Release from polymeric prodrugs: Linkages and their degradation

MINIREVIEW Release from Polymeric Prodrugs: Linkages and Their Degradation AJIT JOSEPH M. D’SOUZA, ELIZABETH M. TOPP Department of Pharmaceutical Chemistry, The University of Kansas, 2095 Constant Ave., Lawrence, Kansas 66047

Received 21 November 2003; revised 12 March 2004; accepted 15 May 2004 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.20096

ABSTRACT: Polymeric prodrugs have evolved into a very useful class of drug delivery agents. Numerous polymeric prodrugs have been prepared for applications ranging from passive drug targeting to controlled release. The mechanistic aspects of the release processes, however, have not been clearly delineated. This review highlights the salient features of the chemical reactions that are responsible for drug release from these systems. The mechanisms of release from polymeric prodrugs employing various chemical linkages, esters, carbonates, carbamates, C – N linkage and amides, are discussed. ß 2004 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 93:1962–1979, 2004

Keywords: polymeric prodrugs; polymer–drug conjugate; passive drug targeting; controlled release; hydrolysis; intramolecular reaction

INTRODUCTION The use of polymers in medicine has evolved into a broad discipline referred to as ‘‘polymer therapeutics,’’1 and encompasses polymeric drugs,2–4 therapeutically active polymer–drug conjugates,5 polymeric prodrugs,6 polymeric micelles to which a drug is covalently bound7 and multicomponent polyplexes for DNA delivery. The versatility of modern synthetic chemistry has allowed pharmaceutical scientists to develop polymer-based therapeutic agents with carefully engineered characteristics such as prolonged plasma half life,5 targetability,8–11 controlled degradation of polymer–drug bonds,12 and enhanced cellular uptake.13 Excellent reviews have been prepared by Duncan1 and Maeda et al.14 on the clinical development in polymer therapeutics and by Veronese15 on bioconjugation in pharmaceutical Correspondence to: Ajit Joseph M. D’Souza (Telephone: 785-864-3010; Fax: 785-864-5875; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 93, 1962–1979 (2004) ß 2004 Wiley-Liss, Inc. and the American Pharmacists Association

1962

chemistry. The macromolecular components used in macromolecular prodrugs have been reviewed by Ouchi and Ohya.16 In polymer–drug conjugates, the stability of the drug–polymer linkage is crucial. Based on the stability of this bond, polymer conjugates may be classified into two broad categories: ‘‘permanent’’ conjugates, and prodrugs. In permanent polymer– drug conjugates, a half-life of cleavage of this linkage appreciably greater than the plasma half-life of the conjugate is desirable. In contrast, cleavage half-lives shorter than plasma halflives are desirable for polymer–drug conjugates intended as prodrugs. Generally, a prodrug is a biologically inactive derivative of a parent drug molecule designed to circumvent problems associated with the delivery of the parent drug. Prodrugs usually require the transformation of the prodrug to the drug within the body to elicit therapeutic action. Inactive polymer–drug conjugates intended as prodrugs are therefore also known as polymeric prodrugs. Although a few reviews have discussed polymeric prodrugs1,17,18 the mechanistic aspects of the

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 8, AUGUST 2004

RELEASE FROM POLYMERIC PRODRUGS

release processes have not been clearly delineated. The purpose of this review is not to enumerate the hundreds of polymeric prodrugs that have been investigated, but to highlight the salient features of the chemical reactions that are responsible for drug release from these systems. Because the desired use of these prodrugs can greatly affect the choice of the release mechanism, the uses of polymeric prodrugs are briefly discussed before delving into the mechanistic aspects of drug release. Examples of polymeric prodrugs and their mechanisms of release are then discussed according to the type of drug–polymer linkage employed. USES OF POLYMERIC PRODRUGS Traditionally, prodrugs have been used to enhance the solubility, improve transport properties, or enhance the stability of the drug molecule.19 With polymeric prodrugs there exist additional opportunities for extended and targeted drug delivery. Polymeric Prodrugs for Controlled Release Conjugation of a drug with a polymer leads to a macromolecular prodrug. If the molecular weight of this conjugate is such that its hydro˚ ,20 the renal threshold, dynamic radius is 45 A the elimination of the conjugate via glomerular filtration, is slowed considerably. The renal threshold molecular weight has been identified to be 45,000 Da for HPMA,21 30,000 Da for PEG,22 and 40,000 Da for dextran.23 Thus, by coupling a drug with a polymer, the half-life of the polymer–drug conjugate can be significantly enhanced.5 If the polymer–drug conjugate is retained in the body for a long period of time it can serve as a depot. The nature of the polymer–drug linkage and the stability of the drug conjugate linkage can then be controlled to influence the rate of drug release, and therefore, the effectiveness of the prodrug. As a general rule, if the conjugate is designed as a depot, the drug must be liberated according to the prescribed regimen without immediate total release of the drug (i.e., a burst effect) following administration. Polymeric prodrugs intended for controlled release under physiological conditions are designed to release the drug by using one of two mechanisms for cleavage of the polymer– drug bond: (1) enzymatic hydrolysis and (2) nonenzymatic hydrolysis. This category also includes polymeric prodrugs that are synthesized to circumvent the limited solubility of the drug or

1963

those that are intended for prolonged circulation but may be inactive when conjugated with the polymer. Polymeric Prodrugs for Targeted Delivery Targeting using polymeric prodrugs can be achieved by active and passive means. For active targeting, a ligand intended to bind a specific site is usually attached to the polymer along with the drug. For example, in a HPMA–doxorubicin– galactosamine intended for the treatment of liver cancer, HPMA (N-(2-Hydroxypropyl)methacrylamide) is the polymer, doxorubicin is the antitumor drug, and galactosamine is the targeting ligand that is intended to bind to the asialoglycoprotein receptor found on hepatocytes.24,25 Other targeting moieties like antibodies that bind to specific cell surface proteins have also been used for sitespecific delivery, e.g., immunoconjugates of HPMA copolymer—DOX targeted with either anti-EL4 antibody, polyclonal antithymocyte globulin (ATG), monoclonal anti-Thy 1.2 antibody or its F(ab0 )(2) fragment toward Thy 1.2 (CDw90) cell surface receptor expressed on neoplastic T cells.26 It is well established that large molecules that circulate for extended periods of time passively accumulate in tumors.27 The underlying physiological mechanism appears to be a combination of increased tumor vascular permeability and insufficient lymphatic drainage resulting in what is termed the ‘‘enhanced permeation and retention effect’’ (EPR effect). Because this is thought to be universal to all solid tumors, polymeric prodrugs have been developed to achieve site-specific delivery to cancerous solid tumors.8 This approach of passive targeting, although limited by the heterogeneity of the vasculature and irregular blood flow to the tumor, offers a window of opportunity for effective therapy.28 In polymeric prodrugs administered for tumor specific delivery, the drug–polymer bond must be sufficiently stable to maintain its chemical integrity under physiological conditions until the tumor is reached, where it should cleave rapidly. Because metastatic tumors express proteolytic enzymes including the thiol protease Cathepsin B,29,30 metalloproteinases such as collagenases and stromelysins,31 and serine proteases like plasminogen activator and plasmin32 on their cell surface, prodrugs intended for tumorotropic delivery are required to release the drug using proteolytic cleavage of the polymer–drug linker by these enzymes. Alternatively, acid activated cleavage JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 8, AUGUST 2004

1964

D’SOUZA AND TOPP

of the polymer–drug bond can also be used for tumorotropic delivery33 because a polymeric prodrug encounters a significant decrease in pH as it passes from extracellular fluid into the endosomes (pH 6.5–5.0) and lysosomes (pH
4.0).

RELEASE OF THE DRUG FROM THE POLYMER–DRUG CONJUGATE General Considerations Drugs and polymers can be conjugated using a variety of chemical linkages as reviewed by Veronese.15 The hydrolysis rate constants of these linkages depend on the chemical nature of the linkage, the structure of the polymer, and surrounding conditions. For example, the oximino bond is more susceptible to hydrolysis than an ester bond, which is more labile than the amide bond. As discussed below, the rate of hydrolysis of the linkage also depends on a variety of other factors such as its distance from the polymer backbone, the hydrophilicity of the surrounding groups and steric crowding around the center of reaction. Generally, direct linkages between the polymer backbone and the drug do not undergo hydrolysis under mild conditions. Hence, sometimes the drug is separated from the backbone with a spacer unit. Typically, a spacer is a bifunctional molecule that links the drug to the polymer. It is selected such that the hydrolysis of the spacer–polymer bond is rate limiting in the release of the drug from the polymeric prodrug. The cleavage of the spacer–polymer bond activates a trigger on the spacer that causes a rapid intramolecular reaction to release the drug from the spacer. An example of such a system employing the benzyl elimination system is shown in Scheme 1.34 Several other reactions have been reviewed by Shan et al.35 Thus, as will be seen subsequently, the spacer can control the rate of conversion of the prodrug to drug. Conjugates in which the drug is linked to the polymer using a spacer are referred to as tripartite, while those without a spacer are referred to as bipartite. Very often the choice of conjugating bonds is limited by the derivatizable functional groups present on the drug and the polymer. This limitation also can be overcome by introducing an appropriate spacer between the drug and the polymer. Thus, drugs can be conjugated to the polymer using a host of bonds, the choice of the appropriate bond always being dependent on the application. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 8, AUGUST 2004

Scheme 1. The benzyl elimination system.34

Carboxylic Esters (esters) Nonenzymatic Hydrolysis Because of their relative ease of hydrolysis at physiological pH compared to carbonates, carbamates, and amides, esters have been the linkages of choice when developing polymeric prodrugs for controlled release. Ester-linked polymeric prodrugs intended for controlled release have been developed as homogeneous (single phase) depots like soluble polymer–drug conjugates as well as heterogeneous depots like hydrogels36 and polymeric micelles.7 Enzymes usually cannot access the interior of heterogeneous systems; these polymer–drug conjugates depend primarily on aqueous hydrolysis for their release. Therefore, most of the initial literature focuses on nonenzymatic hydrolysis of the polymer–drug linkage. As expected of ester compounds, aqueous hydrolysis of ester-linked polymeric prodrugs is sensitive to the electron withdrawing and donating substituents close to the reaction center.37 In polymeric prodrugs, however, the electronics of the ester linkage are not the only factors that affect the rate of release. Several other factors have been identified, the most important of which is the hydration of the polymeric prodrug. It has been reported by several researchers that the rate of hydrolysis of esters is dependent on the extent of hydration. In fact, hydrophilic neighboring

RELEASE FROM POLYMERIC PRODRUGS

groups enhance hydrolysis,38–40 while hydrophobic groups can retard hydrolysis.40,41 These observations are consistent with the effect of dielectric constant on the rate of hydrolysis of ester. It can be speculated that hydrophilic neighboring groups increase the local dielectric constant and hence the rate of hydrolysis, whereas hydrophobic groups result in the opposite. Pitt and Shah have used this dependence of the hydrolytic rate on hydration to engineer zero-order release kinetics.42 They tested four hydrogel-forming copolymers with varying proportions of 2-p-nitrobenzoxyethyl methacrylate (NBEMA) and methoxydiethoxyethyl methacrylate (MDEEMA) for the release of p-nitrobenzoic acid. It was observed that the water content of the hydrogels decreased from 62 to 32% w/w as the proportion of NBEMA, the more hydrophobic component, increased from 5 to 20 mol %. At low NBEMA content the release followed first-order kinetics, while at higher concentration followed near zero-order kinetics. Thus, changing the ratio of the hydrophilic to hydrophobic pendant groups on the polymer can influence hydration of the polymeric prodrug, and the degree of hydration can be used to manipulate the rate of release from hydrogels. Building on this approach, Shah et al.43 have described another strategy to control the degree of polymer hydration, which involves the use of water-soluble polymers that exhibit a lower critical solution temperature (LCST).44,45 The polymeric prodrug is soluble below an ideal LCST but insoluble above the LCST; furthermore, as the drug is released by hydrolysis, the LCST of the polymer increases (Fig. 1) leading to solubiliza-

Figure 1. An illustration of the increase in lower critical solution temperature of the polymeric prodrug with progress in reaction.

1965

tion, and hence hydration. Thus, a polymeric prodrug that has a LCST of less than 378C is initially insoluble at 378C but is solubilized with progressing hydrolysis. This increased hydration leads to an increased reactivity, which when coupled with the decrease in hydrolysis rate due to decreased concentration of the ester results in near zero-order release kinetics. Rajewski et al. reported a similar observation.46 Li and Kwon observed that the rate of hydrolytic release of methotrexate was limited in stable micelles where the ester linkage was predominantly present in the hydrophobic core of the micelle. However, rapid release of methotrexate was seen from micelles that were unstable and the ester linkage was quite hydrated.47 The strategies cited above can be used for heterogeneous systems, but they cannot be used to manipulate release from soluble polymeric prodrugs. Pitt and Shah48 have outlined an approach that takes advantage of the dependence of secondary structure of polyelectrolytes on their degree of ionization. Polyacrylates exist as random coils when their side chains are esterified, but adopt a more extended conformation when converted to their ionized form upon hydrolysis of the ester.49 It was hypothesized that this change in conformation, characterized by an increase in the expansion coefficient of the polymer, relieves the steric hindrance to the hydrolysis of the pendant p-nitrophenyl acrylate as the hydrolysis proceeds. In accordance with this hypothesis, the expansion coefficient and the rate of hydrolysis increased as the reaction progressed. The increased reactivity is compensated for by the decrease in the rate because of the decreasing concentration of the p-nitrophenyl acrylate. The sum of these two counteracting processes is reflected in the near zero-order kinetics observed for the release of p-nitrophenol. Another factor influencing the rate of hydrolysis of ester linkages in polymeric prodrugs is steric hindrance as illustrated by Larsen et al.50 The t1/2 of release of naproxen from a dextran–naproxen conjugate increased from 4.0 h in conjugates with a glycolic acid spacer to 180 h in conjugates without a spacer. Tang et al. have described a similar effect for the release of norethindrone from norethindrone-poly-a,b-(hydroxyalkyl)-DL-aspartamide conjugates.51 The rate of release increased with the length of the alkyl spacer. Because the release rate from heterogeneous systems is influenced by both the hydrolysis rate of the polymer–drug linkage and diffusion rate of the JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 8, AUGUST 2004

1966

D’SOUZA AND TOPP

drug from the matrix, inclusion of both processes may be required in models of release kinetics. Kondo et al.52 have derived the equation for the release of 5-fluorouracil from a 1-(2-methacryloyloxy)ethyl carbamoyl-5-fluorouracil matrix, using a simple model of a heterogeneous system. Their results indicate that both the diffusion rate constant and the hydrolysis rate constant are necessary to describe the kinetics of drug release. Pitt and Schindler53,54 have derived the kinetic expressions for release of a drug from a drug–polymer conjugate in a solid polymer film using Fick’s second law. They have shown that nearly zeroorder kinetics can be achieved with certain combinations of the drug diffusion coefficient, the hydrolytic rate constant, and the film dimensions. Despite this ease of hydrolysis of esters, the rate of hydrolysis is often cited as the rate-limiting step during release.55 Therefore, several researchers have tried to engineer anchimeric assistance (neighboring group participation) to enhance the rate of release.56–59 Karmalkar et al. suggested modulation of the organization of the polymer structure to bring the catalytic groups in proximity to the polymer–drug conjugate.57 A charge transfer complex between N-vinyl imidazole and pnitrophenyl p-vinyl benzoate with 2-hydroxyethyl methacrylate was polymerized to yield an imidazole nitrogen close to the benzoate carbonyl. When evaluated for release, polymers with the imidazole group released faster than those without, suggesting base catalysis by the imidazole at physiological pH. Because the catalytic activity of imidazole is a function of pH, it was suggested that such hydrogelsˇould be exploited as pH responsive delivery systems. To enhance the applicability of this approach to polymers that do not form charge transfer complexes, Karmalkar et al. demonstrated the use of metal–ion coordination and molecular imprinting techniques to position a catalytic species close to the reaction center.58 In addition to the factors above, there is evidence to suggest that polymeric prodrugs having pendant drugs distributed uniformly along the backbone release faster than those that have them in clustered blocks.36 Although crosslinking can retard the rate of hydrolysis,60 hydrophilicity of the backbone is more influential in determining the hydrolysis rate than the extent of crosslinking.36 Thus, the release kinetics from polymeric prodrugs employing aqueous hydrolysis are complicated, and are a function of the reaction kinetics as well as the rate of diffusion of the drug out of the hydrogel. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 8, AUGUST 2004

Enzymatic Hydrolysis In recent years, considerable interest has been generated by soluble polymeric prodrugs. With soluble polymeric prodrugs, there is the potential for enzymatic catalysis of the hydrolytic reaction necessary to release the drug. Carboxyl esterases that catalyze the hydrolysis of esters to yield carboxylic acid and alcohol are distributed widely in vertebrate tissue and blood, and humans are rich in enzymes capable of hydrolyzing esters. The rate of enzymatic hydrolysis has been observed to be a function of steric crowding. Larsen et al. have studied the hydrolysis of benzoate esters of dextran in 80% human plasma and in 0.05 M phosphate buffer, pH 7.40. Similar rates of hydrolysis were observed in both media, suggesting a lack of enzymatic hydrolysis. This lack of enzymatic hydrolysis was attributed to steric hindrance by the dextran backbone.61,62 It has been proposed that a spacer separating the drug from the polymer is essential for enzymatic hydrolysis. Greenwald et al. have designed watersoluble PEG-based tripartite prodrugs whose rate of conversion to drug is a function of the ester linkage.63 They suggest that the rate of enzymatic hydrolysis of these esters is affected by steric crowding, with increased steric crowding decreasing the rate of hydrolysis. Thus, by varying the stereochemistry around the reaction center, the hydrolytic half-life in plasma can be manipulated to meet the drug release rate requirements. Some polymeric prodrugs illustrating the effect of the steric crowding on the enzymatic hydrolytic rate are listed in Table 1.63 There is also evidence suggesting that the hydrophilicity/hydrophobicity of the system is significant in determining the rate of hydrolysis.64 Although no quantitative models for the enzymatic hydrolysis of ester-linked polymeric prodrugs have been developed, the structure/rate relationship seems to follow the model proposed by Buchwald and Bodor for the hydrolysis of ester prodrugs of small molecular weight drugs.65–67 They have proposed the use of the inaccessible solid angle, ‘‘Oh,’’ as a measure of steric crowding. The inaccessible solid angle is the solid angle at which access to a reaction center is hindered by substituents and can be calculated as reported by Seeman et al.68 Briefly, for any atom a direction was considered hindered (Fig. 2) if a ray emitted from the center of the atom intersected with the van der Waal’s surface of another atom; and Oh

RELEASE FROM POLYMERIC PRODRUGS

1967

Table 1. Effect of Steric Crowding on the Rate of Enzymatic Hydrolysis of PEG-Based Prodrugs of Daunorubicin (DNR) in Rat Plasma63

values represent the percentage of hindered direction points. Oh ¼

Nhindered  100 Ntotal

In these studies it was observed that, of all the factors that are known to influence enzymatic hydrolysis, stereochemistry has the most influence on the rate. The half-life of the reaction increases with increasing steric hindrance. Typically, the rate of aqueous hydrolysis of esters correlates best

with the steric hindrance at the reaction center, the carbonyl carbon. Surprisingly, however, the reaction rates of enzymatic hydrolysis of esters correlated better with the steric hindrance at the sp2 carbonyl oxygen than that at the sp2 carbonyl carbon. This suggests that, unlike aqueous hydrolysis, the carbonyl oxygen plays a more important role in enzymatic hydrolysis than the carbonyl carbon. This is consistent with the proposed mechanism of hydrolysis that involves the stabilization of the oxyanion tetrahedral intermediate JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 8, AUGUST 2004

1968

D’SOUZA AND TOPP

the colon, however, dextranases of bacterial origin reduce the molecular weight of dextran,72 making the drug–dextran linkage more accessible, and hence susceptible to esterase cleavage. Other conjugates like poly(L-glutamic acid)-paclitaxel have also been prepared from biodegradable poly(L-glutamic acid). However, in this conjugate the biodegradability of the polymer does not seem to influence the release kinetics or its site.73 Figure 2. A representation illustrating the unhindered direction (A) and the hindered direction (B) with respect to an atom.

by the enzyme69,70 (Fig. 3). Besides steric crowding, the rate of hydrolysis was influenced by the oil/water partition coefficient of the prodrug. The alcohol and the acyl groups of the ester also influence the rate of enzymatic reaction but not as much as the stereochemistry. In the case of straight-chain alcohol substituents, butyl is observed to have the fastest rate of hydrolysis. Chains shorter and longer than butyl resulted in slower rates. Increased branching on the alcohol also resulted in slower reaction rates. Although the structure of the acyl portion of the ester did not correlate with the reaction rates, a chain length of three to four carbon atoms was deemed necessary for fast hydrolysis. Additionally, the polymer portions of polymeric prodrugs have been known to sterically hinder esterases. Researchers have taken advantage of this hindrance along with the biodegradability of dextran to achieve colon-specific delivery of dextran–NSAID conjugates after oral administration. It has been suggested that because of the high molecular weight of dextran, esterases in the upper part of GI tract cannot access and hydrolyze the drug–dextran ester linkage.71 Upon reaching

Figure 3. Stabilization of the tetrahedral oxyanion intermediate during enzymatic hydrolysis as proposed by Satoh et al.70 JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 8, AUGUST 2004

Carbonate Esters (carbonates) Carbonate esters in aqueous solutions are normally resistant to nonenzymatic hydrolysis at physiological pH. In analogous structures, they are known to hydrolyze nearly threefold slower than esters.74 But under heterogeneous conditions, carbonate linkages have been found to hydrolyze faster than esters.75 Some researchers have successfully employed the hydrolysis of the carbonate linkages to yield release from biodegradable hydrogels.76–78 In vivo, carbonates may also undergo enzymatic hydrolysis,36 and only a few reports exist on the enzymatic lability of cabonate linkages. The structure–reactivity relationship of carbonate esters is expected to be similar to that of carboxylic esters. Some researchers have reported facile hydrolysis of the carbonate bond in rat plasma (Table 2). Carbamate Esters (carbamates) Like carbonates, carbamates are not very susceptible to nonenzymatic hydrolysis at neutral pH, but are quite labile at acidic and basic extremes.79 Taking advantage of the increase in acidity associated with the degradation of PLGA microspheres, Yoo et al.80 demonstrated the release of water-soluble doxorubicin and doxorubicin– PLGA oligomers from nanoparticles of a waterinsoluble PLGA–doxorubicin conjugate linked by a carbamate linkage (Fig. 4). This approach may be suitable for carbonates and amides as well. Carbamate esters exhibit less lability to esterases than carbonates. Some esterases, however, can hydrolyze certain carbamate esters. Greenwald et al. have tested a few carbamatelinked PEG–doxorubicin conjugates (Table 3). Only the phenolic carbamate ester underwent enzymatic hydrolysis. This is consistent with the observations of Digenis and Swintosky, who examined the enzymatic stability of carbamate esters and suggested that only N-substituted or monosubstituted carbamates derived from phenols show high lability and strong enzymatic

RELEASE FROM POLYMERIC PRODRUGS

1969

Table 2. Enzymatic Hydrolysis of PEG-Based Prodrugs of Doxorubicin (DOX), Daunorubicin (DNR) and Amphotericin B (AMB) Linked by Carbonate Linkages, in Rat Plasma

The labile bond is marked in bold.

catalysis, whereas most N-disubstituted and aliphatic hydroxy carbamates are highly stable.81 C – N Linkages Of the different C – N linkages, the hydrazones are the most popularly used to conjugate polymers to drugs. Numerous prodrugs containing the hydrazone linkage have been synthesized for targeted delivery. The popularity of the hydrazone linkage stems from the fact that this bond is quite stable at physiological pH (7.4), but hydrolyzes at pH 5.0. The hydrazone linkage has been used

in polymeric conjugates of cytotoxic drugs intended for tumorotropic or lysosomotropic delivery. Examples of such soluble conjugates are N-(2-hydroxy-propyl)methacrylamide)-doxorubicin,82 PEG–doxorubicin,83 PEG–paclitaxel,84 polyglutamine–streptomycin,85 and dextran– streptomycin.85 Doxorubicin was also conjugated to micelle-forming diblock copolymers composed of poly(l-lactic acid) (PLLA) and methoxy-poly (ethylene glycol) (mPEG).86 Various macromolecular prodrugs using this linkage have been surveyed by Katz et al.33 Release from prodrugs employing the hydrazone linkage is represented JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 8, AUGUST 2004

1970

D’SOUZA AND TOPP

Figure 4. PLGA-Doxorubicin linked by a carbamate bond.

in Scheme 2. A hydrolytic t1/2 of 1–2 days was observed between pH 5.0 and 6.0, whereas at pH 7.5 no more than 20% of the hydrazone linkage was hydrolyzed over 30 days.84,86 Hydrazone linkage combined with peptide spacers led to much faster hydrolytic rates due to the substrate specificity of the peptides to lysosomal enzymes.82,87 No conclusions can be drawn regarding the influence of the structure of the nonpeptidyl linker on the hydrolytic rates of hydrazones. However, their sensitivity to acid hydrolysis can be explained as follows. The hydrolysis of C – N takes place in two steps (Scheme 3) and involves a tetrahedral carbinolamine intermediate. Typically, the hydrolysis rate constant initially increases with pH to a maximum, after which it decreases to become pH independent. Thus, in the case of imines, the pHrate profile is bell-shaped. At pH 7.5, the rate of hydrolysis is determined by the attack of hydroxide ion on the protonated imine.88 As the pH decreases to about 5.0, the rate-determining step changes from formation of the carbinolamine intermediate to its decomposition, with a concomitant increase in the rate of hydrolysis. The increase in the rate of hydrolysis of imines at acidic pH (
5) coupled with the low pH encountered by the polymeric prodrugs during uptake into cells has been exploited by researchers to engineer selective lysosomotropic delivery of drugs from polymeric prodrugs that are stable at pH 7.4.33 When the second of these steps is rate limiting, the choice of the leaving group can significantly influence the rate of hydrolysis of the polymer– drug linkage. Electron withdrawing groups attached to the sp2 nitrogen increase the rate of hydrolysis, whereas electron-donating groups are known to decrease the rate. Decreases in the pH JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 8, AUGUST 2004

from 7.4 to 5.0 have caused marked increases in the rate of hydrolysis of hydrazones. Because of this, hydrazone linkages have become popular in polymeric prodrugs intended for lyosomotropic delivery. The functional groups attached to the carbonyl sp2 carbon are known not to have a significant influence on the rate of hydrolysis unless they are involved in resonance stabilization altering the electronic environment around the imine.89 A more thorough treatment of the hydrolytic cleavage of C – N has been given by Bruylants and Feymants.88 PEG based polymeric prodrugs of doxorubicin containing the imine (PegC – NR), have been tested for controlled release by Hoffman et al., but the reaction was incomplete, with only 50% of the drug being released.90 Amides Amide bonds are very stable to aqueous hydrolysis at physiological pH, and to enzymatic hydrolysis by esterases. Therefore, the amide bond is often the bond of choice when linking the polymer to the drug to make permanent polymer drug conjugates. Although the kinetics and mechanism of nonenzymatic hydrolysis of amides have been studied thoroughly,91 they do not appear viable as linkages for polymeric prodrugs. However, some aromatic amides have been shown to be weak substrates for esterases. In these cases, hydrolysis of aromatic amides at reduced rates (relative to esters) is observed in the presence of esterases.92–94 Some researchers have used this specificity to design PEGylated prodrugs employing an acyl aromatic amide bond that is susceptible to esterases. Examples of this approach are shown in Table 4. Under these conditions steric crowding is expected to be rate-determining. Prodrugs using Peptidyl Spacers Besides aromatic amides, the amides of amino acids are known to be substrates for hydrolysis by peptidases and proteases. Because peptidases are expressed in significant amounts in metastatic cancers and are also present in normal lysosomes, investigators have designed tripartite polymeric prodrugs with peptidyl spacers such that the peptide spacer is stable in the circulation but can be cleaved by tumor proteases to release the drug. This cleavage has to occur at the amide bond linking the C-terminal amino acid to the amine group on the drug, as illustrated in Figure 5a. The works of Duncan et al. on HPMA–p-nitroaniline conjugates linked with peptidyl spacers

RELEASE FROM POLYMERIC PRODRUGS

1971

Table 3. Enzymatic Hydrolysis of PEG-Based Prodrugs of Daunorubicin (DNR) and 6-Mercaptopurine (MP) Linked by Carbamate Linkages in Rat Plasma17

The labile bond is marked in bold.

have suggested that, of all the lysosomal proteases the thiol protease cathepsin B was primarily responsible for the cleavage of the drug polymer linkage.95–97 Kopecek has reported a series of HPMA–p-nitroaniline conjugates connected by varying peptidyl linkers that were

Scheme 2. Release from a hydrazone-linked polymeric prodrug.

Scheme 3. Mechanism of hydrolysis of imines. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 8, AUGUST 2004

1972

D’SOUZA AND TOPP

Table 4. PEG-Based Prodrugs of Cytosine Arabinose Daunorubicin (DNR) Linked by Aromatic Amide Linkages

(ARA)

and

The labile bond is marked in bold.

evaluated for their substrate specificity to cathepsin B.98 It was observed that the structure hydrolysis rate relationship could be explained using the nomenclature developed by Schechter and Berger99 for the interaction of the subsite (Si) in an enzyme’s active site with the amino acid residue (Pi) of the substrate. Based on this information, custom synthesis of spacers with a controlled rate of degradation was undertaken by Duncan et al.100 The rate of release of the p-nitroaniline was found to be dependent on JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 8, AUGUST 2004

the specificity of the peptide spacer sequence to cathepsin B, with the fastest rates of hydrolysis being observed for the Gly-Phe-Leu-Gly tetrapeptide sequence.101 Further studies on this tetrapeptide linker have found it to be stable in plasma but cleavable by cathepsin B.102 Since then several researchers have reported the effect of steric hindrance due to the polymer,103,104 steric hindrance due to the drug,105 the chemical nature of the drug,106 and enhanced recognition of the drug by the enzyme active site107 on the rate of

RELEASE FROM POLYMERIC PRODRUGS

Figure 5. Desired cleavage site on the peptidyl spacer (a), cis-aconityl anhydride (b), phthalamic acid (c), and benzamide (d).

enzymatic hydrolysis of peptidyl spacers. Several polymeric prodrugs employing peptidyl spacers have been synthesized for daunomycin,108 puromycin,108 doxorubicin,106 melphalan,109 sarcolysin,109 N,N-di-(2-chloroethyl)-4-phenylene diamine (PDM),110 mitomycin,111 adriamycin,106 and fluorouracil.104 At times, however, such rational design based on the crystal structure of the protease active site is not successful,112 and iterative designs of protease substrate can yield unexpected results.113,114 Therefore, researchers have also adopted a combinatorial strategy to develop cathepsin substrates.115 Soyez et al. have reviewed the spacers that have been used in polymeric prodrugs intended for tumorotropic drug delivery.18 Marre et al. have confirmed the Gly-Phe-Leu-Gly tetrapeptide sequence to be the best substrate.116 Based on the work thus far some trends have been observed. Generally, a peptidyl spacer containing at least three residues is necessary to achieve enzymatic hydrolysis; the rate of hydrolysis increases with increasing spacer length. For drugs linked by the Gly-Phe-Leu-Gly spacer, the rate of drug release was observed to follow the order puromycin > mitomycin > adriamycin > daunomycin,18 indicating that the type of drug and the polymer attached to the spacer greatly influence the rate of cleavage.

1973

mide beads.117 The resulting conjugate released doxorubicin with a t1/2 of 3 h at pH 4.0 and 96 h at pH 7.5. This linkage was stable at physiological pH and could be cleaved easily at the acidic pH encountered in endosomes and lysosomes. Since then, several other polymeric prodrugs employing the cis-aconityl amide linkage like poly(aminopropyl)dextran-daunorobicin,118 alginate-daunomycin,119 chitosan–adriamycin,120 mPEG–PLLA–doxorubicin,86 and HPMA–doxorubicin82 have been synthesized. Release from a polymeric prodrug containing a cis-aconityl amide bond is represented in Scheme 4. Kratz et al. have surveyed the biological effects of conjugates employing the cis-aconityl amide linkage.33 The use of cis-aconityl anhydride (Fig. 5b), a precursor to cis-aconityl amide, as a linker was inspired by the mechanism of hydrolysis of the amide group in phthalamic acid121 (Fig. 5c) and maleamic acid.122 The amide hydrolysis of phthalamic acid under acidic conditions is 105 times faster than that of benzamide (Fig. 5d) at pH 3.121 Bender et al. have attributed this enhanced rate of hydrolysis to intramolecular catalysis by the neighboring carboxyl group (Scheme 5).121 The pH–rate constant profile for this reaction can be described by spontaneous hydrolysis of the unionized species or acid catalyzed hydrolysis of the deprotonated species using the equation:123 kobs ¼

k1 ½H þ  ½H þ  þ Ka

This equation can account for the hydrolysis over a pH of 4 to 8, indicating no change in the ratedetermining step. Therefore, from pH 4 to 8 the logarithmic hydrolysis rate constant is linearly dependent on the pH (slope of 1). This allows a 100-fold increase in the rate of hydrolysis from a pH of 7.5 to 5.5. Thus, cis-aconityl anhydride has

cis-Aconityl Amides Polymeric prodrugs linked by an amide bond have been used only to a limited extent due to the relative stability of amides in vivo. However, certain amides with neighboring nucleophilic groups like cis-aconityl amide that are sufficiently chemically labile have been used. This linkage was initially used by Shen and Ryser to conjugate doxorubicin to aminoethyl polyacryla-

Scheme 4. Release from a polymeric prodrug linked by cis-aconityl aminde. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 8, AUGUST 2004

1974

D’SOUZA AND TOPP

Scheme 5. Mechanism of hydrolysis of maleamic acid.

been used as a spacer to link polymers to drugs that are stable at pH 7.5 but can release the drug rapidly under acidic conditions (pH 5.0). In practice, polymeric conjugates using cis-aconityl amide linkage do not completely release the conjugated drug under acidic conditions. Dinand et al. have attributed this to isomerization about the double bond, leading to the formation of a trans isomer that is incapable of intramolecular catalysis.124 This supports the idea that the aqueous hydrolysis of amide bonds at a rate acceptable for controlled release requires assistance from nucleophilic species.125

SUMMARY Significant progress has been made in the field of polymeric prodrugs. Access to newer chemical synthetic techniques has allowed the synthesis of ‘‘designer’’ polymeric prodrugs for specific applications. Through an understanding of the mechanisms of release, researchers have successfully exploited polymer structure and chemistry, as well as physiological and pathophysiological conditions, to engineer polymeric prodrugs intended for different applications. Polymeric prodrugs have been synthesized for controlled release at pH 7.5 and for lysosomal release at pH 5. The release from polymeric prodrugs employing aqueous hydrolysis depends on a multitude of factors like hydration of the linkage, the nature of the leaving group and steric crowding around the linkage. Substrate specificity, hydrophilicity, and steric crowding primarily influence the release from enzyme susceptible linkages. The development of these polymeric prodrugs into products will require the tailoring of release rates to specific applications, and hence, a quantitative understanding of the mechanisms of release.

REFERENCES 1. Duncan R. 2003. The dawning era of polymer therapeutics. Nat Rev Drug Discov 2:347–360.

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 8, AUGUST 2004

2. Albertsson AC, Donaruma LG, Vogl O. 1985. Functional polymers. Part XXXVIII. Synthetic polymers as drugs. Ann NY Acad Sci 446:105– 115. 3. Donaruma LG, Ottenbrite RM, Vogl O. 1980. Polymeric drugs. Kobunshi 29:541–545. 4. Donaruma LG. 1975. Synthetic biologically active polymers. Prog Polym Sci 4:1–25. 5. Harris JM, Chess RB. 2003. Effect of pegylation on pharmaceuticals. Nat Rev Drug Discov 2:214– 221. 6. Coxon FY, Cassidy J. 2000. The place of polymer prodrugs in cancer therapy. Emerging Drugs 5: 457–463. 7. Yokoyama M, Miyauchi M, Yamada N, Okano T, Kataoka K, Inoue S. 1990. Polymer micelles as novel drug carriers: Adriamycin-conjugated polyethylene glycol-poly(aspartic acid) block copolymer. J Controlled Release 11:269–278. 8. Duncan R. 2002. Polymer–drug conjugates: Targeting cancer. In: Muzykantov V, Torchilin V, editors. Biomedical aspects of drug targeting. Norwell, MA: Kluwer Academic Publishers, pp 193–209. 9. Yokoyama M. 2002. Drug targeting with polymeric micelle drug carriers. In: Yui N, editor. Supramolecular design for biological applications. Boca Raton, FL: CRC Press LLC, pp 245–267. 10. Chilkoti A, Dreher MR, Meyer DE, Raucher D. 2002. Targeted drug delivery by thermally responsive polymers. Adv Drug Del Rev 54:613– 630. 11. Lu Z-R, Shiah J-G, Sakuma S, Kopeckova P, Kopecek J. 2002. Design of novel bioconjugates for targeted drug delivery. J Controlled Release 78: 165–173. 12. Ulbrich K, Pechar M, Strohalm J, Subr V, Rihova B. 1997. Synthesis of biodegradable polymers for controlled drug release. Anna NY Acad Sci 831: 47–56. 13. Murthy N, Campbell J, Fausto N, Hoffman AS, Stayton PS. 2003. Bioinspired pH-responsive polymers for the intracellular delivery of biomolecular drugs. Bioconjug Chem 14:412–419. 14. Maeda H, Kataoka K, Kabanov A, Okano T. 2003. Polymer drugs in the clinical stage: Advantages and prospects, vol. 519. New York: Kluwer Academic/Plenum, p 224. 15. Veronese FM, Morpurgo M. 1999. Bioconjugation in pharmaceutical chemistry. Farmaco 54:497–516. 16. Ouchi T, Ohya Y. 1995. Macromolecular prodrugs. Prog Polym Sci 20:211–257. 17. Greenwald RB, Choe YH, McGuire J, Conover CD. 2003. Effective drug delivery by PEGylated drug conjugates. Adv Drug Del Rev 55:217–250. 18. Soyez H, Schacht E, Vanderkerken S. 1996. The crucial role of spacer groups in macromolecular prodrug design. Adv Drug Del Rev 21:81–106.

RELEASE FROM POLYMERIC PRODRUGS

19. Bundgaard H. 1985. Design of prodrugs. Amsterdam, The Netherlands: Elsevier Science Publishers B.V., p 372. 20. Petrak K, Goddard P. 1989. Transport of macromolecules across the capillary walls. Adv Drug Del Rev 3:191–214. 21. Seymour LW, Duncan R, Strohalm J, Kopecek J. 1987. Effect of molecular weight (Mw) of N-(2hydroxypropyl)methacrylamide copolymers on body distribution and rate of excretion after subcutaneous, intraperitoneal, and intravenous administration to rats. J Biomed Mater Res 21: 1341–1358. 22. Yamaoka T, Tabata Y, Ikada Y. 1994. Distribution and tissue uptake of poly(ethylene glycol) with different molecular weights after intravenous administration to mice. J Pharm Sci 83:601–606. 23. Mehvar R, Shepard TL. 1992. Molecular-weightdependent pharmacokinetics of fluoresceinlabeled dextrans in rats. J Pharm Sci 81:908–912. 24. Julyan PJ, Seymour LW, Ferry DR, Daryani S, Boivin CM, Doran J, David M, Anderson D, Christodoulou C, Young AM, Hesslewood S, Kerr DJ. 1999. Preliminary clinical study of the distribution of HPMA copolymers bearing doxorubicin and galactosamine. J Controlled Release 57:281–290. 25. Pimm MV, Perkins AC, Duncan R, Ulbrich K. 1993. Targeting of N-(2-hydroxypropyl)methacrylamide copolymer-doxorubicin conjugate to the hepatocyte galactose-receptor in mice: Visualisation and quantification by gamma scintigraphy as a basis for clinical targeting studies. J Drug Target 1:125–131. 26. Rihova B, Jelinkova M, Strohalm J, Subr V, Plocova D, Hovorka O, Novak M, Plundrova D, Germano Y, Ulbrich K. 2000. Polymeric drugs based on conjugates of synthetic and natural macromolecules. II. Anti-cancer activity of antibody or (Fab0 )(2)-targeted conjugates and combined therapy with immunomodulators. J Controlled Release 64:241–261. 27. Fang J, Sawa T, Maeda H. 2003. Factors and mechanism of ‘‘EPR’’ effect and the enhanced antitumor effects of macromolecular drugs including SMANCS. Adv Exp Med Biol 519:29–49. 28. Jain RK. 1997. Delivery of molecular and cellular medicine to solid tumors. Adv Drug Del Rev 26:71–90. 29. Thomssen C, Schmitt M, Goretzki L, Oppelt P, Pache L, Dettmar P, Janicke F, Graeff H. 1995. Prognostic value of the cysteine proteases cathepsins B and cathepsin L in human breast cancer. Clin Cancer Res 1:741–746. 30. Mai J, Waisman DM, Sloane BF. 2000. Cell surface complex of cathepsin B/annexin II tetramer in malignant progression. Biochim Biophys Acta 1477:215–230.

1975

31. Davidson A, Drummond AH, Galloway WA, Whittaker M. 1997. The inhibition of matrix metalloproteinase enzymes. Chem Industry 258– 261. 32. Andreasen PA, Egelund R, Petersen HH. 2000. The plasminogen activation system in tumor growth, invasion, and metastasis. Cell Mol Life Sci 57:25–40. 33. Kratz F, Beyer U, Schutte MT. 1999. Drug– polymer conjugates containing acid-cleavable bonds. Crit Rev Ther Drug Carrier Syst 16:245– 288. 34. Greenwald RB, Pendri A, Conover CD, Zhao H, Choe YH, Martinez A, Shum K, Guan S. 1999. Drug delivery systems employing 1,4- or 1,6elimination: poly(ethylene glycol) prodrugs of amine-containing compounds. J Med Chem 42: 3657–3667. 35. Shan D, Nicolaou MG, Borchardt RT, Wang B. 1997. Prodrug strategies based on intramolecular cyclization reactions. J Pharm Sci 86:765–767. 36. Harris FW. 1984. Controlled release from polymers containing pendent bioactive substituents. In: Langer RS, Wise DL, editors. Med Appl Controlled Release, Vol. 1, p 103–128. 37. Heller J, Helwing RF, Baker RW, Tuttle ME. 1983. Controlled release of water-soluble macromolecules from bioerodible hydrogels. Biomaterials 4:262–266. 38. Kondo S, Murase K, Kuzuya M. 1994. Mechanochemical solid-state polymerization. VII. The nature of hydrolysis of novel polymeric prodrugs prepared by mechanochemical copolymerization. Chem Pharm Bull 42:2412–2417. 39. Pitt CG, Cha Y, Shah SS, Zhu KJ. 1992. Blends of PVA and PGLA: Control of the permeability and degradability of hydrogels by blending. J Controlled Release 19:189–199. 40. Harris FW, Arah CO. 1980. Synthesis and hydrolysis of hydrogels containing pendent herbicide substituents. Polym Prep 21:107–108. 41. Kondo S, Sasai Y, Kuzuya M, Furukawa S. 2002. Synthesis of water-soluble polymeric prodrugs possessing 4-methylcatechol derivatives by mechanochemical solid-state copolymerization and nature of drug release. Chem Pharm Bull 50:1434– 1438. 42. Pitt CG, Shah SS. 1995. Manipulation of the rate of hydrolysis of polymer-drug conugates: The degree of hydration. J Controlled Release 33:397– 403. 43. Shah SS, Wertheim J, Pitt CG. 1995. Polymer– drug conjugates: Dependence of the rate of hydrolysis on the LCST. Proc Int Symp Controlled Release Bioact Mater 22:34–35. 44. Feil H, Bae YH, Feijen J, Kim SW. 1993. Effect of comonomer hydrophilicity and ionization on the lower critical solution temperature of N-isopropy-

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 8, AUGUST 2004

1976

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

55.

56.

57.

D’SOUZA AND TOPP

lacrylamide co-polymers. Macromolecules 26: 2496–2500. Schild HG. 1992. Poly(N-isopropylacrylamide): experiment, theory and application. Prog Polym Sci 17:163–249. Rajewski LG, Topp EM, Phillips EM, Stella VJ. 2000. Mechanisms of drug release from hyaluronic acid esters. In: Abatangelo G, Weigel PH, editors. New frontiers in medical sciences: Redefining hyaluronan, 1st ed., vol. 1196. Amsterdam, The Netherlands: Elsevier, pp 149–161. Li Y, Kwon GS. 2000. Methotrexate esters of poly(ethylene oxide)-block-poly(2-hydroxyethylL-aspartamide). Part I: Effects of the level of methotrexate conjugation on the stability of micelles and on drug release. Pharm Res 17:607–611. Pitt CG, Shah SS. 1996. Manipulation of the rate of hydrolysis of polymer–drug conjugates: The secondary structure of the polymer. J Controlled Release 39:221–229. Bekturov EA, Bakauova ZK. 1986. Synthetic water-soluble polymers in solution. Heidelberg: Huthig & Wepf Verlag, pp 11–66. Larsen C. 1989. Macromolecular prodrugs. XII. Kinetics of release of naproxen from various polysaccharide ester prodrugs in neutral and alkaline solution. Int J Pharm 51:233–240. Tang G, Zhu K, Chen Q. 2002. Controlled delivery system for norethindrone based on biodegradable poly-alpha,beta-(hydroxyalkyl)-DL-aspartamide. Drug Del 9:215–222. Kondo S, Hosaka S, Hatakeyama I, Kuzuya M. 1998. Mechanochemical solid-state polymerization. IX. Theoretical analysis of rate of drug release from powdered polymeric prodrugs in a heterogeneous system. Chem Pharm Bull 46:1918– 1923. Pitt CG, Schindler A. 1996. The kinetics of drug cleavage and release from matrixes containing covalent polymer–drug conjugates. [Erratum to document cited in CA122:298833]. J Controlled Release 39:111. Pitt CG, Schindler A. 1995. The kinetics of drug cleavage and release from matrixes containing covalent polymer–drug conjugates. J Controlled Release 33:391–395. Yean L, Meslard JC, Subira F, Bunel C, Vairon JP. 1990. Reversible immobilization of drugs on a hydrogel matrix. 3. Hydrolysis of chloramphenicol precursors. Makromol Chem 191:1131–1142. D’Souza AJ, Schowen RL, Topp EM. 2004. Polyvinylpyrrolidone–drug conjugate: Synthesis and release mechanism. J Controlled Release 94:91– 100. Karmalkar RN, Kulkarni MG, Mashelkar RA. 1996. Pendent chain linked delivery systems: I facile hydrolysis through anchimeric effect. J Controlled Release 42:185–193.

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 8, AUGUST 2004

58. Karmalkar RN, Kulkarni MG, Mashelkar RA. 1997. Pendent chain linked delivery systems: II. Facile hydrolysis through molecular imprinting effects. J Controlled Release 43:235–243. 59. Scholsky KM, Fitch RM. 1986. Controlled release of pendant bioactive materials from acrylic polymer colloids. J Controlled Release 3:87–102. 60. McCormick CL, Fooladi M. 1977. Synthesis, characterization, and release mechanisms of polymers containing pendant herbicides. ACS Symp Series 53:112–125. 61. Larsen C, Johansen M. 1985. Macromolecular prodrugs. I. Kinetics and mechanism of hydrolysis of O-benzoyl dextran conjugates in aqueous buffer and in human plasma. Int J Pharm 27:205–218. 62. Larsen C, Johansen M. 1987. Macromolecular prodrugs. IV. Kinetics of hydrolysis of metronidazole monosuccinate dextran ester conjugates in aqueous solution and in plasma—Sequential release of metronidazole from the conjugates at physiological pH. Int J Pharm 35:39–45. 63. Greenwald RB, Choe YH, Conover CD, Shum K, Wu D, Royzen M. 2000. Drug delivery systems based on trimethyl lock lactonization: poly(ethylene glycol) prodrugs of amino-containing compounds. J Med Chem 43:475–487. 64. Wilkins RM. 1976. Design and evaluation of polymer combinations for the controlled release of herbicides. Proc Int Controlled Release Pestic Symp, pp 7.1–7.15. 65. Buchwald P, Bodor N. 2000. Structure-based estimation of enzymatic hydrolysis rates and its application in computer-aided retrometabolic drug design. Pharmazie 55:210–217. 66. Buchwald P. 2001. Structure–metabolism relationships: Steric effects and the enzymatic hydrolysis of carboxylic esters. Mini Rev Med Chem 1:101–111. 67. Buchwald P, Bodor N. 2002. Physicochemical aspects of the enzymatic hydrolysis of carboxylic esters. Pharmazie 57:87–93. 68. Seeman JI, Viers JW, Schug JC, Stovall JC. 1984. Correlation of nonadditive kinetic effects with molecular geometries. Structure and reactivity of alkyl- and cycloalkenylpyridines. J Am Chem Soc 106:143–151. 69. Cygler M, Schrag JD, Sussman JL, Harel M, Silman I, Gentry MK, Doctor BP. 1993. Relationship between sequence conservation and threedimensional structure in a large family of esterases, lipases, and related proteins. Protein Sci 2:366–382. 70. Satoh T, Hosokawa M. 1998. The mammalian carboxylesterases: From molecules to functions. Annu Rev Pharmacol Toxicol 38:257–288. 71. Larsen C, Harboe E, Johansen M, Olesen HP. 1989. Macromolecular prodrugs. XVI. Colontargeted delivery—Comparison of the rate of

RELEASE FROM POLYMERIC PRODRUGS

72.

73. 74.

75.

76.

77.

78.

79.

80.

81. 82.

83.

84.

release of naproxen from dextran ester prodrugs in homogenates of various segments of the pig gastrointestinal (GI) tract. Pharm Res 6:995–999. Nielsen LS, Weibel H, Johansen M, Larsen C. 1992. Macromolecular prodrugs. XX. Factors influencing model dextranase-mediated depolymerization of dextran derivatives in vitro. Acta Pharm Nord 4:23–30. Li C. 2002. Poly(L-glutamic acid)—Anticancer drug conjugates. Adv Drug Del Rev 54:695–713. Rattie ES, Shami EG, Dittert LW, Swintosky JV. 1970. Acetaminophen prodrugs. 3. Hydrolysis of carbonate and carboxylic acid esters in aqueous buffers. J Pharm Sci 59:1738–1741. Wang LF, Chiang HN, Chen WB. 2002. Synthesis and properties of a naproxen polymeric prodrug. J Pharm Pharmacol 54:1129–1135. Stubbe BG, Horkay F, Amsden B, Hennink WE, De Smedt SC, Demeester J. 2003. Tailoring the swelling pressure of degrading dextran hydroxyethyl methacrylate hydrogels. Biomacromolecules 4:691–695. van Dijk-Wolthuis WN, Tsang SKY, Hennink WE. 1996. Biodegradable dextran hydrogels. Proc Int Symp Controlled Release Bioact Mater 23:755– 756. van Dijk-Wolthuis WN, Hoogeboom JAM, van Steenbergen MJ, Tsang SKY, Hennink WE. 1997. Degradation and release behavior of dextranbased hydrogels. Macromolecules 30:4639–4645. Douglas KT, Williams A. 1976. A rational approach to the design of pesticides with optimal hydrolytic lability: Mechanistic analysis of carbamate ester reactivity. Proc Int Controlled Release Pestic Symp, pp 8.9–8.35. Yoo HS, Oh JE, Lee KH, Park TG. 1999. Biodegradable nanoparticles containing doxorubicin–PLGA conjugate for sustained release. Pharm Res 16:1114–1118. Digenis GA, Swintosky JV. 1975. Drug latentiation. Handbuch Exp Pharmakol 28:86–112. Ulbrich K, Etrych T, Chytil P, Jelinkova M, Rihova B. 2003. HPMA copolymers with pHcontrolled release of doxorubicin. In vitro cytotoxicity and in vivo antitumor activity. J Controlled Release 87:33–47. Rodrigues PCA, Beyer U, Schumacher P, Roth T, Fiebig HH, Unger C, Messori L, Orioli P, Paper DH, Mulhaupt R, Kratz F. 1999. Acid-sensitive polyethylene glycol conjugates of doxorubicin: Preparation, in vitro efficacy and intracellular distribution. Biol Med Chem 7:2517–2524. Rodrigues PCA, Scheuermann K, Stockmar C, Maier G, Fiebig HH, Unger C, Muelhaupt R, Kratz F. 2003. Synthesis and in vitro efficacy of acid-sensitive poly(ethylene glycol) paclitaxel conjugates. Bioorgan Med Chem Lett 13:355– 360.

1977

85. Coessens V, Schacht E, Domurado D. 1996. Synthesis of polyglutamine and dextran conjugates of streptomycin with an acid-sensitive drug-carrier linkage. J Controlled Release 38: 141–150. 86. Yoo HS, Lee EA, Park TG. 2002. Doxorubicinconjugated biodegradable polymeric micelles having acid-cleavable linkages. J Controlled Release 82:17–27. 87. Etrych T, Chytil P, Jelinkova M, Rihova B, Ulbrich K. 2002. Synthesis of HPMA copolymers containing doxorubicin bound via a hydrazone linkage. Effect of spacer on drug release and in vitro cytotoxicity. Macromol Biosci 2:43–52. 88. Bruylants A, Feytmans de Medicis E. 1970. Cleavage of the carbon-nitrogen double bond. In: Patai S, editor. Chemistry of the carbon–nitrogen double bond. New York: Wiley-Interscience, pp 465–504. 89. Bruyneel W, Charette JJ, Hoffmann ED. 1966. Kinetics of hydrolysis of hydroxy and methoxy derivatives of N-benzylidene-2-aminopropane. J Am Chem Soc 88:3808–3813. 90. Hoffman AS, Saito H, Harris JM. 1997. Drug delivery from biodegradable PEG hydrogels with Schiff base linkages. Proc Int Symp Controlled Release Bioact Mater 24:565–566. 91. Brown RS. 2000. Studies in amide hydrolysis: The acid, base, and water reactions. In: Greenberg A, Breneman CM, Liebman JF, editors. Amide linkage. New York: John Wiley & Sons, Inc., pp 85–114. 92. Krisch K. 1963. Isolation of an esterase from swine liver microsomes. Biochem Z 337:531–545 (cited in ref. 128). 93. Benohr HC, Krisch K. 1967. Carboxylesterases from beef liver microsomes. I. Isolation, properties and substrate specificity. Hoppe Seylers Z Physiol Chem 348:1102–1114 (cited in ref. 128). 94. Benohr HC, Krisch K. 1967. Carboxylesterases from beef liver microsomes, II. Dissociation and association, molecular weight, reaction with E 600. Hoppe Seylers Z Physiol Chem 348:1115– 1119 (cited in ref. 128). 95. Duncan R, Pratten MK, Cable HC, Ringsdorf H, Lloyd JB. 1981. Effect of molecular size of 125Ilabelled poly(vinylpyrrolidone) on its pinocytosis by rat visceral yolk sacs and rat peritoneal macrophages. Biochem J 196:49–55. 96. Duncan R, Lloyd JB, Kopecek J. 1980. Degradation of side chains of N-(2-hydroxypropyl) methacrylamide copolymers by lysosomal enzymes. Biochem Biophys Res Commun 94:284–290. 97. Duncan R, Cable HC, Lloyd JB, Rejmanova P, Kopecek J. 1982. Degradation of side-chains of N-(2-hydroxypropyl)methacrylamide copolymers by lysosomal thiol-proteinases. Biosci Rep 2: 1041–1046.

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 8, AUGUST 2004

1978

D’SOUZA AND TOPP

98. Kopecek J. 1984. Controlled biodegradability of polymers—A key to drug delivery systems. Biomaterials 5:19–25. 99. Schechter I, Berger A. 1967. On the size of the active site in proteases. I. Papain. Biochem Biophys Res Commun 27:157–162. 100. Duncan R, Cable HC, Lloyd JB, Rejmanova P, Kopecek J. 1983. Polymers containing enzymically degradable bonds. 7. Design of oligopeptide side-chains in poly[N-(2-hydroxypropyl)methacrylamide] copolymers to promote efficient degradation by lysosomal enzymes. Makromol Chem 184:1997–2008. 101. Rejmanova P, Kopecek J. 1983. Degradation of oligopeptide sequence in N-(2-hydroxypropyl)methacrylamide copolymer by bovine spleen cathepsin B. Makromol Chem 184:2009–2020. 102. Rejmanova P, Kopecek J, Duncan R, Lloyd JB. 1985. Stability in rat plasma and serum of lysosomally degradable oligopeptide sequences in N-(2-hydroxypropyl) methacrylamide copolymers. Biomaterials 6:45–48. 103. Lu ZR, Gao SQ, Kopeckova P, Kopecek J. 2001. Modification of cyclosporin A and conjugation of its derivative to HPMA copolymers. Bioconjug Chem 12:129–133. 104. Nichifor M, Schacht EH, Seymour LW. 1997. Polymeric prodrugs of 5-fluorouracil. J Controlled Release 48:165–178. 105. Subr V, Strohalm J, Hirano T, Ito Y, Ulbrich K. 1997. Poly[N-(2-hydroxypropyl)methacrylamide] conjugates of methotrexate—Synthesis and in vitro drug release. J Controlled Release 49:123–132. 106. Subr V, Strohalm J, Ulbrich K, Duncan R, Hume IC. 1992. Polymers containing enzymically degradable bonds, XII. Effect of spacer structure on the rate of release of daunomycin and adriamycin from poly[N-(2-hydroxypropyl)methacrylamide] copolymer drug carriers in vitro and antitumor activity measured in vivo. J Controlled Release 18:123–132. 107. Kasuya Y, Lu ZR, Kopeckova P, Tabibi SE, Kopecek J. 2002. Influence of the structure of drug moieties on the in vitro efficacy of HPMA copolymer–geldanamycin derivative conjugates. Pharm Res 19:115–123. 108. Duncan R, Kopeckova-Rejmanova P, Strohalm J, Hume I, Cable HC, Pohl J, Lloyd JB, Kopecek J. 1987. Anticancer agents coupled to N-(2-hydroxypropyl)methacrylamide copolymers. I. Evaluation of daunomycin and puromycin conjugates in vitro. Br J Cancer 55:165–174. 109. Duncan R, Hume IC, Yardley HJ, Flanagan PA, Ulbrich K, Subr V, Strohalm J. 1991. Macromolecular prodrugs for use in targeted cancer chemotherapy: Melphalan covalently coupled to N-(2-hydroxypropyl)methacrylamide copolymers. J Controlled Release 16:121–136.

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 8, AUGUST 2004

110. Soyez H, Seymour LW, Schacht E. 1999. Macromolecular derivatives of N,N-di-(2-chloroethyl)-4phenylene diamine mustard. 2. In vitro cytotoxicity and in vivo anticancer efficacy. J Controlled Release 57:187–196. 111. Seymour LW, Soyez H, De Marre A, Shoaibi MA, Schact EH. 1996. Polymeric prodrugs of mitomycin C designed for tumor tropism and sustained activation. Anti-Cancer Drug Design 11:351–365. 112. Majer P, Collins JR, Gulnik SV, Erickson JW. 1997. Structure-based subsite specificity mapping of human cathepsin D using statine-based inhibitors. Protein Sci 6:1458–1466. 113. Gron H, Breddam K. 1992. Interdependency of the binding subsites in subtilisin. Biochemistry 31:8967–8971. 114. Schellenberger V, Turck CW, Rutter WJ. 1994. Role of the S0 subsites in serine protease catalysis. Active-site mapping of rat chymotrypsin, rat trypsin, alpha-lytic protease, and cercarial protease from Schistosoma mansoni. Biochemistry 33:4251–4257. 115. Peterson JJ, Meares CF. 1998. Cathepsin substrates as cleavable peptide linkers in bioconjugates, selected from a fluorescence quench combinatorial library. Bioconjug Chem 9:618–626. 116. Marre AD, Seymour LW, Schacht E. 1994. Evaluation of the hydrolytic and enzymic stability of macromolecular Mitomycin C derivatives. J Controlled Release 31:89–97. 117. Shen WC, Ryser HJP. 1981. cis-Aconityl spacer between daunomycin and macromolecular carriers: A model of pH-sensitive linkage releasing drug from a lysosomotropic conjugate. Biochem Biophys Res Commun 102:1048–1054. 118. Mann JS, Huang JC, Keana JF. 1992. Molecular amplifiers: Synthesis and functionalization of a poly(aminopropyl)dextran bearing a uniquely reactive terminus for univalent attachment to biomolecules. Bioconjug Chem 3:154–159. 119. Al-Shamkhani A, Duncan R. 1995. Synthesis, controlled-release properties and antitumor activity of alginate—cis-aconityl-daunomycin conjugates. Int J Pharm 122:107–119. 120. Son YJ, Cho YW, Lee SC, Kwon IC, Chung H, Jeong SY, Park CR, Kim I-S, Suh SB. 2001. Synthesis of adriamycin-conjugated glycol chitosan and in situ self-association. Polym Prepr 42:129–130. 121. Bender ML, Chow Y-L, Chloupek F. 1958. Intramolecular catalysts of hydrolytic reactions. II. The hydrolysis of phthalamic acid. J Am Chem Soc 80:5380–5384. 122. Kirby AJ, Lancaster PW. 1972. Structure and efficiency in intramolecular and enzymic catalysis. Catalysis of amide hydrolysis by the carboxy group of substituted maleamic acids. J Chem Soc Perkin Transact 2 Phys Organic 1206–1214.

RELEASE FROM POLYMERIC PRODRUGS

123. Bundgaard H, Steffansen B. 1990. Hydrolysis and rearrangement of phthalamic acid derivatives and assessment of their potential as prodrug forms for amines. Acta Pharmaceut Nord 2:333– 342. 124. Dinand E, Zloh M, Brocchini S. 2002. Competitive reactions during amine addition to cis-aconityl anhydride. Au J Chem 55:467–474. 125. Fife TH. 1977. Intramolecular nucleophilic attack on esters and amides. Bioorg Chem 1:93–116. 126. Conover CD, Zhao H, Longley CB, Shum KL, Greenwald RB. 2003. Utility of poly(ethylene

1979

glycol) conjugation to create prodrugs of Amphotericin B. Bioconjug Chem 14:661–666. 127. Choe YH, Conover CD, Wu D, Royzen M, Greenwald RB. 2002. Anticancer drug delivery systems: N4-acyl poly(ethyleneglycol) prodrugs of ara-C. I. Efficacy in solid tumors. J Controlled Release 79:41–53. 128. Banarjee PK, Amidon GL. 1985. Design of prodrugs based on enzyme-substrate specificity. In: Bundgaard H, editor. Design of prodrugs. Amsterdam: Elsevier Science Publishers B.V. (Biomediacl Division), pp 93–133.

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 8, AUGUST 2004