vinylsulfone (VS-PEG) and
`PEG-orthopyridyl-disulfide (OPSS-PEG). Even if the
`thiol addition rate to the first two derivatives is very rapid,
`some addition to the amino group (mainly present in pro-
`teins) may also take place, especially at basic pHs. On the
`other hand, the reaction with OPSS-PEG is very specific
`for thiol groups but the conjugates may be reversed in the
`presence of thiols as reducing agents.
`. Heterobifunctional PEGs [31,32,216]: these derivatives
`present two different functional groups, one for each
`extreme, which simplify the linking of different molecules
`to the same PEG chain. Therefore, it is easier to obtain
`conjugates that carry both a drug and a targeting molecule.
`Among the proposed and commercially available heterobi-
`functional PEGs, H2N-PEG-COOH, HO-PEG-COOH
`and H2N-PEG-OH are the most used.
`. PEG with linkers designed for a controlled release of the con-
`jugated drug: one of the most exploited linkers is a peptide
`sequence, designed to be recognised and cleaved by lysosomal
`enzymes when the conjugates reach the intracellular compart-
`ment. Examples of such peptide linkers are H-Gly-Phe-Leu-
`G1y-OH or H-Gly-Leu-Phe-Gly-OH 133,341. Altematively, a
`linker may respond to pH changes or release the drug by a
`1,6-elimination reaction or by a cyclisation reaction [35].
`Moreover, the linker and the polymer together can form a
`double prodrug system, where the drug released is obtained
`after polymer hydroþis (frst prodrug) that triggers the linker
`(second prodrug), as reported for he drug delivery system
`based on trimethyl lock (TML) lactonisation [36,217].
`. Multiarm or 'dendronised' PEGs (Figure 5): the former are
`compounds prepared by attaching linear PEG to a
`
`Expert Op¡n. Ther. Patenß(2004) 14(6)
`
`863
`
`MPI EXHIBIT 1041 PAGE 5
`
`MPI EXHIBIT 1041 PAGE 5
`
`

`

`Protein, pept¡de and non-peptide drug PEGylation for therapeutic application
`
`,ao'oyt"!^-otu+H2N-protein+pEG-o
`o
`
`H
`\1M"!x-N-Prot"in
`il
`
`BrCN+
`
`- Protein
`
`X = Nle or Ê-Ala
`
`Figure 6. Use of PECrlVlet.Nle-OSu or PEG-tVlet-p-Ala-OSu to introduce a reporter amino acid at the PEGylation site'
`PEG con-¡ugation is followed by polymer moiety release by BrCN to leave Nle or B-Ala that can be identified on the protein by amino acid
`sequence analysis.
`PEG: Polyethylene qlycol.
`
`+ NH¡
`
`HN
`
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`H
`
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`
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`
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`
`ca2*
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`
`o
`
`NH,
`
`Protein
`
`Scheme 1. Reaction between a glutam¡ne residue in a protein and an alkyl amine, catalysed by TGase.
`TGase: Iransglutam¡nase.
`
`multimeric compound 12181, whereas the latter are linear
`PEGs with a dentritic structure at one or both chain
`extremes 126,37,2071. The aim of both derivatives is to
`increase the drug:polymer molar ratio, overcoming prob-
`lems of high viscosity that may occur with monofunctional
`drug conjugate solutions, in particular for therapeutic treat-
`ments that require quantity of drug.
`
`PEG and any polymer conjugate, especially when multifunc-
`tional drugs are involved, face great problems regarding repro-
`ducible synthesis, scaling-up, characterisation and methods of
`analysis, all issues that have been discussed in detail [38,39ì.
`One additional problem in protein conjugation is the localisa-
`tion of the PEGylation site into the primary amino acid
`sequence. So far the classical approach involves proteolytic
`digestion followed by Edman degradation, as reported for
`PEGylated IFN-2cx 1401, where the PEGylated sites are deter-
`mined by the missing amino acids. Beyond the lengthy proce-
`dure, the polymer may interfere v/ith the analysis as cleavage
`by proteolytic enrymes may be incomplete due to steric hin-
`drance. A more direct approach involves the use of two tailor
`made PEGs, PEG-MeI-N1e-COOH or PEG-Met-BAla-
`COOH tzrgl, which possess a labile bond in the peptide
`spacer arm that can be cleaved by treatment with BrCN
`(Figure6). After cleavage at the methionine level, tfre PEG
`chains are removed and the remaining nor-leucine or
`p-alanine tags, linked to the protein, are identified with stand-
`ard sequence investigation methods. The tag can be revealed
`by amino acid anaþis or mass spectrometry [411.
`A PEGylation procedure that differs from all others was
`recently developed by Sato and involves the use of the enzyme
`transglutaminase (TGase) 142,2201. In vivo TGase catalyses
`crosslinking between a protein containing an acceptor
`
`glutamine and another protein with an amino donor group,
`such as lysine t43,44ì. This enzyme catalyses the attachment of
`PEG-alkylamine, which behaves as a new substrate of TGase to
`the glutamine residues of the protein (S.h"-" l). This system
`provides new opportunities in polymer derivatisationi in fact,
`beyond the high homogeneity of the Tcase-catalysed conju-
`gates, recombinant DNA techniques, it may be used in com-
`bination to introduce amino acids sequences that are specific
`and more favourable TGase substrates. Amongst the several
`TGases isolated from natural sources, the guinea-pig liver
`TGase (G-TGase) and the microbial TGase (M-TGase) were
`reported for PEGylation procedures. The former possesses
`more stringent requirements on neighbouring glutamine
`amino acìds for enzyme activity and transglutamination site
`flexibility, although there is no unique amino acid recogni-
`tion sequence in proteins, G-TGase has been used for the
`preparation of site-specific PEGylated conjugates t45,461. With
`TGase, the suggested strategy consists of the introduction of
`a specific sequence, comprising a glutamine residue, at the
`N terminus of proteins, in order to maintain the peptide
`backbone flexibility. Instead, M-TGase does not require high
`specificity in the substrate sequence, although an optimal
`accessibility to the glutamine site is required either through
`peptide flexibility or high solvation of the sequence t¿zl. On
`the other hand, the amino donors (i.e., PEG-NH) do not
`need strict requirements to allow TGase conjugation, and
`high affinity PEG-NHZ has been designed to link the amino
`group to the PEG with a spacer chain of six carbon atoms
`(PEG-(CHr6-NH, l+at). In the case of IL-Z, the TGase-cata-
`lysed PEGylated form preserved a high degree of activity
`because the N terminus is, in this protein like many others, a
`flexible region and so is less involved in peptide recognition.
`The enzyme-catalysed coupling seems to yield more
`
`864
`
`Expert Op¡n. Ther. Patents (2004) 14(6)
`
`MPI EXHIBIT 1041 PAGE 6
`
`MPI EXHIBIT 1041 PAGE 6
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`

`

`Pasut, Guiotto & Veronese
`
`H
`
`----------->
`
`mPEG
`
`Yl
`
`_lå1
`
`Nq
`
`+
`
`o
`
`mPEG/
`
`SC-PEG
`
`His34 of IFN
`
`His34 of PEG-IFN
`
`Scheme 2. Add$t formation at the level of His34 using SC-PEG as PEcylating agerìt. (Reproduced with permission from tszl)
`PEG: Polyethylene glycol; SC: Succinimidyl carbonate.
`
`mPEG-O
`
`mPEG-
`
`- IFN
`
`NH
`
`U>-o
`
`mPEG
`
`PEGr-lFN
`
`o
`
`o
`
`-t\ Y
`
`50mM
`sodium borate
`pH9
`
`IFN-NH2
`---+>
`
`mPEG
`
`PEG2-NHS
`
`Scheme 3. PEcylat¡on of IFN-q-2a by branched nPEG2-COOH (a0 kDa). (Reproduced with permission from tlzl),
`NHS: N-Hydroxysuccinimide; PEG: Polyethylene glycol,
`homogenous derivatives than the chemical PEGylation that
`isknowntooftenleadtoamixtureof isomers.
`
`5. Polyethylene glycol (PEG)-prote¡n conju gates
`
`Many proteins and peptides have short half-lifes when
`administered in vivo, due to other proteolytic enzyme activ-
`ity, fast kidney ultrafiltration and activation of immune sys-
`tem response. PEGylation may overcome all of these
`drawbacks thanks to the increased hydrodynamic volume of
`conjugates and the shielding effect of PEG towards enzymes
`and antibodies. Due to protein multivalence, the PEGs used
`for protein conjugation should have only one reactive group
`to avoid crosslinking reactions; for this reason, batches com-
`pletely devoid or with minimal traces of PEG aiol must be
`employed. Furthermore, to avoid protein denaturation, the
`conjugation chemistry should be gentle, yield stable bonds,
`avoid unmasking of epitopes or sites of proteolysis and,
`where possible, it should lead to homogeneous products or,
`at least, reproducible positional isomers. Far from addressing
`all and each of the above-mentioned requirements, several
`coûugates have been engineered showing interesting
`improvements over the starting materials, and some conju-
`gates have already hit the market.
`
`5.1 lnterferons
`Type 1 interferons (IFN-c, -Þ, -r, -t and -ol) are structurally
`and functionally related proteins belonging to a cytokìne fam-
`ily. Thet activity is mediated by lFN-inducible gene activa-
`tion t49l that occurs when the IFN blnds to a multimeric cell
`surface receptor. The activities of these proteins include a
`wide range of effects, mainly antiviral, antitumour and
`
`immunomodulatory properties tsol. IFN-ct' was first approved
`for hairy-cell leukaemia therapy, then for the treatment of
`hepatitis B and C and later for various dermatological pathol-
`ogies; INF-p was approved for the treatment of multiple scle-
`rosis. The low molecular weight of interferons (- 20 kDa)
`reflects the relatively short serum half-life of these proteins,
`which was improved by PEG conjugation.
`The first study in this direction was the modification of
`IFN-cr-2a with linear succinimidyl carbonate PEG (SC-PEG;
`5 kDa) via a urea linkage. The coupling, performed at equi-
`molar ratio of protein and polymer, mainly led to mono-
`PEGylated isomers and, in small amounts, di-PEGylated
`conjugates as well as free IFN [221ì. Characterisation of the
`conjugates indicated that lysine residues were the site of
`PEGylation 1401. Although a once a week schedule was possi-
`ble, while the unconjugated protein was usually given three
`times a week, the desired pharmacokinetic/activity profile was
`not yet achieved. After conjugation of PEG to IFN-cr-Zb vla a
`carbamate linkage, Enzon researchers proposed a method
`that, by acidification of the conjugate aqueous solution to
`pH 3 or lower, allowed the removal of the few PEG chains
`that were attached to INF's amino acid residues (i.e., His)
`through unstable carbamate bonds [222ì.
`Further investigations demonstrated that derivatives with
`better pharmacokinetic profiles and higher activity could be
`obtained by conjugation of IFN-u-Zb with SC-PEG (12 tDa)
`in sodium phosphate buffer solution at pH 6.5. This reaction
`gave an unexpected conjugate at His34, representing - 47o/o of
`the total PEGylated species (Scheme 2) tsr,zosì. The stability of
`this species was studied by [lH]-nuclear magnetic resonance
`(NMR) analysis following the ppm shift of Het of Hls34
`during its conversion from the PEGylatea to the
`
`Expert Op¡n. Ther. Patents(2004\ 14(6\
`
`865
`
`MPI EXHIBIT 1041 PAGE 7
`
`MPI EXHIBIT 1041 PAGE 7
`
`

`

`Protein, pept¡de and non-pePt¡de drug PEGylation for therapeutic application
`
`kDa
`
`200
`
`1 16.3
`97.4
`
`66.3
`55.4
`
`36.5
`31
`
`21.5
`14.4
`6
`
`kDa
`
`!-
`
`ü
`
`ü
`
`r-
`
`ú
`
`4
`
`iD
`
`b
`
`;
`
`ri=r
`
`120
`
`73.4
`
`41.6
`
`22.8
`
`!¡
`
`-
`
`J
`
`4
`
`Figure 7. SDS-PAGE analysis of the PEGylated IFN-a-2a
`m¡xture. a) Gel specifìcally stained for proteln with Coomassie
`blue, Lane 1 : molecular weight marker protein; Lane 2: PEGylation
`reaction m¡xture; Lane 3: purified PEG,-|FN; Lane 4: IFN-cr-2a.
`b) Gel specifically stained for PEG with iodine, Lane 1: molecular
`weight marker PEGs; Lane 2: PEGylation reaction mixture; Lane 3:
`purified PEGr-|FN; Lane 4: IFN-o-2a. Note that the IFN-a-2a is not
`stained by iodine, (Reproduced with permission from tr ul),
`PEG: Poìyethylene glycol; SDS PAGE: Sodium dodecyl sulfate polyacrylamide gel
`eleLtr ophores¡s.
`
`Table 1. Pharmacokinetic propert¡es of IFN-o-2a and its
`PEcylated form in rats [18¡.
`Half-life (h) Plasma res¡dence
`t¡me (h)
`'l 0
`20.0
`
`Prote¡n
`
`IFN-o-2a
`
`PEG2 (40 kDa)-lFN-a-2a
`
`PEG: Polyethylene glycol.
`
`866
`
`nonPEGylated form t5zl. The higher activity ol this IFN
`preparation was related to the ability to release free and fully
`active IFN by slow hydrolysis of the His-PEG bond t2081.
`Further studies were also carried out to find adequate for-
`mulations to prevent loss of activity and denaturation of
`IFN-PEG during the conjugation and the lyophilisation
`procedure t2231. These investigations allowed marketing
`PEG-Intron in 2000. Although the in vitro potency of this
`IFN-PEG is only one-quarter of the free interferon form, its
`serum life is approximately six times longer, allowing for a
`less frequent administration schedule while maintaining an
`efficacy comparable with unmodified IFN Ilo,ssl.
`A different approach to IFN-PEGylation exploited the
`special properties of branched PEGs. A high molecular
`weight branched PEG (PEG2, 40 kDa) was chosen on the
`basis of several preliminary studies disclosing that: i) the
`protein surface protection at the conjugation site with a sin-
`gle, long chain PEG is better than several small PEG chains
`at different sites [4], ii) branched PEGs have lower distribu-
`tion volumes than linear PEGs of identical molecular weight
`and the delivery to organs such as liver and spleen is faster
`[b4] and iii) proteins modified with branched PEG possess
`greater stability towards enzymes and pH degradation [30].
`The 40 kDa branched succinimidyl PEG (PEG2-NHS) was
`linked to IFN a-2a using a 3:1 PEG:protein molar ratio in
`50 mM sodium borate buffer pH 9 (Scheme 3) tl7l.
`PEGylation under these conditions led to a mixture con-
`taining 45 50% monosubstituted protein, 5 - 10%
`polysubstituted (essentially dimer) and 40 - 50o/o unmodi-
`fied IFN (FigureT). Identification of the major positional
`isomer within the mono-PEGylated fraction was carried out
`by combination of high performance cation exchange chro-
`matography, peptide mapping, amino acid sequencing and
`mass spectroscopy analysis, which showed that PEG was
`attached mainly to either Lys31, Lys121, Lysl31 or Lysl34
`117,2041. Even though lhe in vitro anlivlral activity for
`PEG2-IFN was greatly reduced (only 7o/o of residual activ-
`ity was found) , Itre in vivo activity, measured as the ability to
`reduce the size of various human tumours, was higher than
`that of free IFN. The positive result could be related to the
`extended blood residence time of the conjugated form as
`shown in Täble l. These studies brought into the market a
`long lasting blood IFN conjugate, Pegasys, that is effective
`in eradicating hepatic and extrahepatic hepatitis C virus
`(HCV) infection Ir8l.
`A different cytokine, IFN-P, is approved for the treatment
`of multiple sclerosis in the US, like other cytokines, it suffers
`lrom a short blood residence time, again suggesting that a
`PEGylation strategy might be a solution to the problem. An
`exhaustive study conducted by Pepinsky and colleagues [55]
`resulted in a PEG modification of IFN-B-1a that exploited a
`reductive alkylation of an amine residue in phosphate buffer
`at pH 6 with an excess of 20 kDa PEG aldehyde and sodium
`cyanoborohydride to reduce the intermediate Schiff base. The
`conjugate, after extensive purification by gel-filtration and ion
`
`2.1
`150
`
`Expert Op¡n. Ther. Patents (2004) 14(6)
`
`MPI EXHIBIT 1041 PAGE 8
`
`MPI EXHIBIT 1041 PAGE 8
`
`

`

`Pasut, Guiotto & Veronese
`
`while pharmacokinetic experiments showed a fivefold increase
`in serum halflife. A 20 kDa PEG was chosen, as the 5 kDa
`PEG led to a fully active conjugate but with no improvement
`in the pharmacokinetic profile, while the 40 kDa PEG deriva-
`tive was completely inactive.
`A different approach was followed by Shearwater and
`Serono researchers, taking advantage of a site-specific
`PEGylation at CyslT of IFN-p t2241 using a thiol-reactive
`PEGylating agent, OPSS-PEG. Conjugation could be spe-
`cifically directed to CyslT since the other two cysteines are,
`in the native form of INF-P, involved in a disulfide bridge.
`Derivatisation was carried out with PEGs of different molec-
`ular weights but with a special and original strategy, well
`described in the patent. Low molecular weight PEGs are, in
`general, more reactive than those with a high molecular
`weight and in this special case the low molecular weight
`PEGs can overcome the steric hindrance around the thiol
`moiety at Cys17 IFN-P, yielding polymer-IFN conjugates.
`On the basis of this observation, a modification with high
`molecular weight PEGs was obtained via a two-step proce-
`dure: in the first step the protein was modified with a low
`molecular weight, heterobifunctional PEG oligomer; conju-
`gation with a higher molecular weight PEG possessing spe-
`cific reactivity towards the free terminal end of the first
`oligomer completed the procedure (Figureg). This strategy
`implied the use of a special heterobifunctional PEG oli-
`gomer with a group reactive towards the free cysteine
`(OPSS) at one extreme and a hydrazine group at the other.
`Hydraz