Drug–Polymer Conjugates

5.45 Drug–Polymer Conjugates F M Veronese and G Pasut, University of Padua, Padua, Italy & 2007 Elsevier Ltd. All Rights Reserved. 5.45.1

Introduction

1043

5.45.2

Polymeric Conjugates

1045

5.45.2.1

Conjugates with Low-Molecular-Weight Drugs

1045

5.45.2.2

Polymer–Protein Conjugates

1047

5.45.3

Polymers for Bioconjugation

1047

5.45.3.1

Vinyl Polymers

1048

5.45.3.2

Poly(Amino Acids) and Analogs

1048

5.45.3.3

Polysaccharides

1050

5.45.3.4

Proteins

1051

5.45.3.5

Poly(Styrene-co-Maleic Acid/Anhydride)

1051

5.45.3.6

Poly(Ethylene Glycol)

1051

5.45.4

Considerations for PEGylation

1055

5.45.5

Controlled Release of Conjugated Proteins or Drugs

1055

5.45.6

PEGylation Chemistry for Proteins

1056

5.45.6.1

Amino Group PEGylation

1056 1058 1058

5.45.6.1.1 5.45.6.1.2

Random PEGylation of asparaginase Random PEGylation of interferon a-2a

5.45.6.2

Thiol PEGylation

1059

5.45.6.3

Carboxy PEGylation

1059

5.45.7

Strategies in Protein PEGylation

1060

5.45.8

Enzymatic Approach for Protein PEGylation

1062

5.45.9

PEGylated Protein Purification and Characterization

1063

5.45.10

Conclusion

1064

References

5.45.1

1064

Introduction

Proteins and peptides have always been seen as potent and specific therapeutic agents, either to rescue the activity of native biomolecules or to treat important diseases like cancer. Insulin and growth hormone are the most well-known examples of the former group,1–4 while in the latter group some microbial enzymes used as anticancer drugs, such as asparaginase or methioninase,5,6 have been reported to deplete essential nutrients for cancer cells. Despite the interesting opportunity that these therapeutic agents offer, unfortunately they have intrinsic limitations ascribed to low stability in vivo, a short period of residence in the body, and immunogenicity, the last being common for nonhuman proteins. In the case of low-molecular-weight drugs, a short in vivo half-life (due to rapid clearance by the kidneys or enzyme degradation) is often observed and sometimes there are physicochemical drawbacks, like low solubility or instability. Several drug delivery systems have been developed in the last few years to improve the pharmacokinetic and pharmacodynamic profiles of many drugs7 with high or low molecular weight and with different chemical structure. These approaches can be based on tailor-made formulation of the drug, such as liposomal preparation or controlled release formulations, or on a covalent modification of the drug molecule itself, such as polymer conjugation. When modified in this way, the drug can often achieve a prolonged body residence, an increased resistance to degradation, and decreased side effects, and there is generally improved patient compliance. In this chapter we will focus on the area of polymer conjugation to drugs, a technique that is growing fast and that has already resulted in several products available in the marketplace8 (Table 1).

1043

1044

Drug–Polymer Conjugates

Table 1 Several polymer conjugates grouped according to the molecular weights of the conjugated drug Conjugates

Indication

Year to market or status

Company

SMANCS (Zinostatin, Stimalamer)74

Hepatocellular carcinoma

1993

Yamanouchi Pharmaceutical

PEG-adenosine deaminase (Adagen)83

SCID syndrome

1990

Enzon

PEG-asparaginase (Oncaspar)84

Acute lymphoblastic leukaemia

1994

Enzon

Linear PEG-interferon a2b (PEG-Intron)81,151

Hepatitis C, clinical evaluation on cancer, multiple sclerosis and HIV/AIDS

2000

Schering Plough/Enzon

Branched PEG-interferon a2a (Pegasys)36

Hepatitis C

2002

Roche/Nektar

PEG-growth hormone receptor antagonist (Pegvisomant, Somavert)93

Acromegaly

2002

Pfizer (Pharmacia)

PEG-G-CSF (Pegfilgrastim, Neulasta)94

Prevention of neutropenia associated with cancer chemotherapy

2002

Amgen

Branched PEG-anti-VEGF aptamer (Pegaptanib, Macugen)95

Age-related macular degeneration

2004

EyeTech

PEG-anti-TNF Fab (CDP870; Certolizumab pegol, Cimzia)152,153

Rheumatoid arthritis and Crohn’s disease

Phase III

UCB (formerly Celltech)

A PEGylated diFab antibody. Targets VEGFR-2 (CDP791)

Solid tumors

Phase II

UCB-ImClone System

HPMA copolymer-doxorubicin (PK1; FCE28068)43

Cancer, in particular lung, breast cancers

Phase II

Pfizer (CRC/Pharmacia)

HPMA copolymer-doxorubicingalactosamine (PK2; FCE28069)45

hepatocellular carcinoma

Phase I/II

Pfizer (CRC/Pharmacia)

HPMA copolymer-camptothecin (MAG-CPT; PNU166148)49

Clinical evaluation on several solid cancers

Phase I

Pfizer (Pharmacia)

HPMA copolymer-paclitaxel (PNU166945)50

Clinical evaluation on several solid cancers

Phase I

Pfizer (Pharmacia)

HPMA copolymer-platinate (AP5280)51

Clinical evaluation on several solid cancers

Phase II

Access Pharmaceutical

HPMA copolymer-platinate (AP5346)

Clinical evaluation on several solid cancers

Phase I/II

Access Pharmaceutical

Polyglutamate-paclitaxel (XYOTAX; CT-2103)55–58

Cancer, in particular lung, ovarian and esophageal cancers

Phase II/III

Cell Therapeutics

Polyglutamate-camptothecin (CT-2106)61

Clinical evaluation on colorectal, lung, ovarian cancer

Phase I/II

Cell Therapeutics

PEG-camptothecin (PROTHECAN)97

Clinical evaluation on several solid cancers

Phase II

Enzon

PEG-paclitaxel101

Clinical evaluation on several solid cancers

Phase I

Enzon

High-molecular-weight drugs

Low-molecular-weight drugs

Adapted from Duncan, R. Nat. Rev. Drug Disc. 2003, 2, 347–360.

Drug–Polymer Conjugates

Table 2 Most common advantages achieved using drug–polymer conjugation Prolonged half-life and less frequent administrations Increased water solubility (especially for insoluble small drugs) Protection from proteolysis and chemical degradation Reduction of immunogenicity and antigenicity Reduced toxicity Targeting to tumor tissue by ‘EPR effect’ Modification of biodistribution Drug release under specific conditions using proper spaces between the drug and the polymer Reproduced with permission from Pasut, G.; Guiotto, A.; Veronese, F. M. Exp. Op. Ther. Patents 2004, 14, 859–894.

Many classes of therapeutic agents are amenable to polymer conjugation; however, almost all of the conjugated drugs so far available on the market are proteins, since these were the first to be studied with this technology. A number of different diseases can be treated with conjugated proteins, hence polymer conjugation is not limited to a small number of therapeutic areas. Several compounds produced by conjugation of polymers with nonpeptide drugs with low molecular weight are currently under clinical evaluation and are expected to be on the market in the near future. These are mainly antitumor drugs, as such drugs have the most side effects and limitations that might, at least in part, be solved by polymer conjugation. Recently, this technology has been applied in the field of gene delivery and antisense nucleotides. Polymer conjugation conveys several advantages to drugs (Table 2) by deeply changing their in vitro and in vivo properties. By conjugating drugs with hydrophilic polymers, it is possible to achieve: (1) an increased solubility of the conjugated drug, even for those with very low solubility (e.g., taxol); (2) an enhanced drug bioavailability, which is the result of reduced kidney clearance due to the increased hydrodynamic volume of conjugates; (3) protection from degrading enzymes; (4) prevention or reduction of several drawbacks such as aggregation, immunogenicity, and antigenicity; and (5) specific targeting in organs, tissues, or cells, or exploitation of the ‘enhanced permeability and retention (EPR) effect’ of solid tumor tissues. One limitation of this technology involves patient compliance; conjugates cannot be orally administered because their size prevents absorption through this route, and they have to be parenterally administered. Research in the field of low-molecular-weight drug–polymer conjugation moved up a gear in the 1950s and 1960s when Peptamin was conjugated to the blood plasma expander polyvinylpyrrolidone.9 Before then, the research in this area mainly involved the chemistry of conjugation. In the 1960s the first clinical trials of the polymeric anticancer agent divinylethermaleic anhydride/acid copolymer (DIVEMA) were conducted; unfortunately, a severe toxicity was demonstrated, which prevented its use.10 Since then the concept of a polymer–drug conjugate has been better rationalized11 and many interesting derivatives have been developed to enhance the activity of various drugs–peptides and proteins in particular, but also molecules such as oligonucleotides or chelating agents for diagnostic purposes. The first studies on protein–polymer conjugation appeared in the 1960s and 1970s when dextran was investigated as a coupling polymer.12 However, the real breakthrough came when other synthetic polymers were employed, and poly(ethylene glycol) (PEG) emerged as the best candidate for protein modification.13 Its superiority is reflected in the fact that there are a number of interesting products already on the market containing PEG, and there are many more in advanced clinical trials (Table 1). Other synthetic polymers investigated for polymer conjugation include poly(styrene-co-maleic acid/anhydride) (SMA) and poly(N-(2-hydroxypropyl) methacrylamide) copolymers (HPMA), and polyglutamic acid (PGA). The first application of PEG as a bioconjugation polymer was proposed in the late 1970s by Professor F. Davis and A. Abuchowski at Rutgers University.31,32 Following his pioneering study a large number of drugs with different structure (proteins, peptides, low-molecular-weight drugs, polynucleotides) have been PEGylated, thus creating a new class of drugs,13 some of which rapidly became blockbuster products. The first polymers studied for drug delivery had a linear structure and were composed of one monomer, but polymer chemistry was soon expanded yielding a selection of interesting new structures such as dendrimers,14–16 dendronized polymers,17 graft polymers,18,19 block copolymers,20 branched polymers,21 multivalent polymers,22 stars,23 and hybrid glycol24 and peptide derivatives (Figure 1).25

5.45.2 5.45.2.1

Polymeric Conjugates Conjugates with Low-Molecular-Weight Drugs

The term ‘low-molecular-weight drugs’ is used here to describe not only the nonpeptide drugs but also small peptides and oligonucleotides.

1045

1046

Drug–Polymer Conjugates

(e) (a)

(f) (b)

(c)

(d)

(g)

Figure 1 Different polymer structures: (a) dendrimers, (b) dendronized polymers, (c) graft polymers, (d) block copolymers, (e) multivalent polymers, (f) branched polymers, and (g) stars.

An important milestone in the development of these conjugates was the rationalization of the polymeric carrier proposed by Ringsdorf in 1975.11 According to his model (Figure 2), the drug is linked to a polymeric backbone (biodegradable or not), directly or through a spacer; a targeting residue and eventually a solubilizing group may also be present. The spacer between the polymer and drug may control the release rate or target the release to certain cellular compartments. This may be achieved by the use of spacers cleavable under special conditions, such as acidic medium or lysosomal enzymes. The increased hydrodynamic volume of the conjugate generally extends the blood residence time since the kidney clearance rate is reduced and less frequent administrations are therefore needed. Moreover, hydrophobic drugs can be easily solubilized in water by coupling with hydrophilic polymers, which helps the formulation. The high hydrodynamic volume of the macromolecular carriers offers a passive targeting to solid tumor by the known ‘enhanced permeability and retention (EPR) effect’26 due to the anatomical and physiological modifications in such tissues, in particular the increased vascular density (the result of highly active angiogenesis), the presence of vessels with both wide fenestrations and lack of smooth muscle layer, and finally a decreased lymphatic drainage. In solid tumor there is also extensive production of vascular mediators, which again leads to increased vascular permeability. The EPR effect can therefore lead to concentrations of a macromolecular carrier in tumor tissue 5–10-fold higher than those in blood plasma, a situation that is difficult to reach with unmodified low-molecular-weight drugs. In addition, the polymeric prodrug can enter cells only through endocytosis,27,28 a process that is increased in tumor cells, further enhancing drug specificity.29 In the last few years active targeting has been receiving great attention for the improvements that may come from a selective therapy toward selected cells, tissues, or organs.30 An important requirement for drug–polymer conjugates is the absence or limited presence of free drug as impurity (1–2% with respect to the total amount of drug), which otherwise might lead to an overestimation of the conjugate’s potency in a biological evaluation.

Drug–Polymer Conjugates

Spacer

Polymeric backbone

Drug

Solubilizing residue

Targeting moiety

Figure 2 Ringsdorf’s small drug–polymer model.

Degrading enzymes or antibodies

Protein

Polymer

Figure 3 Protein surface protection offered by conjugated polymer chains.

5.45.2.2

Polymer–Protein Conjugates

Although successful results have been obtained in the clinic using streptokinase or neocarcinostatin conjugates, coupled to oxidized dextran and poly(styrene-co-maleic acid/anhydride), respectively, the research on protein–polymer conjugation really took off following two reports on the modification of bovine serum albumin and bovine liver catalase with PEG.31,32 Polymer–protein conjugates have all the benefits described above for low-molecular-weight drug conjugates, but some additional advantages can be achieved, namely the protection of sites of proteolysis and the reduction or prevention of immunogenicity and antigenicity (Figure 3).33–35 These are the result of the shielding effect of polymer chains on the protein surface, which prevents the approach of other proteins or enzymes by steric hindrance. In general this also influences the activity of conjugated protein, which is lower compared to the native protein in in vitro tests, but is more than compensated for in vivo thanks to the great improvement of pharmacokinetic parameters.36,37

5.45.3

Polymers for Bioconjugation

Many polymers have been investigated as candidates for drug delivery,38 for example, in the modification of a protein or as a carrier for low-molecular-weight drugs, with linear or branched structures of natural or synthetic origin. These include: *

synthetic polymers: PEG, HPMA copolymers, poly(ethyleneimine) (PEI), poly(acroloylmorpholine) (PacM), poly(vinylpyrrolidone) (PVP), polyamidoamines, divinylethermaleic anhydride/acid copolymer (DIVEMA), poly(styrene-co-maleic acid/anhydride) (SMA), polyvinylalcohol (PVA);

1047

1048

Drug–Polymer Conjugates

* *

natural polymers: dextran, pullulan, mannan, dextrin, chitosans, hyaluronic acid, proteins; and pseudosynthetic polymers: PGA, poly(L-lysine), poly(malic acid), poly(aspartamides), poly((N-hydroxyethyl)L-glutamine) (PHEG).

A short description is given below of most of these polymers because of their limited application, while PEG is described in more detail.

5.45.3.1

Vinyl Polymers

These polymers are prepared by radical polymerization of the respective vinyl monomer or by copolymerization of two or more different monomers, to modulate the properties of the final product. Copolymerization in fact allows a tailor-made polymer that is better at responding to the specific requirements of drug delivery to be designed. This kind of carrier can reach high levels of drug loading since, in theory, it is possible to prepare a polymer in which each monomer can carry a drug molecule. Vinyl polymers are not biodegradable and therefore must be limited in size to allow body clearance, mainly by kidney filtration, thus avoiding accumulation in the body. Usually, their molecular weight needs to be below the limit for renal filtration (40–50 kDa). Among all synthesized vinyl polymers, HPMA emerged as the best candidate and led to a number of important conjugates.39–41 The most studied HPMA conjugate is its derivative with the antitumor agent doxorubicin (FCE28068). The drug was linked to the polymeric backbone through a peptidyl spacer (Gly-Phe-Leu-Gly) specifically designed to be cleaved by lysosomal cathepsin B,42 yielding a conjugate known as PK1 (Figure 4a).43 PK1 was optimized to have certain features such as: (1) a molecular weight of 30 000 Da to ensure body clearance and, at the same time, to allow tumor targeting44; (2) a spacer sequence that was selected to be stable in plasma, but promptly released in lysosomes; and (3) an amount of bound drug (B8.5 wt%) was required to reach therapeutic concentration in tumor cells. From the PK1 studies the PK2 conjugate was produced (FC28069, Figure 4b), which, like the pure drug, possesses an N-acylated galactosamine residue as the targeting group.45 This molecule conveys targeting to the liver thanks to its interaction with hepatocyte asialoglycoprotein receptors. PK2 has a molecular weight of 25 000 Da, a doxorubicin content of B7.5 wt%, and a galactosamine content of B1.5–2 wt%. PK1 reached phase II clinical trials for the treatment of breast, colon, and small-cell lung cancer, and it was the first HPMA conjugate to be administered to patients.46,47 PK2 was the first targeted polymer to enter clinical trials, and it was used for the treatment of primary and secondary liver cancer.48 PK1 and PK2 showed a two- to five-fold reduction in anthracycline toxicity, and despite the high cumulative doses of doxorubicin administered, no cardiotoxicity was observed. HPMA copolymer was studied as a potential carrier for two other anticancer drugs apart from anthracyclines, namely, camptothecin (MAG-CPT49; Figure 4c) and paclitaxel (PNU16694550; Figure 4d). Both these drugs suffer from low solubility in water; polymer conjugation provides a solution to this problem while allowing the EPR effect for solid tumor targeting to be exploited. Camptothecin is linked to HPMA copolymer through a H-Gly-NH-(CH2)6-NH-Gly-OH spacer that forms an ester linkage with the C-20 hydroxyl group of the drug. The conjugate has a molecular weight ranging from 20 000 to 30 000 Da depending on the drug loading (5–10 wt%). Paclitaxel, on the other hand, is conjugated at the level of the C-2 hydroxyl group (drug loading B5 wt%) by an ester bond with the spacer Gly-Phe-Leu-Gly. The drug solubility in water is increased from 0.0001 mg mL 1 of the free drug to 2 mg mL 1 of the conjugate. Both HPMA conjugates entered phase I clinical trials, but the results indicated the need for optimization of the derivatives’ design, in particular regarding the stability of the linkage between the polymer and the drugs (ester) and the drug loading. In this case, the products showed the toxicity of the respective free drugs due to the rapid hydrolysis of the ester bond. Another intensively studied and promising conjugate is the HPMA–platinate conjugate (AP5280, Access Pharmaceuticals, Figure 4e), where the metal is chelated by a malonate molecule, which is linked to a 25 000 Da HPMA chain by a Gly-Phe-Leu-Gly spacer. The loading of platinum is about 7 wt%. To be effective platinum has to be released from the conjugate and the malonate derivatives showed the best rate of hydrolysis. The conjugate has now entered phase II clinical trials.51,52 Many studies were carried out to investigate the potential toxicity of the HPMA copolymer alone or as a conjugate. For example, it was found that PK1 could be administered in clinical trials up to cumulative doses of 420 g–2 without problems of immunogenicity or toxicity.46,53,54

5.45.3.2

Poly(Amino Acids) and Analogs

PGA, poly(L-lysine), poly(aspartamides), poly(N-hydroxyethyl-L-glutamine) (PHEG), are easily synthesized and possess a peptidyl structure. They are biodegradable when obtained from the natural L-amino acids but are not if

Drug–Polymer Conjugates

CH3

CH3 CH2 C

CH2 C

O 95

O x

O

HN

O

HO

CH3 CH2 C

CH C

O 5 HN

HN

CH3

CH3

CH2 C

O

y

HN

HO

z

HN

O

O

NH

NH O

NH

O

HN

O

HN

HN

O O

NH

O NH

OH

NH OH

O

O

O

NH

O

OH

O

OCH3

OH

HO

O

O

NH OH

O

OH

O

OCH3

OH HO

HO

HO

HO O

HO

O

OH

O

(a)

(b)

CH3

CH3

CH3

CH2 C

CH2

C

O m

CH3

CH2 C O n

CH2

C O n

O m

HN

HN

HO

O

O

NH

HN

HN

O

CH2

O

HO

NH

NH 6

NH O

H2C HN

O O

O

CH3 O

O NH

N

(c)

N

O

O

O

O O

HO

O

O

O

O O

NH

O O

O

OH

O

(d) Figure 4 Several polymer conjugates of low-molecular-weight drugs: (a) HPMA copolymer-doxorubicin (PK1; FCE28068), (b) HPMA copolymer-doxorubicin-galactosamine (PK2; FCE28069), (c) HPMA copolymer-camptothecin (MAG-CPT; PNU166148), (d) HPMA copolymer-paclitaxel (PNU166945), (e) HPMA copolymer-platinate (AP5280), (f) polyglutamate-paclitaxel (XYOTAX; CT-2103), and (g) PEG-camptothecin (PROTHECAN).

1049

1050

Drug–Polymer Conjugates

CH3

CH3

CH2 C

CH2 C O 90

O 10

HN

HN

HO

NH CH

O NH

NH

NH CH

CH y

x COO−Na+

O

O

O

O

z O

O

O O

HN O O

O

O

NH

O

HO O

O O

O

O

O

O

NH O O Na+O−

N Pt

(e)

O

O−

O O

OH

O

HO

NH3

O O

NH

O O

O OH O

O O

NH3

(f)

O N N O O

O

CH3

O O

O n

O

H3C

O

O O

N N

(g)

O

Figure 4 Continued

prepared from the D-enantiomers. The drug loading of these polymers, similarly to HPMA, is high since any monomer possesses a side reactive group (amine, carboxyl, or hydroxyl) that can be derivatized for drug coupling. Of this class of polymers, the conjugate PGA-paclitaxel (CT-2103, XYOTAX,55–58 Figure 4f) from Cell Therapeutics is at the most advanced clinical stage (phase II/III). In this case, a PGA with B40 000 Da molecular weight was linked to paclitaxel through an ester bond reaching a loading of B37 wt%. An interesting property of this polymer is its biodegradability by cathepsin B to form glutamyl-paclitaxel.59 In early trials it was administered every 3 weeks in the treatment of mesothelioma, renal cell carcinoma, nonsmall cell lung cancer, and a paclitaxel-resistant ovarian cancer disease. A significant number of patients with partial response or stabilized disease was observed. Phase II confirmed its efficacy in patients with recurrent ovarian cancer, but in this case the conjugate displayed a more frequent neurotoxicity than predicted from phase I trials, probably related to the fact that the patients were heavily pretreated with platinates. Further investigations are ongoing.60 PGA was also conjugated to camptothecin through an ester bond leading to the product CT-2106 (Cell Therapeutics) containing 33–35 wt% of drug and a molecular weight of 50 000 Da. The conjugate has entered a phase I trial.61

5.45.3.3

Polysaccharides

Several polysaccharides have been used in pharmaceutical applications. In terms of pharmacokinetics of the carriers themselves, molecular weight, electric charge, chemical modifications, and degree of polydispersity and/or branching largely determine their fate in vivo. Generally, large molecular weight (440 kDa) polysaccharides have low clearance and relatively long plasma half-life, resulting in reticuloendothelial or tumor tissue accumulation. As with other

Drug–Polymer Conjugates

macromolecular carriers the tumor accumulation is due to the EPR effect, but through the additional linking to the polymer of targeting molecules it is possible to reach specific cells. The most important application as polymeric carrier has been for the preparation of macromolecular prodrugs that are inactive as such, but after release of the free drug at the site of interest they perform their action.62 Polysaccharides have also been used in the preparation of protein conjugates that, while still maintaining the activity of the starting proteins, increase the duration of effect and decrease the immunogenicity.62 The most commonly used polymer of this class was dextran38 (mainly 1,6 poly a-D-glucose with some 1,4 branching links), which was first approved as a plasma expander. Its conjugate with doxorubicin (AD-70; mol.wt. dextran B70 000 Da) entered phase I clinical trials, but displayed a toxicity attributed to uptake of dextran by the liver reticuloendothelial cells.63 Dextran was also used as a carrier of proteins, in particular to improve the pharmacokinetics. The most important results were obtained with streptokinase (Streptodekase),64 but superoxide dismutase was also studied.65 For the coupling the polymer was oxidized by periodate yielding aldehyde groups that in turn were reacted with protein amino groups, mainly from lysines. This method, however, was abandoned due to the fact that the multiplicity of binding groups in the polymer might yield to undesirable cross-linking in the reaction with the protein, beyond the high heterogeneity of the products.

5.45.3.4

Proteins

Among all proteins, albumin and transferrin are the most studied as macromolecular carriers. Although the tissue distribution of these proteins is influenced by their functional role in the body, a series of investigations have shown that the anatomical and physiological characteristics of tumors can promote, by the EPR effect, the uptake in these tissues of drugs conjugated to serum proteins. Antitumor and antiviral agents have been conjugated to serum proteins and early clinical trials have been performed with transferrin conjugates of cisplatin and doxorubicin,66 with adenine arabinoside monophosphate conjugated to lactosaminated albumin,67 and with methotrexate bound to albumin.68 Clinical trials with an albumin-doxorubicin prodrug and a long-acting opioid component are under way.69 A limitation for the use of proteins is their structural complexity with several groups having different functionalities (amino, carboxylic, hydroxyl, and thiol groups). This may be an advantage where they are linked to simple drugs, since high loading is possible, but it also presents a great problem when conjugating to peptides or proteins, since the presence of many reactive sites may lead to cross-linking and heterogeneous products. The use of albumin as a carrier of therapeutic proteins was exploited 20 years ago with good results being achieved in the case of superoxide dismutase.70 However, in the case of albumin, it is possible to overcome the problem of the reactive group multiplicity by exploiting the lone thiol group,71 which can react with specific thiol-reactive molecules. Thanks to the great potential of genetic engineering it is now possible to obtain the desired protein or peptide fused with albumin,72 avoiding the risks of cross-linking.

5.45.3.5

Poly(Styrene-co-Maleic Acid/Anhydride)

The protein neocarcinostatin (NCS), which exhibits cytotoxicity against mammalian cells and Gram-positive bacteria at concentrations as low as 0.01 mg mL 1, was conjugated to SMA: two small polymer chains (1.6 kDa) were linked to the amino group of Lys-20 and Ala-1. The conjugate, named SMANCS, showed a half-life 10–20 times higher than the native protein and by the EPR effect the accumulation in tumor was 30-fold that of muscle.73 Thanks to its increased hydrophobicity, SMANCS can be solubilized in lipid media such as Lipiodol and administered intra-arterially for treatment of tumors. The derivative was marketed in Japan in 1993 (Zinostatin Stimalamer) for the treatment of hepatocellular carcinoma.74 SMA conjugation, however, did not become a general method for protein coupling because, like dextran and HPMA, it possesses many sites of attachment along the polymer backbone, which can give rise to complex mixtures of products.

5.45.3.6

Poly(Ethylene Glycol)

As a modifying polymer PEG has the benefit of three decades of studies littered with important milestones, which has already resulted in seven products on the market (Table 1). This polymer is chosen for: (1) the lack of immunogenicity, antigenicity, and toxicity; (2) its high solubility in water and in many organic solvents; (3) the high hydration and flexibility of the chain, at the basis of the protein rejection properties; and (4) its approval by the FDA for human use. PEG is synthesized by ring opening polymerization of ethylene oxide. The reaction, which is initiated by methanol or water, gives polymers with one or two end chain hydroxyl groups termed monomethoxy-PEG (mPEG-OH)

1051

1052

Drug–Polymer Conjugates

or diol PEG (HO-PEG-OH), respectively. The polymerization process produces a family of PEG molecules with a wide range of molecular weights. In this form PEG is not suitable for use in drug conjugation, although it is largely employed in pharmaceutical technology as excipient. As with any polymer, the biological properties of PEG, such as kidney excretion or protein surface coverage, are based on its size, so it is necessary to use samples that are as homogeneous as possible. Fractionation techniques (e.g., gel filtration chromatography) need to achieve narrow polydispersivity and must also exclude the presence of the diol form (formed from traces of water in the polymerization reaction) as an impurity in monomethoxy PEG batches. A wide series of activated PEGs have been developed to address different chemical groups in the drugs. Furthermore, PEGs with various shapes (branched, multifunctional, or heterobifunctional) are now available. Monofunctional polymers (mPEG-OH), linear or branched, are most suitable for protein modification since the diol or multifunctional polymers can give rise to cross-linking. Those with multiple reactive groups on the chain are useful to obtain an increased drug to polymer ratio. Increased loading is particularly needed in therapeutic agents with low biological activity, which would otherwise require the administration of a large amount of conjugate with consequent high viscosity of the solution. The optimization of both the polymerization procedure and the purification process is now allowing the preparation of PEGs with low polydispersivity, Mw/Mn ranging from 1.01 for PEG with molelcular weights below 5 kDa to 1.1 for PEG as big as 50 kDa. PEG has unique solvation properties that are due to the coordination of 2–3 water molecules per ethylene oxide unit.75 This, together with the great flexibility of the molecule backbone, is responsible for the biocompatibility, nonimmunogenicity, and nonantigenicity of the polymer;31 it also possesses an apparent molecular weight 5–10 times higher than that of a globular protein of comparable mass, as verified by gel permeation chromatography.76 Owing to this large hydrodynamic volume, a single PEG molecule covers a large area of the surface of the conjugated protein, preventing the approach of proteolytic enzymes and antibodies.77–79 In vivo PEG undergoes limited chemical degradation and the body clearance depends upon its molecular weight: below 20 kDa it is easily secreted into the urine, while higher molecular weight PEGs are eliminated more slowly and the clearance through liver becomes predominant. The threshold for kidney filtration is about 40–60 kDa ˚ 80), which represents the albumin excretion limit. Over this limit the (a hydrodynamic radius of approximately 45 A polymer remains in circulation and mainly accumulates in liver. Alcohol dehydrogenase can degrade low-molecularweight PEGs, and chain cleavage can be catalyzed by cytochrome P450 microsomial enzymes.81 Some branched PEGs may also undergo a molecular weight reduction when the hydrolysis and loss of one polymer chain is catalyzed by anchimeric assistance.82 However, regarding safety concerns when choosing the PEG’s molecular weight in the design of a new drug–polymer conjugate ‘it is commonly accepted to use PEGs below 40 kDa’ to avoid accumulation in the body. This is the molecular weight employed in two successful products, Pegasys and Macugen (Table 1) belonging to the PEG–protein and PEG–small drug conjugate classes, respectively. Finally, several years of PEG use as an excipient in foods, cosmetics, and pharmaceuticals, without toxic effects, is clear proof of its safety.76 The first generation of PEG conjugates was based on low-molecular-weight polymers (r12 kDa), most commonly in their linear monomethoxylated form (mPEG). Batches of this polymer often contained a significant percentage of PEG diol, an impurity that, once activated, could act as a potential cross-linking agent. Furthermore, the chemistry employed in mPEG synthesis often resulted in side reaction products or led to weak and reversible linkages. The drugs PEGadenosine deaminase (Adagen)83 and PEG-asparaginase (Oncaspar)84 belong to this generation of PEG conjugates. The second generation of PEGs is characterized by improved polymer purity (reduction of both polydispersivity and diol content for high-molecular-weight PEGs) and by a wide selection of activated PEGs that allow selectivity of reaction toward different protein sites. The availability of several new PEGs is now widening the opportunities in PEGylation technology. Second-generation PEGs currently on the market include: *

*

*

PEG-propionaldehyde, also in the form of the more stable acetal: the reaction with the amino group leads to a Shiff ’s base that, once reduced by sodium borohydride, yields a stable secondary amine that maintains the same neat charge of the parent drug. Several PEG-succinimidyl derivatives: highly reactive toward amino groups. The reaction rate of these derivatives may significantly change depending upon the length and the composition of the alkyl chain between PEG and the succinimidyl moiety.85 ‘Y’ shaped branched PEG86 (see Figure 1f): provides a higher surface shielding effect and is more effective in protecting the conjugated protein from proteolytic enzymes and antibodies (Figure 5). Moreover, proteins modified with this PEG retain higher activity than the same enzyme modified by linear PEGs. This effect is probably due to the branched polymer that prevents the PEG from entering the enzyme active site cleft or other sites involved in biological activity (Figure 6).

Drug–Polymer Conjugates

*

*

*

PEGs reactive toward thiol groups: PEG-maleimide (MAL-PEG), PEG-vinylsulfone (VS-PEG), PEG-iodoacetamide (IA-PEG), and PEG-orthopyridyl-disulfide (OPSS-PEG) have been specifically developed for this conjugation, but only the last one strictly reacts with the thiol groups, avoiding any amino modification that might occur with the other three (Figure 7). Heterobifunctional PEGs87,88: these derivatives have different reactive groups at the two polymer ends that allow two different molecules to be linked separately to the same PEG chain. It is therefore possible to obtain conjugates that carry both a drug and a targeting molecule. Among the proposed and commercially available heterobifunctional PEGs, the most commonly used are H2N-PEG-COOH, HO-PEG-COOH, and H2N-PEG-OH. Multiarm or ‘dendronized’ PEGs (Figure 1b and g): the former are prepared by linking a linear PEG to a multimeric compound, whereas the latter are linear PEGs with a dentritic structure at one or both chain ends.89,90 The aim of both derivatives is to increase the drug/polymer molar ratio, overcoming problems of high viscosity that may occur with solutions of monofunctional drug–polymer conjugates. This is particularly true for drugs that require a high amount of product for the therapeutic treatments. Most of these PEGs, also in the activated form, are now commercially available.

Linear PEG Branched PEG

Protein surface Figure 5 Shielding effect of different PEG structures – linear and branched. The higher steric hindrance of branched PEG compared with linear PEG explains its enhanced capacity to reject the approach of other proteins (e.g., degrading enzymes) or cells. (Reproduced with permission from Pasut, G.; Guiotto, A.; Veronese, F. M. Exp. Op. Ther. Patents 2004, 14, 859–894.)

Branched PEG Enzyme cleft

Linear PEG

Figure 6 Effect of PEG hindrance on the enzyme active site access. The high steric hindrance of branched PEG, which makes it difficult for the active site cleft to be accessed, may explain the lower inactivation of enzymes as compared to linear PEG of the same size. (Reproduced with permission from Pasut, G.; Guiotto, A.; Veronese, F. M. Exp. Op. Ther. Patents 2004, 14, 859–894.)

1053

1054

Drug–Polymer Conjugates

O

O (a)

+

PEG N

HS R

PEG N S R O

O

(b)

PEG S S R

+

HS R

I

+

HS R

PEG

CH2

+

HS R

PEG S

PEG S S N

O

O (c)

PEG

(d)

PEG S

NH

S R

O

O O

NH

CH

CH2 CH2 S R

O

Figure 7 Examples of activated PEG molecules reactive toward thiol groups: (a) PEG maleimide, (b) PEG orthopyridyldisulfide, (c) PEG iodoacetamide, and (d) PEG vinylsulfone.

Several protein bioconjugates derived from this second generation of PEGs have reached the market, such as linear PEG-interferon a-2b (PEG-Intron),91 branched PEG-interferon a-2a (Pegasys),36,92 PEG-growth hormone receptor antagonist (Pegvisomant, Somavert),93 and PEG-granulocyte colony stimulating factor (G-CSF) (pegfilgrastim, Neulasta).94 More recently also a PEG conjugate of a 24-mer oligonucleotide, a branched PEG-anti-VEGF aptamer (pegaptanib sodium injection, Macugen)95 is also marketed, while many other peptide, protein, and oligonucleotides products are under clinical trials and hopefully will be available in the near future. In PEGylation of low-molecular-weight drugs, PEG faces the limit of low drug loading since in its standard form the polymer can be derivatized only at the two chain extremes. However, several derivatives have been prepared and the most advanced is the PEG-camptothecin conjugate (PROTHECAN or Pegamotecan, Enzon), obtained by linking two drug molecules (using C-20 hydroxyl group) at the termini of a 40 000 Da polymer chain (Figure 4g). The drug loading corresponds to 1.7 wt%, which is rather low if compared with other multivalent polymers (i.e., PGA, HPMA copolymers). The product passed phase I trials96; the maximum tolerated dose is 100 mg m–2 (camptothecin equivalent) and the recommended dose for phase II studies is 7000 mg m–2 administered for 1 h i.v. every 3 weeks. Phase II studies are ongoing and preliminary data shows Pegamotecan to be a promising treatment for adenocarcinoma of the stomach and gastroesophageal (GE) junction. The conjugate appears to be well tolerated, with a low incidence of toxicities. Grade 2 cystitis, a known toxicity of camptothecin, was observed in 2 out of 15 patients.97 PEG-camptothecin conjugates with tri- or tetrapeptide linkers, to exploit the drug release inside the cell only, have been shown to be highly stable in plasma but rapidly hydrolyze in the presence of lysosomal enzyme cathepsin B.98,99 In vitro and in vivo results are encouraging and indicate that the polymer bound camptothecin derivative SN-392 (10-amino-7-ethyl camptothecin) is less toxic than the free drug and the camptothecin analog CPT-11, but is still highly effective against Meth A fibrosarcoma cell lines. From pharmacokinetic studies it was immediately evident that the maximal plasma concentration (Cmax) after administration of 10-amino-7-ethyl camptothecin is eightfold higher than that after PEG-SN392 injection, although AUC data are comparable. The mean residence time (MRT, a dose-independent measure of drug elimination expressing the average time a drug molecule remains in the body after intravenous injection) after PEG-peptide-camptothecin injection is threefold higher than that after injection of both forms of 10-amino-7-ethyl camptothecin. The data indicate that the conjugate acts as a circulating reservoir of the antitumor agents, since the conjugate itself it is not active until it enters the cells and the drug is released, by lysosomal enzymes, to exploit its action. This method of release, often employed for antitumor drugs, is discussed further in Section 5.45.5. An interesting study reports the preparation of PEG-doxorubicin conjugates where several parameters, such as polymer molecular weight (5 000–20 000 Da), the structure of PEG (linear or branched), and the sequence of a peptide linker between drug and carrier (i.e., H-GFLG-OH, H-GLFG-OH, H-GLG-OH, H-GGRR-OH, or H-RGLG-OH),

Drug–Polymer Conjugates

were taken into consideration.100 Surprisingly, the conjugate with the greatest antitumor activity in rats had the lowest molecular weight PEG (mPEG5000-GFLF-doxorubicin). Apparently, this contradicts the need to have conjugates with molecular weights above 20–30 kDa for a blood residence time long enough to allow accumulation in tumors by the EPR effect. It was demonstrated by light scattering that this low-molecular-weight PEG-doxorubicin conjugate forms micelles in solution, and consequently its apparent molecular weight rises to 120 000 Da, which ensures an effective EPR effect and explains the antitumor activity. Enzon researchers also studied the preparation and optimization of PEG-paclitaxel conjugates using several PEG molecular weights.101 In this case, the authors reported the importance of a molecular weight Z30 kDa in order to prevent rapid elimination of the conjugates by the kidneys. A PEG derivative of this drug entered phase I clinical trials and preliminary data are encouraging; however, neutropenia, as with the free drug, is a predominant hematological toxicity.102

5.45.4

Considerations for PEGylation

To obtain conjugates with improved pharmacological properties several parameters of the polymer must be taken into consideration. In PEGylation the mass of the PEG chain and the binding site on the protein or drug are important parameters to be considered, as well as the chemistry of linkage and the use of a proper spacer. In the modification of proteins it is better to use few high-molecular-weight chains than a higher number of low-molecular-weight ones. A multipoint attachment of PEG or the use of too large a PEG is expected to reduce or prevent a protein’s bioactivity by interfering with receptor binding; however, a low total mass of bound PEG cannot ensure protection from degradation and results in poor pharmacokinetic profiles. The effect of both number and mass of linked PEG chains on recognition and pharmacokinetic parameters are well documented in the literature.103,104 The site of PEGylation is also critical since when this resides in, or close to, the protein recognition area or active site of enzymes, the polymer affects the biological activity. Furthermore, the modification of certain residues may cause conformational changes ending in denaturation or exposure of buried amino acids, which may lead to aggregation or facilitate proteolytic degradation.

5.45.5

Controlled Release of Conjugated Proteins or Drugs

In general, with nonprotein drugs a stable bond between the polymer and drug is not convenient since many molecules exploit their activity only in the free form. A chemical bond that releases the native molecule is therefore welcome, especially if the release can be triggered under specific controlled conditions. The polymeric prodrug of antitumor agents or substances that possess systemic toxicity should be stable in the bloodstream, but release the drug from the carrier in the desired tissue or cell. The only way to obtain such controlled release is by the use of a proper spacer or linkage between drug and polymer that can be hydrolytically or enzymatically cleaved. Examples of how to achieve a prompt or retarded release of conjugated drugs include: *

*

*

Linkers that respond to pH changes.105 Following cell internalization by endocytosis the conjugates are exposed to the acidic pH of endosomes and lysosomes; furthermore, the pH of tumor tissue is slightly more acidic than that of healthy tissues.106 This situation can be exploited using acid-labile spacers, such as N-cis-aconityl acid, which was the first approach used to reversibly link daunorubicin to aminoethyl polyacrylamide or poly(D-lysine).107 In this case a prompt drug release was evident at pH 4 or lower, while in blood or at pH 6 the linker was stable. A hydrazon linkage can also take advantage of the fact that a low pH is needed to free the drug; this linker was exploited in several conjugates to release adriamycin108 or streptomycin109 from the carrier. Exploitation of lysosomal enzymes. In this case the macromolecular carriers enter the cell by endocytosis and the newly formed endosome fuses with the lysosome, exposing the polymer–drug to a series of degrading enzymes. A spacer (usually an oligopeptide) that is specifically designed to be cleaved by the lysosomal enzymes allows a lysosomotropic drug delivery. This strategy also takes advantage of the fact that lysosomes are overexpressed in tumor cells where cathepsins B or D and other metalloproteinases play a role in tumor growth. Examples of such oligopeptide linkers include those studied by Duncan and Kopecek for optimization of PK1; among these H-Gly-Phe-Leu-Gly-OH and H-Gly-Leu-Phe-Gly-OH appear to be the most effective.42,110 Drug release by anchimeric-assisted hydrolysis. This sophisticated strategy uses linkers that are designed to form a double prodrug system. The drug-linker is first released from the polymer by hydrolysis (first prodrug), which triggers the linker (second prodrug) that finally releases the free and active drug. Examples of these double drug delivery systems include the 1,6-elimination reaction or trimethyl lock lactonization (Figure 8).111,112

1055

1056

Drug–Polymer Conjugates

(a) PEG

H N Protein

O O

COOH

PEG

In vivo cleavage

O

+

O

H N Protein O

HO

O

Hydrolysis and decarboxylation OH −

OH

O

HO

CH2

+

CO2

+

H2N Protein

(b) O PEG Spacer O

O

R1 R2

N Drug H CH3

In vivo ester cleavage by enzyme

OH PEG

+

Controllable rate

O

R1

N Drug H

R2

CH3 Fast

Amide cleavage by lactonization

O O R1 R1 = R2 = H or CH3

R2

+

Drug

NH2

CH3

Figure 8 Controlled release of active molecules from PEG based on the (a) 1,6-elimination system and (b) trimethyl lock lactonization system.

5.45.6

PEGylation Chemistry for Proteins

To appreciate the potential of PEGylation in drug delivery, it is important to understand the chemistry involved in the linking. A brief description is given below.

5.45.6.1

Amino Group PEGylation

PEGylation at the level of protein amino groups may be carried out with PEGs having different reactive groups at the end of the chain and often, although the coupling reaction is based on the same chemistry (for instance acylation), the obtained products are different. The difference may reside in the number of PEG chains linked per protein molecule, in the amino acids involved, and in the chemical bond between PEG and drug. The most common methods for random PEGylation are reported here, while procedures for site-direct modification will be discussed later. Products available on the market so far mainly come from random PEGylation (Adagen, Oncaspar, PEG-Intron, and Pegasys) since Food and Drug Administration (FDA) authorities approve these conjugate mixtures upon demonstration of their reproducibility. The activated PEG for amino linking can be chosen from a range of commercially available polymers, the most common being PEG succinimidyl succinate (SS-PEG), PEG succinimidyl carbonate (SC-PEG), PEG p-nitrophenyl carbonate (pNPC-PEG), PEG benzotriazolyl carbonate (BTC-PEG), PEG trichlorophenyl carbonate (TCP-PEG), PEG carbonyldiimidazole (CDI-PEG), PEG tresylate, PEG dichlorotriazine, PEG aldehyde (AL-PEG), and a branched form of PEG (PEG2-COOH) (Figure 9). The difference between these PEGs lies in the kinetic rate of amino coupling and in the resulting link between polymer and drug. The derivatives with slower reactivity, such as the carbonate PEGs (pNPC-PEG, CDI-PEG, and

Drug–Polymer Conjugates

O O PEG O

C

O

O CH2 CH2

R

+ H2N

N

O

C

PEG O

C

O CH2 CH2

C

NH

R

O

(a) O O PEG

O

C

O

N

(b)

+

O O PEG O

C

NO2 +

O

(c) O PEG O

C

N O

O

N

N

H2N

+

R

PEG O

C

NH R

(d) Cl O PEG O

C

Cl

O

(e)

+

Cl O N PEG

O

C

O

N

+

(f)

(g) PEG O

SO2 CH2CF3 + H2N

PEG

R

NH

Cl

Cl

N

N

PEG O

N + H2N

R

PEG O

N (h)

R

N N

Cl

NH

R

O NaCNBH3 PEG

H + H2N

R

R PEG

N H

(i) Figure 9 Examples of activated PEG molecules reactive toward amino groups: (a) PEG succinimidyl succinate, (b) PEG succinimidyl carbonate, (c) PEG p-nitrophenyl carbonate, (d) PEG benzotriazol carbonate, (e) PEG trichlorophenyl carbonate, (f) PEG carbonylimidazole, (g) PEG tresylate, and (h) PEG dichlorotriazine, and (i) PEG aldehyde (AL-PEG). (Adapted from Pasut, G.; Guiotto, A.; Veronese, F. M. Exp. Op. Ther. Patents 2004, 14, 859–894.)

1057

1058

Drug–Polymer Conjugates

TCP-PEG) or the aldehyde PEGs, allow a certain degree of selective conjugation within the amino groups present in a protein, according to their nucleophilicity or accessibility.35 An important difference in reactivity is usually observed between the e-amino and the a-amino group in proteins due to their pKa: 9.3–9.5 for the e-amino residue of lysine and 7.6–8 for the a-amino group. This was exploited for the a-amino modification reached by a conjugation at pH 5.5–6.0, as is the case for G-CSF with PEG-aldehyde.94 The e-amino groups of lysine, which possess high nucleophilicity at high pH, are instead the preferred site of conjugation at pH 8.5–9. It is noteworthy that the conjugation performed using PEG dichlorotriazine, PEG tresylate, and PEG aldehyde (the latter after sodium cyanoborohydride reduction) maintains the same total charge on the native protein surface, since these derivatives react through an alkylation reaction, yielding a secondary amine. In contrast, PEGylation conducted with acylating PEGs (i.e., SS-PEG, SC-PEG, pNPC-PEG, CDI-PEG, TCP-PEG, and PEG2-COOH) gives weakly acidic amide or carbamate linkages with loss of the positive charge. The PEG derivatives described above may sometimes give side reactions involving the hydroxyl groups of serine, threonine, and tyrosine and the secondary amino group of histidine. These linkages, however, are generally hydrolytically unstable yielding the starting residue. The reaction conditions or particular conformational disposition may enhance the percentage of these unusual PEGylations; for example, a-interferon was found to be conjugated by SC-PEG or BTC-PEG also at His34 under slightly acidic conditions113 (the pKa value of histidine is between those of the a- and e-amino groups). PEG was found to be linked to the hydroxyl groups of serine in the decapeptide antide or those of tyrosine in epidermal growth factor (EGF).114,115 Two examples of random amino PEGylation are reported here.

5.45.6.1.1 Random PEGylation of asparaginase Asparaginase was one of the first PEGylated enzymes; it catalyzes the hydrolysis of asparagine to aspartic acid and ammonia. The resulting depletion in asparagine is fatal to leukemic lymphoblasts and certain other tumor cells, which by lacking or having very low levels of asparagine synthetase are unable to synthesize asparagine de novo and rely on asparagine supplied in the serum for survival. The free enzyme, however, is cleared too quickly from the body and it is immunogenic, which limits its therapeutic use. When asparaginase from Escherichia coli was modified with a PEG of 5000 Da the conjugate was shown to cause tumor regression in transplanted mice and to possess less immunogenicity than the native Escherichia coli form.116–118 PEGylation also improves the enzyme chemical stability and the resistance to plasma proteases.119 PEG-asparaginase first entered clinical trials in 1984 and since then it has been administered to thousands of patients with acute lymphoblastic leukemia (ALL).120 The PEG-conjugated enzyme can be safely administered to most patients, even in cases where there is an allergic reaction to E. coli or Erwinia asparaginases. The longer serum half-life of PEG-asparaginase allows a longer interval between administered doses. PEG-asparaginase was developed by Enzon and was approved by the FDA in 1994 for the treatment of patients with ALL who are hypersensitive to native forms of the enzyme. It is now available commercially from Enzon as Oncaspar. The mean serum half-life of PEG-asparaginase is about 15 days as opposed to the 24 h of the nonmodified E. coli enzyme and the 10 h of the Erwinia form. The rate of total clearance of PEG-asparaginase was found to be 17-fold lower than that of the unmodified enzyme, whereas the volume of distribution was similar for the two preparations. L-Asparagine levels were undetectable immediately following the 1-h infusion of peg-asparaginase and remained low during the 14-day interval between doses. Interestingly, a recent pharmacoeconomic study121 demonstrated that despite the higher cost of PEG-asparaginase versus the unmodified enzymes, the overall expense of treatment is comparable to that of unmodified enzymes. Since FDA approval in 1994, drug monitoring has been performed by several phase IV clinical studies and detailed recent reviews are available in the literature.84,122 Recent studies have been carried out on the rational basis that immunological, pharmacokinetic, and pharmacodynamic factors have a considerable impact on the efficacy of asparaginase therapy. Therefore, investigations are now aimed at defining the optimum dose and dosing schedule of the different asparaginase preparations that are used in the clinic123 or correlating antibody levels with pharmacological response.124

5.45.6.1.2 Random PEGylation of interferon a-2a Interferon a-2a (IFNa-2a) was modified with an N-hydroxysuccinimide activated PEG having a branched structure. A high-molecular-weight PEG (PEG2, 40 kDa) was chosen on the basis of several preliminary studies disclosing the fact that: (1) the protein surface protection achieved with a single, long and hindered chain PEG is higher than that obtained with several small PEG chains linked at different sites13; (2) branched PEGs have lower distribution volumes than linear PEGs of identical molecular weight, and the delivery to organs such as liver and spleen is faster125; (3) proteins modified with branched PEG possess greater stability toward enzymes and pH degradation.86

Drug–Polymer Conjugates

Table 3 Pharmacokinetic properties of interferon a-2a and its PEGylated form in rats92

Protein

Half-life (h)

Interferon a-2a PEG2 (40 kDa)-interferon a-2a

Plasma residence time (h)

2.1

1.0

15.0

20.0

Adapted from Reddy, K. R., Modi, M. W.; Pedder, S. Adv. Drug Delivery Rev. 2002, 54, 547–570.

The 40 k Da branched succinimidyl PEG (PEG2-NHS) was linked to interferon a-2a using a 3:1 PEG:protein molar ratio in 50 mM sodium borate buffer pH 9 yielding a few protein isomers.34 PEGylation under these conditions led to a mixture containing 45–50% monosubstituted protein, of 5–10% polysubstituted protein (essentially dimer), and 40–50% unmodified interferon. Identification of the major positional isomer within the mono-PEGylated fraction was carried out by a combination of high-performance cation exchange chromatography, peptide mapping, amino acid sequencing, and mass spectroscopy analysis. It was demonstrated that this branched PEG, thanks to its more hindered structure, was attached mainly to one of the following lysines: Lys-31, Lys-121, Lys-131, or Lys-134.36 Even though the in vitro antiviral activity of PEG2-IFNa-2a was greatly reduced (only 7% was maintained with respect to the native protein) the in vivo activity, measured as ability to reduce the size of various human tumors, was higher than that of free IFNa-2a. The positive result could be related to the extended blood residence time of the conjugated form as shown in Table 3. These studies resulted in the release of the interferon conjugate Pegasys, which has a long period of residence in the blood and is effective in eradicating hepatic and extrahepatic hepatitis C virus (HCV) infection.92

5.45.6.2

Thiol PEGylation

The presence of a free cysteine residue represents an optimal opportunity to achieve site direct modification because it rarely occurs in proteins. PEG derivatives having specific reactivity toward the thiol group, i.e., MAL-PEG, OPSS-PEG, IA-PEG, and VS-PEG (Figure 7), are commercially available and allow thiol coupling with a good yield; however, differences among these polymers in terms of protein–polymer linkage and reaction conditions. Even if the thiol reaction rate of IA-, MAL-, or VS-PEGs is very rapid, some degree of amino coupling may also take place, especially if the reaction is carried out at pH values higher than 8. On the other hand, the reaction with OPSS-PEG is very specific for thiol groups, but the obtained conjugates may be cleaved in the presence of reducing agents as simple thiols or glutathione (present in vivo). PEGylation at the level of cysteine allows easier purification of the reaction mixture since the presence of only one or a few derivatizable sites (free cysteines) avoids the formation of a large number of positional isomers or products with different degrees of substitution, this being a common problem for amino PEGylation. The potential of thiol PEGylation may be further exploited by genetic engineering, which allows the introduction of an additional cysteine residue in a protein sequence or the switching of a nonessential amino acid to cysteine. The use of this strategy with human growth factor was extensively studied. To overcome the common problems of random amino PEGylation, cysteine muteins were synthesized by recombinant DNA technology. The cysteine addition at the C-terminus of hGH leads to a fully active mutein that allows a site-specific PEGylation using the thiol-reactive PEG-maleimide (PEG-MAL, 8 kDa). It was necessary to treat the rhGH mutein with 1,4-dithio-DL-threitol (DTT) before the coupling step, to maintain the C-terminal cysteine in the reduced form and to prevent the formation of scrambled disulfide bridges. After removal of DTT excess by gel filtration, the conjugation leads to a mono-PEGylated derivative with a yield of over 80%.126 Hemoglobin (Hb) is another important example of thiol conjugation; in fact, PEGylation of this protein prevents the vasoactivity as a consequence of its extravasation.76 After several unsuccessful attempts through random PEGylation, a site-specific modification was performed at Cys-93 (of the b-chain) with maleimidophenyl PEG (MAL-Phe-PEG; 5, 10, and 20 kDa), leading to PEGylated Hb carrying two polymer chains per Hb tetramer.127 This product was found to be more efficient than polymerized Hb, the Hb-octamer, or Hb-dodecamer.

5.45.6.3

Carboxy PEGylation

PEGylation at the level of protein carboxylic groups needs their activation for the reaction with an amino PEG. This procedure, however, is not without its limitations since undesired intra- or intermolecular cross-links may occur by reaction with the amino groups of the protein itself.

1059

1060

Drug–Polymer Conjugates

O H2N

PROTEIN

COOH

O

+ MeO

N3

NH PEG Ph2P

PBS pH 7.4 36 h, 37 °C

H2N

PROTEIN

COOH H N

O

O

NH

Ph2P

PEG

O

Figure 10 Staudinger ligation leading to a C-terminal mono-PEGylated protein by reaction of a mutated protein, containing a C-terminal azido-methionine, with an engineered PEG derivative, methyl-PEG-triarylphosphine.

To circumvent this problem it is possible to use PEG-hydrazide (PEG-CO-NH-NH2) instead of the usual PEGamino and to carry out the coupling at pH 4–5 thanks to the low pKa of PEG-hydrazide. In this case, the protein’s COOH groups, activated by water-soluble carbodiimide, do not react with the protein amino groups, which at low pHs are protonated, but with the amino group of PEG-hydrazide only.128 An alternative method for specific C-terminal PEGylation is based on the Staudinger ligation.129 The protocol, using a truncated thrombomodulin mutant,130 begins with the expression by E. coli of a mutated protein containing a C-terminal azido-methionine. This reacts specifically with an engineered PEG derivative, methyl-PEG-triarylphosphine, leading to a C-terminal mono-PEGylated protein (Figure 10). This method, however, involves the preparation of a gene encoding a protein with a C-terminal linker ending with methionine. Expression in E. coli is induced when the transformed bacteria are suspended in a medium where methionine is replaced by the azido-functionalized analog. Unfortunately, this method is applicable only to the rare case of proteins lacking methionine in the sequence; otherwise a methionine in the middle of the protein sequence will stop the protein transduction because the azido-analog does not permit the linking of the following amino acid.

5.45.7

Strategies in Protein PEGylation

To better exploit the potential of PEGylation several strategies have been developed with the purpose of: (1) obtaining homogeneous products; (2) forming PEGylated conjugates with high retention of activity; and (3) performing PEGylation under gentle conditions that are compatible with easily degradable proteins. Site-selective conjugation is always preferred as it allows easy purification and characterization of products and, most importantly, better retention of biological activity. As described above, selective PEGylation may be achieved taking advantage of a free cysteine, but this is possible only when this amino acid is present in the native protein or is introduced by genetic engineering. Alternatively, it is possible to take advantage of the lower pKa value of the a-amino group at the N-terminus with respect to the pKa of e-amine of lysines; in fact, performing the reaction under neutral or mildly acidic conditions, prevents the PEGylation at the level of lysine, but leaves the N-terminal amino group reactive.131 The most successful example of this strategy is the alkylation of r-metHuG-CSF with PEG-aldehyde, proposed by Kinstler. The reaction was carried out at pH 5.5 in the presence of sodium cyanoborohydride to reduce the Shiff ’s base initially formed.94,132 The conjugate obtained with a molecule of a 20 kDa PEG showed an improved pharmacokinetic profile due to the reduced excretion by the kidneys. The PEG-G-CSF conjugate Pegfilgastrim has been on the market since 2002. Site-specific PEGylation can also be achieved by exploiting the different accessibility of protein amino groups, as reported for a truncated form of growth hormone-releasing hormone (hGRF1–29). It was demonstrated that by using an appropriate solvent it is possible to alter the accessibility and reactivity of the three available amino groups. Nuclear magnetic resonance (NMR) and circular dichroism analysis indicated that the percentage of a-helix in hGRF1–29, which

Drug–Polymer Conjugates

is only 20% in water, rises to 90% in structure-promoting solvents such as methanol/water or 2,2,2-trifluoroethanol (TFE), thus facilitating a region-selective modification. When PEGylation was performed in TFE the monoPEGylated conjugate at the level of Lys-12 reached 80% for all PEGylated isomers;133 however, the same reaction conducted in DMSO yielded an almost equimolar mixture of mono-PEGylated Lys-12 and Lys-21 isomers.134 Alternatively, specific PEGylation may be performed by blocking some of the reactive groups with a reversible protecting group as reported for insulin. This protein is formed by two polypeptide chains, A and B, and its three amino groups (Gly-A1, Phe-B1, and Lys-B29) are all candidates for PEGylation. Hinds proposed a site-directed PEGylation procedure involving the preliminary preparation of N-BOC (tert-butyl carbamate)-protected insulin.135 As an example, in order to synthesize NaB1-PEG-insulin the intermediate NaA1, NeB29-BOC-protected insulin was prepared prior to conjugation with PEG-SPA at the level of free NaB1. The final conjugate was obtained upon BOC removal with TFA treatment, forming the NaB1-PEG2000-insulin conjugate with 83% of the native insulin activity. In general, in the PEGylation of an enzyme a requirement for high retention of the activity is that the PEG chains do not modify or obstruct the active site. Many strategies have been developed to achieve this goal: (1) the use of branched PEGs that, thanks to their higher hindrance with respect to linear polymers, have reduced accessibility to the active site (Figure 6); (2) to perform PEGylation in the presence of a substrate or an inhibitor that blocks polymer access to the active site; and (3) to conduct the modification after the enzyme is captured on an insoluble resin by substrates or inhibitors linked on it. In the last case, the obtained conjugate is eluted from the resin by changing the pH or adding denaturants, thus leading to a derivative that does not have linked PEG chains at the level of active site and its closer surroundings (Figure 11).136 A problem that may occur during protein PEGylation is the production of a low yield, especially when the modification is directed toward a buried or less accessible amino acid. This inconvenience is enhanced when the reaction is performed with high-molecular-weight PEGs due to the high steric hindrance. In the case of interferon beta (IFN-b), conjugation at cysteine 17 could only be achieved with a low-molecular-weight OPSS-PEG oligomer, but not with a high-molecular-weight polymer.137 Modification with high-molecular-weight PEGs could be successfully attempted via a two-step procedure: in the first reaction, the protein is modified with a short-chain heterobifunctional PEG oligomer, while in the second, the obtained conjugate is linked to a higher molecular weight PEG, possessing specific reactivity toward the terminal end of the first oligomer (Figure 12). The heterobifunctional PEG oligomer had

Ligand

Binding

+ Protein

Insoluble resin

Amino group

Conjugation

Activated PEG

Elution

Figure 11 PEGylation strategy for the protection of an enzyme active site from polymer conjugation: firstly the enzyme is loaded into an affinity resin functionalized with the appropriate ligand. The enzyme’s active site binds the ligand, thus protecting the active site itself and the area close to it from PEG modification. After reaction under heterogeneous conditions the modified enzyme is eluted from the column.

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Protein

High-molecular-weight PEG Low-molecular-weight PEG

Buried active site

Step I

Step II

Final conjugate

Steric hindrance High-molecular-weight PEG Figure 12 Two-step tagging PEGylation strategy for a buried SH group in a protein. Smaller PEG molecules are more reactive than high-molecular-weight PEGs toward the buried cysteine. (Reproduced with permission from Pasut, G.; Guiotto, A.; Veronese, F. M. Exp. Op. Ther. Patents 2004, 14, 859–894.)

O AA

n N H

O

H C

O

Ca2+

H N

AA n

+ RN

AA

H2 TGase

n N H

H C

H N

O

Protein

n

+ NH3

O

O

NH2

AA

NH R Adduct

R = lysine of protein, polymer, etc. Figure 13 Reaction catalyzed by Tgase between a glutamine residue in a protein and an alkyl amine. (Reproduced with permission from Pasut, G.; Guiotto, A.; Veronese, F. M. Exp. Op. Ther. Patents 2004, 14, 859–894.)

a thiol reactive group at one end of the chain and a hydrazine group at the other (OPSS-PEG-Hz, 2 kDa). As mentioned above, hydrazine is still reactive at low pHs when a protein’s amino groups are usually protonated and not reactive, thus preventing unwanted amino PEGylation of the protein. The INF-SS-PEG-Hz conjugate could therefore be selectively modified with PEG-aldehyde (30 kDa) by reductive alkylation. The overall yield was higher than 80%. A recent study demonstrated that specific PEGylation at the lone, but not accessible, thiol groups of G-CSF could be achieved upon its exposure to partially denaturant conditions. After modification with OPSS-PEG, the native conformation of G-CSF was recovered by removal of the denaturant.138

5.45.8

Enzymatic Approach for Protein PEGylation

Recently, as an alternative solution to obtaining homogeneous PEG-protein conjugates under mild reaction conditions, some researchers have been investigating the possibility of exploiting the specificity of enzymes to catalyze PEGylation, which might preserve the protein activity better than with chemical methods. Since the first report, proposed by H. Sato involving the enzyme transglutaminase (Tgase),139 other researchers have developed interesting approaches with different enzymes. From the data obtained so far it is predicted that these procedures will lead to further results of great interest and applicability. Sato studied two methods for interleukin-2 (IL2) PEGylation using transglutaminases (Tgase) from guinea-pig liver (G-Tgase) or from Streptoverticillium sp. strain s-8112 (M-Tgase). Both enzymes catalyze the transfer of an amino group from a donor (for example PEG-NH2) to a glutamine residue present in a protein (Figure 13). The two enzymes differ

Drug–Polymer Conjugates

Table 4 Comparision of IL2 conjugate activities between random PEGylation and site-directed PEGylation by Tgase139

Proteins

% Activitya

rhIL2

100

PEG10-rhIL2 (random PEGylation)

74

(PEG10)2-rhIL2 (random PEGylation)

36

rTG1-IL2 (chimeric protein for enzymatic PEGylation)

72

PEG10-rTG1-IL2 (enzymatic PEGylation)

69

(PEG10)2-rTG1-IL2 (enzymatic PEGylation)

72

The rTG1 abbreviation represents the N-terminal fused Pro-Lys-Pro-Gln-Gln-Phe-Met sequence introduced as TGase subtrate. Adapted from Sato, H. Adv. Drug Delivery Rev. 2002, 54, 487–504. a The amount of activity was expressed as the per cent residual bioactivity as compared to the rhIL2.

in the required amino acid sequence neighboring the glutamine of the substrate. Several tailor-made linear PEGs, differing in molecular weight and type of alkylamine present at the polymer end, have been synthesized, the best reactivity being shown by polymers terminating with a -(CH2)6-NH2 group. IL2 contains six glutamines, but none of them is a suitable substrate for the more specific G-Tgase. The problem was overcome by preparing chimeric IL2 proteins that had at the N-terminus a peptide sequence that is a good G-Tgase substrate, like the Pro-Lys-Pro-GlnGln-Phe-Met (derived from Substance P140) or the Ala-Gln-Gln-Ile-Val-Met (derived from fibronectin141). In the former case, a mono-PEGylated conjugate was obtained while a mixture of mono- and di-PEGylated forms resulted in the latter case. The enzymatic coupling was carried out under mild conditions: 0.1 M Tris–HCl buffer, pH 7.5 at 25 1C for 12 h in the presence of CaCl2 10 mM.136 The derivatives maintained almost the same activity of the native protein, whereas the classical chemical conjugation with mPEG-NHS yielded products with decreased activity (Table 4). Using the less specific M-Tgase, mPEG12000-(CH2)6-NH2 could be directly incorporated into rhIL2 at the level of Gln-74.142 Compared to others site-specific chemical PEGylation, such as cysteine coupling or N-terminus modification at acidic pHs, the enzymatic method produces less undesired products, i.e., protein–protein dimers (due to cysteine oxidation) or eNH2 lysine PEGylation. A two-step enzymatic PEGylation, called GlycoPEGylation, was developed by Neose Technologies. In this case, E. coli-expressed proteins were glycosylated at the level of specific serine and threonine with N-acetylgalactosamine (GalNAc) by in vitro treatment with the recombinant O-GalNAc-transferase. The obtained glycosylated proteins were subsequently PEGylated using the O-GalNAc residue as the acceptor site of the cytidine monophosphate derivative of a sialic acid-PEG, a reaction selectively catalyzed by a sialyltransferase.143 The great advantage of this technology is the possibility of PEGylating proteins produced in E. coli in order to mimic the mammalian ones, since the PEG chains replace the native sugar moiety at the precise site of glycosylation, forming conjugates that retain the correct structure for receptor recognition and an extended plasma half-life. This method, however, is still awaiting large-scale application.

5.45.9

PEGylated Protein Purification and Characterization

Theoretically, in a conjugation reaction conducted with an excess of PEG, one could expect all of the reactive groups of the protein to be modified, thus yielding mainly a single product. However, in order to avoid loss of biological activity, a smaller amount of PEG is generally employed even if this gives rise to a mixture of positional isomers.144 Often the mixture can be fractionated by ionic exchange chromatography145 due to the suitable differences in isoelectric point of each isomer. Reverse-phase chromatography was found to be less efficient in this respect, and gel filtration separates only species with different masses. Once the isomers are separated it is necessary to identify the PEGylation site in the primary sequence. The usual approach involves enzymatic digestion of the conjugate, purification of the peptides, and their identification by mass spectroscopy or amino acid analysis. A good example is reported in the characterization of PEGylated interferon a-2a,146 where the comparison of the peptide fingerprint of the conjugated protein with that of the native protein allowed the region of PEGylation to be identified on the basis of the disappeared peptide signal. Besides the lengthy procedure, the conjugated polymer may interfere by steric hindrance with the proteolytic enzymes, resulting in an incomplete cleavage that complicates the interpretation of the peptide finger printing. An alternative procedure exploits the use of

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Drug–Polymer Conjugates

O

O PEG O C Met

X

X = Nle or βAla

OSu + H2N

protein

PEG O C Met

X HN protein

CNBr

X HN

protein

Figure 14 Use of PEG-Met-Nle-OSu or PEG-Met-bAla-OSu to introduce a reporter amino acid at the PEGylation site: PEG conjugation and release of PEG by CNBr. Nor-leucine (Nle) or b-alanine (bAla) can be identified on the protein by enzymatic digestion of the protein and mass spectrometry analysis of the obtained peptides.

PEG-Met-Nle-COOH or PEG-Met-bAla-COOH in the conjugation step. These possess a chemically labile bond in the peptide spacer at the level of methionine, which can be cleaved by treatment with CNBr (Figure 14) leaving only norleucine or b-alanine tags on the protein. The amino acid tagged peptides can be easily identified by standard sequence methods or by mass spectrometry analysis in the enzymatically digested mixture.147

5.45.10

Conclusion

In this chapter only a few drug conjugates out of the many described in the literature or patents have been discussed. The intention was to give examples of the potential of the method and how different problems were identified and solved. Polymer conjugation is often employed not only to improve the pharmacokinetic profiles of drugs, but also to give new properties to the modified drug, such as a higher solubility and stability, specific targeting, and for proteins removal or reduction of immunogenicity. Many different polymers have been used in this field, both from natural or synthetic sources. Multifunctional polymers are preferred for low-molecular-weight drugs, since higher loading can be achieved, while monofunctional polymers are the essential choice for proteins to avoid unwanted cross-linking. Among all polymers, PEG emerged as the most successful, and it has already led to several marketed products (Table 1). In fact, PEGylation, which was initially used to improve the therapeutic value of peptide and protein drugs, has now been extended to nonprotein compounds and, more recently, oligonucleotides. PEG has several useful properties that are finding application in other therapeutic approaches, such as the surface modification of liposomes (stealth liposome148), micro- and nanoparticles,149 and, last but not least, cells,150 making them biocompatible when implanted in vivo. At the basis of this technology lies an in-depth understanding of the chemistry of polymers, which despite the number of advances made over the years is still open to new advancements.

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Biographies

Francesco M Veronese has a degree in Pharmacy from the University of Padova, Italy. In 1962–1970 he was a researcher for the Italian National Council of Researches, 1971–1972 an Associated Research Professor at the Department of Biochemistry, University of California-Los Angeles, CA, 1973–1981 an Assistant Professor of ‘Pharmaceutical Industry,’ University of Ferrara, and of ‘Applied Biochemistry,’ University of Padova, Italy, and from 1982 Full Professor of ‘Applied Pharmaceutical Chemistry,’ School of Pharmacy, University of Padova. His past research activities have included: specific chemical modification of peptides and proteins for structure–activity relationships; induction, purification, sequence, enzymatic, and structural properties of glutamate dehydrogenases; structural and

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enzyme activity investigation of thermostable enzymes; drug–protein interaction: covalent and noncovalent linkage between furocoumarins and proteins; covalent modification by amphiphylic polymers of polypeptides and proteins to improve their therapeutic potential and their use in biocatalysis; entrapment of bioactive substances by nonbiodegradable matrices (acrylate polymers obtained by g-ray induced polymerization) or polyvinyl alcohol, and by biodegradable polymers (polyphosphazenes) for controlled release; and polymeric prodrugs with targeting properties. Currently, Prof Veronese is carrying out research in the following areas: covalent binding of polymers to low-molecularweight drugs, peptides, and proteins for improved delivery and increased therapeutic index; modification of enzymes with amphiphylic polymers for biocatalysis in organic solvents; and noncovalent entrapment of drugs by insoluble nonbiodegradable or biodegradable polymers for slow and controlled release of bioactive compounds. He was chairman of the PhD program in Pharmaceutical Sciences at the University of Padova 1983–2003; Director of the ‘Seminar of Pharmaceutical Sciences’ 1982–2000; co-organizer of the ‘Advanced School of Pharmaceutical Chemistry’ 1980–1992; co-organizer of the ‘National Seminars in Pharmaceutical Sciences’ 1980–1992; and Past President of the ‘Controlled Release Society,’ Italian Chapter, since 2004. Author of over 190 peer research papers, 15 reviews, 11 book chapters, and 19 US or European patents.

Gianfranco Pasut has a degree in Pharmaceutical Chemistry from the University of Padova, Italy. He spent some time as an exchange student at the University of Pennsylvania, PA before becoming a researcher at the School of Pharmacy, University of Padova, in 2006. Dr Pasut’s research activities have included: studies on doxorubicin conjugated to PEG and macromolecular carriers; modification of cis platinum and Ara-C with PEG; synthesis and characterization of dendronized PEGs as a high-loading carrier for low-molecular-weight drugs and development of a new labeling method with technetium of PEGylated chelating agent for diagnostic application. Currently, Dr Pasut is carrying out research in the following areas: modification of proteins with PEG; preparation of specific targeted macromolecular molecules for diagnostic and therapeutic purposes; and synthesis of PEG drugs releasing nitric oxide.

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Comprehensive Medicinal Chemistry II ISBN (set): 0-08-044513-6 ISBN (Volume 5) 0-08-044518-7; pp. 1043–1068