Advanced Drug Delivery Reviews 54 (2002) 695–713 www.elsevier.com / locate / drugdeliv
Poly( L-glutamic acid)–anticancer drug conjugates Chun Li* Department of Experimental Diagnostic Imaging, Box 59, The University of Texas, M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030, USA
Abstract Chemotherapy has had limited success in the treatment of cancer over the years, due, in part, to the untoward toxicity of the therapeutic agent to normal cells. The design of tailor-made polymer conjugates provides a synthetic approach that can overcome some of the problems. Several synthetic polymer-based anticancer drug conjugates have entered clinical studies. This report reviews the chemistry, physicochemical properties, and therapeutic applications in cancer therapy of polymeric chemotherapeutic agents based on poly( L-glutamic acid). Targeted delivery of anticancer agents using poly( L-glutamic acid) as the drug carrier is also discussed with emphasis on the design of innovative polymeric constructs. 2002 Elsevier Science B.V. All rights reserved. Keywords: Poly( L-glutamic acid); Polymer–drug conjugates; Anticancer drugs; Enhanced permeability and retention effect; Drug targeting
Contents 1. Introduction ............................................................................................................................................................................ 2. Synthesis, characterization, and biological properties of poly( L-glutamic acid)............................................................................. 2.1. Synthesis and characterization........................................................................................................................................... 2.2. Biodegradability .............................................................................................................................................................. 2.3. Biodistribution and biocompatibility .................................................................................................................................. 3. Poly( L-glutamic acid)–drug conjugates ..................................................................................................................................... 3.1. Anthracyclines ................................................................................................................................................................. 3.2. Antimetabolites................................................................................................................................................................ 3.3. DNA-binding drugs.......................................................................................................................................................... 3.4. Paclitaxel......................................................................................................................................................................... 3.5. Camptothecin .................................................................................................................................................................. 4. Targeted delivery using PG as the carrier .................................................................................................................................. 5. Modulation of the EPR effect ................................................................................................................................................... 6. Novel poly( L-glutamic acid)-based polymers ............................................................................................................................. 7. Conclusions ............................................................................................................................................................................ References ..................................................................................................................................................................................
695 696 696 699 699 700 700 703 703 703 705 706 708 709 710 711
1. Introduction *Corresponding author. Tel.: 11-713-792-5182; fax: 11-713794-5456. E-mail address:
[email protected] (C. Li).
Chemotherapy for cancer, in particular for recurrent and metastasis disease, has had limited
0169-409X / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0169-409X( 02 )00045-5
696
C. Li / Advanced Drug Delivery Reviews 54 (2002) 695 – 713
therapeutic effect due mostly to dose-limiting toxicity. Limited aqueous solubility, in vivo instability, and nonselectivity of promising anticancer drug candidates have long been stumbling blocks in cancer drug development. In the past, much effort has been made to develop novel anticancer drug formulations that would ensure the injectability, stability, and safety of these drug candidates. In the mid-1970s, Ringsdorf proposed a polymer–drug conjugate model that could enhance the delivery of an anticancer drug to a tumor [1]. He envisioned that when an anticancer drug is conjugated to a polymeric carrier, its pharmacological properties could be manipulated by changing the physicochemical properties of the polymer. For example, an insoluble drug can be made water-soluble by introducing solubilizing moieties into the polymer. Likewise, active targeting is possible if a targeting moiety is introduced into the polymer. It was later recognized that polymer–drug conjugates tend to have enhanced uptake and persist longer in tumors than in normal tissues even in the absence of a targeting moiety. Coined by Maeda as ‘the enhanced permeability and retention effect’, or the EPR effect, the phenomenon is attributed to the greater permeability of disordered capillary endothelia in malignant tumors towards macromolecules than normal tissue and the lack of functional lymphatics in solid tumors [2–4]. This view is clearly supported by the electron microscopic observation that peripheral tumor vascular endothelium has quantitatively more fenestrations and open junctions than normal vessels [5]. In addition to the EPR effect, tumor cells show a higher degree of uptake of macromolecules by endocytosis than do normal cells, as a result of the enhanced metabolic activity of cancer cells. To date, several synthetic polymers have been successfully introduced into clinical practice, including polyethylene glycol (PEG), polystyrene–maleic anhydride copolymer (SMA), N-(2hydroxypropyl)–methacrylamide copolymer (HPMA), and poly(a,L-glutamic acid) (PG). Excellent reviews have documented the use of some of these polymers in cancer therapy [6–9]. In this review we discuss the applications of PG in the delivery of anticancer agents. [Note the distinction between PG and the more commonly used PGA, which is frequently used as an abbreviation for poly(glycolic acid).]
Unlike other synthetic polymers that have been tested in clinical studies, PG is unique in that it is composed of naturally occurring L-glutamic acid linked together through amide bonds rather than a nondegradable C–C backbone. The pendent free gcarboxyl group in each repeating unit of L-glutamic acid is negatively charged at a neutral pH, which renders the polymer water-soluble. The carboxyl groups also provide functionality for drug attachment. As will be seen later, PG is biodegradable and nontoxic. These features make PG a promising candidate as a carrier of polymer–drug conjugates for selective delivery of chemotherapeutic agents. The cellular uptake of negatively charged polymers can be hindered due to electrostatic repulsion forces between the polymers and the rather negatively charged surface of the cells [10]. Although PG is no exception to this rule, it does not diminish the EPR effect and the accumulation and retention of PG–drug conjugates in solid tumors (Sections 3 and 3.4). Specific receptor-mediated interactions of PG– drug conjugates containing targeting ligands may also increase the rate of polymer uptake into the target cells (Section 4).
2. Synthesis, characterization, and biological properties of poly( L-glutamic acid)
2.1. Synthesis and characterization PG is usually prepared from poly(g-benzyl-L-glutamate) (PBLG) by removing the benzyl protecting group with the use of hydrogen bromide (Fig. 1) [11]. Alkaline hydrolysis of poly( L-methyl glutamate) has also been used to prepare PG, but this method is less popular because of the racemization observed during alkaline hydrolysis [12]. Other esters, such as piperonyl, were also chosen as the protecting group for polyglutamate because it could be removed under gentler conditions using trifluoroacetic acid rather than hydrogen bromide [13]. A sequential copolymer of PBLG may be synthesized by peptide coupling reactions [14]. However, for the preparation of high-molecular-weight homopolymers and block or random copolymers of PBLG, triethylamine-initiated polymerization of the N-carboxyanhydride (NCA) of g-benzyl-L-glutamate is the
C. Li / Advanced Drug Delivery Reviews 54 (2002) 695 – 713
697
Fig. 1. Reaction scheme for the synthesis of poly(a,L-glutamic acid).
most frequently used method. Various solvents, including toluene, dioxane, chlorinated alkanes, and DMF, have been used as reaction medium. Two types of initiators have been identified in the ring-opening polymerization of the NCA of gbenzyl-L-glutamate, corresponding to two mechanisms of polymerization [15]. In the protic mechanism, initiators such as primary amines are acylated by attack on the 5-position of the NCA (Fig. 2A). In the aprotic mechanism, polymerization occurs with aprotic bases such as tertiary amines or alkoxides acting as a general base (Fig. 2B). In the absence of chain transfer and chain termination reactions, the number-average degree of polymerization [ pn ] is given by [ pn ] 5 [M] 0 / [I] 0 where [M] 0 and [I] 0 are the initial NCA and initiator concentrations, respectively. Furthermore, such a polymerization would provide a molecular weight distribution of the Poisson type with a polydispersity of pw /pn , 1.1, where pw is the weight-average degree of polymerization. In aprotic polymerization, the polydispersity is greater than 1.1 because of the diverse initiation and propagation steps involved. Due to the presence of bifunctional intermediates in aprotic polymerization, the final stages of polymerization are diffusion controlled, and it has been found that aging of the polymerization reaction leads to an enhancement of the molecular weight [16]. To achieve a high molecular weight, the removal of chloride ion and moisture becomes of prime importance, because any impurities reacting with either the
amine initiator or NCA at the end of the propagating chains will inhibit the polymerization to high molecular weight. The NCA of g-benzyl-L-glutamate is readily prepared in a single step from commercially available g-benzyl-L-glutamate with phosgene or trichloromethyl chloroformate [17,18]. In a typical procedure, the amino acid or its hydrochloride is suspended in an inert and dry solvent such as ethyl acetate or tetrahydrofuran (THF) and allowed to react heterogeneously with the cyclizing reagent in refluxing solvent. In our experience, the use of triphosgene yields excellent results with the advantage that the compound is a solid and can be handled conveniently in place of toxic gas. We have found that, when THF is used as the solvent, it is desirable to use THF distilled over sodium metal to obtain NCA with high purity. The synthetic route to PG from the amine-initiated ring-opening polymerization of NCA affords polymers with relatively broad distributions of molecular weights. Deming [19] recently reported the polymerization of NCAs by the zero-valent nickel catalyst bipyNi(COD), where bipy is 2,29-bipyridyl and COD is 1,5-cyclooctadiene. The number-average molecular weight of the PBLG formed using bipylNi(COD) was found to increase linearly as a function of the initial monomer-to-initiator ratio, indicating the absence of chain transfer and chain termination reactions. The polymers possessed a narrow molecular-weight distribution (Mw /Mn 5 1.05–1.15) and were obtained in excellent yields. Because of its ‘living’ feature, this polymerization method makes it possible to synthesize well-defined
698
C. Li / Advanced Drug Delivery Reviews 54 (2002) 695 – 713
Fig. 2. Mechanism of the polymerization of NCA glutamate.
block copolymers composed of PG and other amino acids. The potential application of these copolymers in chemotherapeutics remains to be investigated. Tirrell and colleagues [20,21] reported a general biosynthetic route to monodisperse derivatives of PG, in which artificial genes encoding the polymer are expressed in bacterial vectors. PG–drug conjugates derived from monodisperse PG are expected to be more reproducible and predictable with regard to their in vivo pharmacological behavior. However, because of the heterogeneity of the tumor vasculature and permeability, it may be advantageous to use polydisperse PG–drug conjugates to maximize the EPR effect. Recombinant DNA technology may be more suitable for producing fusion protein products linking PG of a defined size to therapeutic proteins such as interferon-a2 and granulocyte colony stimulating factor [22].
A related, but structurally different, polymer comprised of glutamic acid is poly(g-glutamic acid). In this polymer, L-glutamic acid monomers are linked via amide bonds between g-carboxyl and a-amino groups of adjacent monomers (Fig. 3). This naturally occurring, water-soluble polymer can be isolated from bacteria, particularly from different strains of Bacillus and from eucaryotes [23,24]. The polymer
Fig. 3. Structures of poly(g-glutamic acid) and poly(a-glutamic acid).
C. Li / Advanced Drug Delivery Reviews 54 (2002) 695 – 713
is not degraded by various proteases, but is cleaved to glutamic acid monomers under mild acid conditions, where poly(a-glutamic acid) remains mostly intact [24]. Perhaps because of its limited availability, little consideration has been given to poly(gglutamic acid) in the preparation of polymer–drug conjugates. The viscosity molecular weight, Mv , of PG can be determined by viscometry using the relationship [h ] 5 4.1 3 10 25 (Mv )0.94 [25]. The exponent a values in the Mark–Houwink equation, [h ] 5 KM va , gives an indication of the molecular shape of the polymer. Values of 0.5–0.8 are associated with coiled threads, and values of 1.7–2.0 are associated with stiff rods. Viscosity data with PG in a salt-free solution at pH 7.0 suggest that it still behaves as random coils, although with reduced conformational freedom [26]. With the introduction of gel permeation chromatography combined with on-line differential viscometer, differential refractometer, light scattering detectors, detailed polymer characteristics, including molecular weight, molecular weight distribution, intrinsic viscosity, Mark–Houwink constants, and branching information, can be conveniently acquired without the use of calibration [27].
2.2. Biodegradability PG shows a conformational change that passes from a rod-like form in the a-helix state to a more random coil structure with increasing pH at a midpoint of pH 5.5 [28]. At neutral pH, PG is expected to exist as a random coil [29]. It is therefore not surprising to observe a strong effect of pH on the rate of enzymatic degradation of PG [30,31]. Miller suggests that enzymatic attack of peptide bonds occurs in random coil regions of the polymer with adjacent side-chains uncharged [31]. Indeed, a high helix content (at pH ,5) and increasing charge density (at pH .5) decreased the degradation rate of PG by papain [32]. Using a copolymer of L-aspartic acid and L-glutamic acid as the substrate, Hayashi and Iwatsuki [33] found that the degradation rate was controlled by the composition as well as by the sequential distribution of comonomers in the copolymer chains. Conjugation of drug molecules to PG can affect the overall biodegradation of the PG polymer. For example,
699
introducing p-nitroanilide through tripeptide spacers to the side-chains of PG decreased the degradability of the polymer conjugates [34]. Depending on the nature of the bonds used to link drug molecules to PG, degradation of the polymer backbone may not necessarily always lead to the release of free drug. Digesting a PG–doxorubicin conjugate directly linked via amide bonds with papain resulted in the formation of oligomers of glutamic acid. No measurable free doxorubicin was detected in the incubation medium [35]. Several studies have used isolated tissue lysosomal enzymes to investigate the degradability of PG and its derivatives [34,36,37]. PG was found to be more susceptible to lysosomal degradation than poly( Laspartic acid) and poly( D-glutamic acid) [37]. Kopecek and colleagues concluded that both the incorporation of hydrophobic co-monomers and modification of the carboxylic groups of glutamic acid side-chains with hydroxyalkylamine increase the lysosomal degradability of the copolymers. They further show that the main degradation products of the nonionic PG derivative poly(2-hydroxyethyl Lglutamine) were tri- and tetrapeptides, although PG itself was not included in this study [34]. Recent results from Cell Therapeutics (Seattle, WA, USA) demonstrate that monomeric L-glutamic acid is formed in the lysosomal degradation of PG [38]. Evidence obtained from these studies suggests that cystein proteases, particularly cathepsins B, play key roles in the lysosomal degradation of PG [34,36,38].
2.3. Biodistribution and biocompatibility Detailed studies on the biodistribution and biocompatibility of PG and its copolymers and derivatives are scarce. Akamatsu et al. reported the biodistribution of PG using PG labeled with 111 InDTPA through a hydrazine linker [39]. At a molecular weight of about 11,000, the polymer was largely recovered in the kidney and urine, and hardly any accumulates in other tissues. More than 20% of the injected dose per milliliter of blood was circulating in the blood pool 60 min after injection of 111 Inlabeled PG. McCormick-Thomson and Duncan [40] reported the biodistribution of copolymers of glutamic acid with other hydrophobic amino acids
700
C. Li / Advanced Drug Delivery Reviews 54 (2002) 695 – 713
and found that their biodistribution patterns were markedly influenced by the composition of the copolymers. On the other hand, the introduction of a hydrophilic component to PG copolymers had less of an effect on their biodistribution behavior. When PG copolymers are sufficiently hydrophilic, they are eliminated primarily through the renal route with limited deposition in the cells of the reticuloendothelial system [41]. Based on these data, it is reasonable to speculate that the biodistribution of PG–drug conjugates would vary with the drugs used and the degree of modification of the carboxyl groups of PG. PG has been shown to be less effective in activation of the fibrinolytic system than other amino acids, as tested by the whole blood clot lysis time method [42]. A copolymer of L-glutamic acid with tyrosine showed neither general toxicity nor hepatotoxicity at a single intravenous dose of 100 mg / kg when the molar ratio of L-glutamic acid in the copolymer exceeded 4:1 [40]. PG is nontoxic in the quantities required of drug carriers. In various in vivo studies where PG was used as a control, PG has consistently been shown to be well tolerated at a single dose of up to 800 mg / kg or multiple doses at an accumulated dose of 1.8 g / kg [43,44]. PG itself is nonimmunogenic in man, rabbit, and guinea pig [45]. However, a weak immunogenic response can be elicited when PG is conjugated to a drug, the magnitude of which depends on the chemical structure of the conjugate [46].
3. Poly( L-glutamic acid)–drug conjugates The carboxyl groups on the side-chains of PG offer attachment points for the conjugation of chemotherapeutic agents to the polymeric carrier. Table 1 summarizes the various anticancer agents that have been conjugated to PG. The structures of these drugs are shown in Fig. 4. The points of attachment to PG are highlighted in the structures. Several drug conjugates have also been described using derivatives of PG such as poly(hydroxypropylglutamine) and poly(hydroxyethylglutamine) as carriers [47–50]. These conjugates are not within the scope of the current review.
3.1. Anthracyclines Doxorubicin (Dox) and other anthracyclines are perhaps one of the most extensively studied groups of drugs with regard to their conjugation to polymeric carriers. Dox has been conjugated to PG under the assumption that greater selectivity may be achieved if the conjugates only degrade and release Dox after they are endocytosed by tumor cells. Thus, Dox was conjugated to PG either directly or through oligopeptide spacers via amide bonds [51]. Conjugates with oligopeptide spacers readily yielded free Dox upon digestion with a protease such as papain, whereas the release of Dox was not observed in conjugates where Dox was directly coupled to PG [35]. The relative cytotoxic activities of PG– oligopeptide–Dox conjugates were consistent with the release of Dox or Dox-bound peptide from the conjugates. PG–Dox was practically non-cytotoxic [35]. This appears to be a general observation, as most PG–drug conjugates are less cytotoxic than their parent unconjugated drugs (Table 1). In vivo, the conjugate without oligopeptide spacer was completely inactive, whereas conjugates with degradable spacers were active. Anticancer activity increased with increasing oligopeptide length and degradation rate [46]. These results underscore the importance of introducing an enzymatically degradable spacer between PG and drug in this type of design. Dox and daunorubicin (Dau) have also been attached to PG via hydrolytically labile ester bonds [52] and hydrazone bonds [48] (Fig. 4). The ester linkage was formed by the reaction of 14-bromodaunorubicin with the carboxylic group of PG via a nucleophilic substitution reaction [52]. The resulting conjugates yielded a dose-dependent increase in life span, although the effects were less profound than for free Dox when the drugs were given in a single intravenous dose a day after intravenous transplantation of leukemia cells. Increased antitumor activity was observed when the molecular weight increased from 14,000 to 60,000 at an equivalent Dox dose of 30 mg / kg [52]. These data can be interpreted based on the renal clearance threshold and the longer circulation of the higher-molecular-weight conjugate, because glomerular filtration is highly dependent on the molecular weight of macromolecules injected intravenously. The hydrazone linkage was formed by
C. Li / Advanced Drug Delivery Reviews 54 (2002) 695 – 713
701
Table 1 Poly( L-glutamic acid)–drug conjugates synthesized and tested Drug
Drug action
Types of bonds
In vitro activity
In vivo activity
Ref.
Doxorubicin (Dox)
DNA intercalating
Amide, directly or through oligopeptide spacers
Conjugates are less cytotoxic than free drug in L1210 leukemia and B16 melanoma cells
PG–Dox is inactive, PG–oligopeptides –Dox are active. Activity increases with increasing degradability of spacer. Model: i.p. L1210 leukemia with i.p. injection of drug; end point: survival
[35,46]
Doxorubicin (Dox)
DNA intercalating
Ester
N/A
PG–COO–Dox is active. Activity increases with increasing molecular weight. Model: i.v. Gross leukemia with i.v. injection; end point: survival
[52]
Daunorubicin
DNA intercalating
Hydrazone
Conjugate is less cytotoxic than free drug in Yac leukemia cells
Conjugate is more active than free drug. Model: i.p. Yac lymphoma with i.v. injection; end point: survival
[48]
Ara-C
Antimetabolites
Amide, introduced directly at N-4 or through C-59 aminoalkyl-phosphoryl side-chain of Ara-C
Markedly less cytotoxic than free drug
Conjugate linked at N-4 is more active than Ara-C. Conjugate linked via aminoalkyl-phosphoryl side-chain is equally as active as Ara-C. Model: i.p. L1210 with i.p. injection; end point: survival
[47]
Uracil and uridine derivatives
Antimetabolites
Ester, through cyclic derivatives of uracil or uridine
N/A
N/A
[53]
Cyclophosphamide
Covalent DNA binding
Ester, through hydroxyhexylthio linker
N/A
N/A
[54]
Melphalan
Covalent DNA binding
Amide
Less active than free drug
Significantly more efficacious than free Melphalan. Model: s.c. Yoshida sarcoma in rats with s.c. injection of drug; end point: tumor growth delay
[55]
Mitomycin C (MMC)
Covalent DNA binding
Amide
Less cytotoxic than MMC
Less active than MMC. Model: i.p. P388 mouse leukemia with i.p. injection of drug; end point: survival
[56]
Paclitaxel (TXL)
Microtubule assembly
Ester
Less active than free drug
Significantly more efficacious than free paclitaxel in a variety of tumor models. Model: syngeneic tumors or xenografts inoculated s.c., i.p., or i.m. in rats or mice with i.v. injection of drug; end points: tumor growth delay, survival
[43,44]
Camptothecin (CPT)
Topoisomerase I inhibitor
Ester
Less active than free drug
Significant antitumor activity. Model: human lung H3222 tumor xenografts s.c., or intratracheally inoculated in mice with i.v. injection of drug; end points: tumor growth delay, survival
[64]
Camptothecin (CPT)
Topoisomerase I inhibitor
Ester, through glycin linker
N/A
Antitumor activity optimized when molecular weight of PG increased to 50 kDa, CPT loading increased to 37%, and attachment point is at the 20(S)-OH site. Model: s.c. murine B16 melanoma with i.p. injection of drug; end point: tumor growth delay
[65,66]
702
C. Li / Advanced Drug Delivery Reviews 54 (2002) 695 – 713
Fig. 4. Structures of the anticancer drugs that have been conjugated to PG. The attachment points on the drugs are highlighted.
C. Li / Advanced Drug Delivery Reviews 54 (2002) 695 – 713
condensing the methylketone in Dau with hydrazidederivatized PG [48]. The conjugate was active, although less potent than the free drug, as determined by a [ 3 H]uridine incorporation assay. When injected intravenously into mice bearing Yac lymphoma inoculated intraperitoneally, PG–hydrazone– Dau showed significant antitumor activity. Unfortunately, it is not possible to critically evaluate its antitumor activity and compare the results with that of free drug because the data were not obtained at equally toxic doses or at equal dose levels. A common drawback of in vivo studies performed with Dox or Dau conjugates is that the use of solid tumor models was not included in the evaluation process. The contribution of the EPR effect to the antitumor activity of these conjugates, particularly those with hydrolytically labile bonds between PG and drug, may be sufficiently significant to yield more impressive results.
3.2. Antimetabolites Kato et al. [47] prepared two types of conjugates of 1-b-D-arabinofuranosylcytosine (Ara-C) with PG via amide bonds: a conjugate in which Ara-C is directly linked at N-4 of Ara-C to the carboxyl groups of PG and a conjugate in which Ara-C is linked via the aminoalkylphosphoryl side-chain introduced at C-59 of Ara-C (Fig. 4). In vitro, these conjugates had markedly decreased cytotoxicity against murine leukemia L1210 cells when compared with that of free Ara-C. However, they exhibited antitumor activity that was greater than, or equal to, that of free Ara-C in mice bearing L1210 tumor inoculated intraperitoneally after a single intraperitoneal injection [47]. The authors attributed the observed in vivo activity to the slow cleavage of free Ara-C from the conjugates and protection of Ara-C from deactivation by cytidine deaminase. Interesting chemistry has also been developed to synthesize PG conjugates with derivatives of uracil and uradine [53]. The in vitro and in vivo activities of these conjugates were not evaluated further.
3.3. DNA-binding drugs Several DNA-binding drugs, including cyclophosphamide [54], L-phenylananine mustard (Melphalan)
703
[55], and mitomycin C (MMC) [56], have been conjugated to PG. Melphalan was conjugated to PG with the use of water-soluble carbodiimide [55]. Because the carboxyl group in melphalan was not protected, the yield was quite low (Fig. 4). The antitumor activity of PG–melphalan was demonstrated in rats bearing subcutaneously inoculated sarcoma. Using PG– 3 H-phenylananine (PG– 3 H-Phe) as the model compound, it was shown that the free drug could be released from the conjugate, and that PG– 3 H-Phe had a tendency to be absorbed through the lymphatic routes compared with free 3 H-Phe after subcutaneous administration [55]. MMC was also conjugated to PG through its aziridine amine using carbodiimide (Fig. 4) [56]. MMC was released from the conjugates via a non-enzymatic mechanism. The release rate, in vitro cytotoxicity, and in vivo antitumor activity of PG–MMC conjugates were affected by the extent of MMC substitution. Decreased activity was observed when the drug payload increased. Several conclusions can be made from studies of conjugates of various chemotherapeutic agents with PG. First of all, it is clear that in order for PG–drug conjugates to be active in vivo, the linkages between the drug and the polymer must be hydrolytically or enzymatically degradable. Secondly, the use of appropriate tumor models, e.g. ectopically or orthotopically inoculated solid tumors, is necessary in order to adequately evaluate the EPR effect of macromolecules on the antitumor activity of PG– drug conjugates. It is surprising that none of the above studies used such a model in which the conjugate is administered systemically as an intravenous injection. Finally, detailed studies on the pharmacokinetics and biodistribution of polymer– drug conjugates are needed in order to fully assess the potential of PG–drug conjugates in the treatment of solid tumors.
3.4. Paclitaxel Based on the above considerations, we synthesized a PG–paclitaxel conjugate in which the drug is attached to PG via ester bonds. Paclitaxel (Taxol, TXL) represents a class of antitumor agents that exert their action by promoting tubulin polymerization and microtubule assembly [43]. PG–TXL was
704
C. Li / Advanced Drug Delivery Reviews 54 (2002) 695 – 713
less potent than TXL, both in its ability to promote microtubule assembly and to rescue TXL-dependent CHO mutant cells in vitro. However, PG–TXL demonstrated significantly reduced systemic toxicity and remarkable antitumor efficacy, including complete regression of well-established solid tumors in vivo [43]. The maximum tolerated dose of TXL in rats and mice was 20 and 60 mg / kg, respectively. In contrast, the maximum tolerated dose of a single intravenous injection of PG–TXL in rats and mice was 60 and 160 mg eq. / kg, respectively, representing a three- and two-fold improvement in toxicity. To examine whether PG–TXL has a broad spectrum of antitumor activity, we evaluated the therapeutic activity of PG–TXL against four syngeneic murine tumors (breast MCa-4, breast MCa-35, hepatocarcinoma HCa-1, and sarcoma FSa-II) inoculated intramuscularly into C3Hf / Kam mice. In addition, the antitumor and anti-metastatic activities of PG– TXL were also studied using a human SKOV3ip1 ovarian tumor injected intraperitoneally into nude mice, and a human MDA-MB-435Lung2 breast tumor grown in the mammary fat pad of nude mice [44]. Treatments with PG–TXL showed significantly better antitumor activities than treatments with TXL in all tumor models examined. The observation that PG–TXL possesses antitumor activity superior to free TXL in preclinical animal studies suggests that PG–TXL might have favorable pharmacokinetic properties and / or it has a mechanism of action different from that of TXL. A series of studies were performed to compare the pharmacological actions of PG–TXL with that of free TXL. Morphological analysis and biochemical characterizations demonstrated that both drugs were able to induce apoptosis in cells expressing wild-type p53 or mutant p53, to arrest cells in the G 2 / M phase of the cell cycle, and to down-regulate HER-2 / neu expression [57]. Furthermore, when PG–TXL was compared with other water-soluble derivatives of TXL, including small-molecular-weight sodium pentetic acid–TXL and polyethylene glycol–TXL conjugate (molecular weight |5 kDa), all showed the same effects on telomeric association, mitotic index, chromatin condensation, and formation of apoptotic bodies [58]. These results indicate that PG–TXL has the same mechanisms of action as TXL. Is enhanced cellular uptake of PG–TXL a contri-
buting factor responsible for its improved antitumor activity? To investigate the release of free TXL from PG–TXL and its cellular uptake, we used three radioactive compounds, [ 3 H]PG–TXL (labeled at the PG polymer), PG–[ 3 H]TXL (labeled at TXL), and [ 3 H]TXL, for incubation with MDA-MB453 cells in culture. No significant amount of radioactivity was transported into the cells when [ 3 H]PG–TXL was used. In contrast, there was a time-dependent uptake of radioactivity by the cells when PG–[ 3 H]TXL was added to the cell culture, indicating that it was the released portion of the conjugate that had been taken up by the cells. When free [ 3 H]TXL was incubated with the cells under the same conditions, the free drug was rapidly transported into the cells, reaching maximum uptake within 30 min. Conversely, the cellular uptake of TXL from PG–[ 3 H]TXL was slower, but persisted for a longer period of time [57] (Li, unpublished data). Thus, the ability of PG–TXL to release the active drug extracellularly in the vicinity of tumor cells over a prolonged period of time appears to be an important feature for PG–TXL to exhibit its superior action. We believe that PG–TXL exerts its action through the EPR effect of macromolecules. PG–[ 3 H]TXL has a much longer half-life in plasma than [ 3 H]TXL. Whereas TXL has an extremely short half-life in the plasma of mice (t 1 / 2 5 29 min), the apparent half-life of PG–TXL is prolonged (t 1 / 2 5 317 min) [43]. Based on the area under the tissue concentration– time curve (AUC) values, tumor exposure to [ 3 H]TXL was five times greater when administered as PG–TXL than as TXL. Furthermore, concentrations of free TXL released from PG–[ 3 H]TXL remained relatively constant up to 6 days after dosing [59]. Thus, enhanced tumor uptake and slow, sustained release of TXL from PG–TXL in the tumor is one of the major factors contributing to its markedly improved in vivo antitumor activities. Polymer–drug conjugates are designed to possess relatively stable linkages between the polymeric carriers and the chemotherapeutic agents in order to achieve improved selectivity. After internalization, either by the nonspecific pinocytic pathway or receptor-mediated endocytosis, they are ultimately directed into the lysosomal compartment where the conjugates are degraded and the free drug is released. Several polymer–drug conjugates that have
C. Li / Advanced Drug Delivery Reviews 54 (2002) 695 – 713
reached the stage of clinical application, i.e. PK1 [8] and SMANCS [7], are thought to operate by this mode. However, this model requires that the conjugates be taken up by tumor cells efficiently. As shown in the case of PG–TXL, pinocytosis of macromolecules can be a slow and inefficient process when compared with the cellular uptake of low-molecular-weight, liposoluble free drugs. Alternatively, polymer–drug conjugates can be designed in such a way that the drugs may be gradually released into the extracellular fluid and in the vicinity of the tumor cells by virtue of hydrolysis or by specific enzymes present on the surface of the tumor cell membrane. The advantages of this approach include simplified conjugation chemistry, less variation in antitumor activity because drug release is no longer dependent on intratumoral enzyme activity [60], and improved antitumor efficacy. A prerequisite for this approach to work, however, is that the conjugates are relatively stable while in the blood circulation in order to minimize systemic toxicity [59]. In initial clinical trials conducted in the United Kingdom, PG–TXL (CT-2103) was given as a 30min infusion every 3 weeks. Patients have been entered at dose levels ranging from 30 to 720 mg / m 2 . CT-2103 was detectable in the plasma of all patients and had a long plasma half-life of up to 185 h, consistent with preclinical findings. Importantly, peak plasma concentrations of free TXL were less than 0.1 mM 24 h after CT-2103 administration at doses up to 480 mg / m 2 (176 mg / m 2 TXL equivalents) [61]. Based on these results, CT-2103 has subsequently entered two Phase I / II trials in the United States. In one study, CT-2103 was given as a single agent as a 10-min intravenous infusion every 3 weeks at an equivalent TXL dose of 175 mg / m 2 [62]. In the second study, CT-2103 (175 and 210 mg / m 2 ) was given in combination with cisplatin (75 mg / m 2 ) [63]. Clinical experiences so far have confirmed several advantages of CT-2103 in the management of cancer patients. CT-2103 is easier to administer than Taxol , since it can be delivered rapidly in 10 min rather than hours. Treatment with CT-2103 only results in infrequent and mild hypersensitivity reactions. Therefore, unlike Taxol therapy, no premedication is necessary. Furthermore, patients undergoing CT2103 therapy have a better
705
quality of life, because there is no significant hair loss, nerve damage, or neutropenia at the current dose. Finally, and most significantly, activity in patients who have failed prior chemotherapy, including Taxol treatment, is observed with CT-2103.
3.5. Camptothecin Camptothecin (CPT) and some of its analogs have shown a broad spectrum of antitumor activity against many solid tumors in xenografts. At the target level, CPT specifically inhibits the breakage / rejoining reaction of DNA topoisomerase I. Numerous studies have shown that prolonged contact between the drug and tumor cells is necessary in order to obtain an optimal therapeutic effect and that the presence of intact E-ring lactone is necessary for its biological activity (Fig. 4). We have conjugated CPT directly to PG at the C20(S)-position of CPT through ester bonds with the goal of obtaining a CPT formulation with reduced toxicity and improved efficacy [64]. PG–CPT delayed the growth of established human lung H322 tumors grown subcutaneously in nude mice when given intravenously every 4 days for four doses at an equivalent CPT dose of 40 mg / kg per dose. When H322 cells were inoculated intratracheally, the same treatment schedule prolonged the median survival of treated mice by four-fold compared with that of untreated control mice. Significantly, mice with intratracheally inoculated H322 tumors were resistant to both CPT and cisplatin treatments. These studies suggest that PG may be used as an effective solubilizing carrier for CPT and that PG protects the lactone structure in CPT from the rapid ring-opening process. Singer and colleagues have performed systematic studies examining the effect of linkers between CPT and PG, the point of attachment on the CPT molecule, polymer molecular weight, and drug loading on the antitumor activity of conjugates of CPT and PG [65–67]. They used B16 murine melanoma tumor inoculated subcutaneously to screen for PG–CPT conjugates with optimized properties for further clinical development. It has been found that coupling through the 20(S)-hydroxy group, with or without the use of a glycine linker, yielded the most active conjugates [65]. Because the 20(S)-hydroxyl group is located in close proximity to the lactone ring, it is
706
C. Li / Advanced Drug Delivery Reviews 54 (2002) 695 – 713
speculated that the E-ring lactone is better protected by PG chains in PG–CPT (20-OH conjugated) or PG–Gly–CPT (20-OH conjugated). When CPT is directly conjugated to PG, the maximum drug payload achievable is 15% due to steric hindrance. With a glycine linker, the loading could reach as high as 50% by weight [66]. Increasing the molecular weight of PG from 33 to 50 kDa resulted in enhanced antitumor activity, probably due to decreased renal clearance and increased plasma halflife. Increasing the loading of CPT also increased the antitumor efficacy without substantially altering the maximum tolerated dose. Based on these results, PG–Gly–CPT with a molecular weight of 49 kDa and 37% loading (w / w) was used for further testing [66]. Improved antitumor activity in nude mice with human colon and human lung carcinomas were seen with the optimized PG–Gly–CPT conjugate as compared with irinotecan (CPT-11) [67].
4. Targeted delivery using PG as the carrier The targeting efficiency of polymer–drug conjugates may be further enhanced when targeting moieties recognizing tumor cell surface receptors are introduced to facilitate cellular uptake of the conjugates that have already entered tumors by the EPR effect. Compared with direct coupling of drug to homing moieties, conjugation through polymer intermediates can substantially increase the drug payload without a significant detrimental effect on the binding activity of the targeting effect. Obviously, blood clearance of the conjugate must be minimized in comparison with the rate of extravasation at the target sites in order to achieve efficient extravasation and a meaningful targeting effect. Due to the substantial increase in the molecular weight of the targeted conjugates containing antibody, the EPR effect may be increased when antibody is introduced to polymer–drug conjugates. To ascertain that the antitumor activity of immunoconjugates observed in vivo is a direct result of enhanced binding rather than an increased EPR effect, control substances such as conjugates containing nonspecific antibody should also be tested under the same experimental conditions. Unfortunately, many reported studies failed
to include this type of control in their animal experiments. Rowland et al. [68] were the first to propose the use of PG as an intermediate polymer carrier. The polymer was loaded with p-phenylenediamine mustard and the conjugate was then linked to an immunoglobin (Ig) against mouse lymphoma EL4 cells through a ‘concentration-dilution’ technique to minimize antibody cross-linking (Table 2). The two coupling reactions for the attachment of antibody and drugs were both effected by water-soluble carbodiimide with the formation of stable amide bonds [68]. About 66% of the anti-EL4 activity was retained in the conjugate. The conjugate induced a marked increase in survival of mice bearing intraperitoneally inoculated EL4 lymphoma compared with PG–mustard or PG–mustard plus Ig controls after daily intraperitoneal injection (days 1–4). Unfortunately, the contributions of variables such as distribution, clearance, and biological barriers were inherently omitted from this model. Other linkages between PG and antibodies were also explored. In one study, MMC was linked to the carboxyl groups on PG through aziridinyl amide bonds. Protected disulfide groups were simultaneously introduced into the side-chains of PG. The PG– MMC conjugate was then attached to mAbs through thioether bonds using thio groups on PG and maleimide-derivatized mAbs [69]. High molar ratios of drug per antibody (up to 1:48) were achieved. The rate of release of MMC from the conjugates was significantly higher in serum media than in PBS, suggesting that the aziridinyl amide bonds are susceptible to enzymatic hydrolysis. The cytotoxicity of the immunoconjugates directly correlated with the binding of the immunoconjugates to target cells. Antibody-mediated cell killing was demonstrated when conjugates were incubated with target cells for short periods of time (1–2 h) and only with mAbs that internalized from the cell surface. A longer exposure time (24 h) of target cells to the noninternalizing immunoconjugates leads to nonspecific cytotoxicity [69]. The results of this study underline the importance of cellular binding and internalization of immunoconjugates to achieve selective cytotoxicity. It would be interesting to compare the antitumor activity of these conjugates in relevant tumor models.
C. Li / Advanced Drug Delivery Reviews 54 (2002) 695 – 713
707
Table 2 Targeted poly( L-glutamic acid)–drug conjugates Drug
Types of bonds between drug and PG
Types of bonds between targeting device and PG
In vitro activity
In vivo activity
Ref.
p-Phenylenediamine mustard
Amide
Side-chain amide
N /A
Immunoconjugates more active than PG–drug conjugate used alone or in combination with the antibody. Model: i.p. EL4 lymphoma with i.p. injection
[68]
Mitomycin C
Azaridinyl amide, hydrolyzable
Side-chain thioether
Antibody-mediated cytotoxicity observed when incubated with target cells for short periods
N /A
[69]
Doxorubicin
Hydrazone
Side-chain hydrazone
Immunoconjugate was less active than free Dox, equally active as plain PG–Dox
Dox–PG–anti-AFP was more active than PG–Dox up to day 17. Model: s.c. hepatoma with i.v. injection
[71]
Chlorine e 6 photosensitizer
Amide
Side-chain hydrazone, further reduced by NaCNBH 3 to C–N
Strong phototoxicity against ovarian cancer ex vivo; no activity against non-ovarian cancers
N /A
[72]
Daunomycin
Amide
Terminal thioether
More cytotoxic than free drug and PG–Dau
Immunoconjugates more active than PG–Dau or free Dau. Model: i.p. AH66 hepatoma in rats with i.p. injection of drugs
[73,74]
Doxorubicin
Amide
Terminal thioether via PEG spacer
Enhanced cellular uptake. More cytotoxic than PEG–PG–Dox against target cells when exposed for a short period (6 h)
N /A
[76]
In the above studies, antibodies were linked to PG through the lysine amino groups, which are distributed along the whole antibody molecule. PG could potentially block the binding site on the antibody, leading to the loss of the antibody’s binding affinity. A popular method to conjugate antibody to PG in a more controlled fashion is to introduce hydrazide functional groups to the side-chains of PG, which, in turn, react with aldehyde groups introduced in the sugar moiety of the antibodies by periodic acid oxidization to form a hydrazone linkage [48,70–72]. Galun et al. [71] introduced adipic dihydrazide first to PG side-chains, followed by condensation of PG– hydrazide with Dox via its ketone and with an oxidized anti-a-fetoprotein monoclonal antibody (anti-AFP). The resultant conjugate maintained the full binding activity of native mAb in vitro. However, about 10–15% of the conjugate was high-molecularweight aggregates. Furthermore, the cytotoxic activi-
ty of the immunoconjugates determined by [ 3 H]thymidine and [ 3 H]-leucine incorporation assays did not show significant enhancement compared with plain PG–Dox conjugate. In vivo, effective targeting of the immunoconjugates was demonstrated only in the initial treatment period. No advantage could be demonstrated over the use of Dox–PG–anti-AFP 17 days after the initial treatment. These results were explained on the basis of a reduction in the number of cell-surface-associated AFP receptors in the later stage of the study [71]. The hydrazone formed between PG and mAb can be further reduced to a stable C–N bond [72]. This method was used to conjugate the murine mAb OC125 recognizing the antigen CA125 expressed in human ovarian carcinomas to hydrazine-derivatized PG. The formation of aggregates due to cross-linking and some loss of affinity for the immunoconjugates, possibly reflecting the effects of polymer folding on
708
C. Li / Advanced Drug Delivery Reviews 54 (2002) 695 – 713
the mAb conformation, were seen in these immunoconjugates. When a photosensitizer, chlorine e 6 , was conjugated to PG–OC125, the resulting conjugate exhibited high cytotoxicity against a number of human ovarian carcinoma cells obtained from patients, whereas little cytotoxicity against nonovarian cells was observed. Because ovarian cancer in the majority of patients is confined to the peritoneal cavity, it is proposed that phototherapy combined with intraperitoneal injection of targeted PG–chlorine e 6 conjugates may be a promising approach for the treatment of ovarian cancer. Various side reactions, including coupling among antibody molecules and cross-linking, are possible when mAb are coupled to the side-chains of PG. To overcome these limitations, an anti-a-fetoprotein antibody to PG–daunomycin through a single terminal thio group located at the chain end [73,74], PG–Dau, was covalently linked to the antibody through a stable thioether bond formed between its chain end –SH group and the maleimide groups introduced to the antibody. The chain end –SH group was introduced by polymerization of the NCA of g-benzyl glutamate using a cystamine initiator. The resulting conjugate retained most of the antigenbinding activity of the parent antibody and was more cytotoxic than unconjugated daunomycin and the PG–Dau conjugate. In vivo studies demonstrated that the conjugates were significantly more efficacious in prolonging the lives of rats with AH66 hepatoma expressing a-fetoprotein than free daunomycin, antibody or a mixture of daunomycin and antibody [74]. To improve the yield and purification scheme, Palacios et al. [75] developed a solidphase synthetic method to introduce –SH at the end of the PG chain. We have recently synthesized a PEG–PG block copolymer, which contains a vinyl sulfone (VS) group at the end of the PEG block of the copolymer [76]. Dox was conjugated to VS-PEG–PG via amide bonds. The reaction of sulfhydryl-containing C225, a mAb directed against epidermal growth factor receptor (EGFR), with VS-PEG–PG–Dox yielded C225–PEG–PG–Dox. Immunoprecipitation and Western blot analysis confirmed the binding of C225–PEG–PG–Dox to EGFR. Confocal fluorescent microscopic studies showed that C225–PEG– PG–Dox, but not PEG–PG–Dox, selectively bound
to human vulvar squamous carcinoma A431 cells overexpressing EGFR. Receptor-mediated uptake of C225–PEG–PG–Dox occurred rapidly (within 5 min), whereas nonspecific uptake of PEG–PG–Dox through adsorptive endocytosis required an extended period of time to be internalized. C225–PEG–PG– Dox was 10-fold more potent than PEG–PG–Dox in inhibiting the growth of A431 cells when the cells were exposed to the drugs for 6 h, followed by washing and an additional 72 h of incubation. These results suggest that conjugation of a receptor-homing ligand to the end of a PG chain through a PEG linker may enhance the targeted delivery of anticancer agents. Further study of the pharmacokinetics and in vivo antitumor activity will be needed to fully assess the advantages of this system.
5. Modulation of the EPR effect In the treatment of many solid tumors, combining chemotherapy and radiotherapy has significantly improved response and survival rates. Many chemotherapeutic agents are able to increase the sensitivity of the tumor to radiation, therefore potentiating the tumor response to radiation-caused damage. Due to the EPR effect of polymer–drug conjugates, drug molecules are released over a prolonged period of time from the polymeric conjugates at the tumor sites. We hypothesize that a stronger radiosensitizing effect may be achieved when polymer–drug conjugates are used in combination with tumor irradiation than when free drugs are used. We further postulate that tumor irradiation can in turn potentiate the tumor response to polymer–drug conjugates by increasing tumor vascular permeability and thus their uptake into solid tumors. To test these hypotheses, we used PG–TXL as a model polymer–drug conjugate. Delays in the growth of syngeneic murine ovarian OCa-1 tumors grown intramuscularly in C3Hf / Kam mice were used as the treatment end point. PG–TXL given 24 h before tumor irradiation increased the efficacy of tumor radiation by a factor of more than 4. PG–TXL not only produced a much stronger radiopotentiating effect than TXL, the kinetics of its radiopotentiating effect were also different from that of TXL [77]. When the treatment end point was tumor cure, enhancement factors of 8.4 and 7.2 were
C. Li / Advanced Drug Delivery Reviews 54 (2002) 695 – 713
documented after fractionated and single dose radiation, respectively [78]. These values, to our knowledge, are greater than those produced by other taxanes or by any other chemotherapeutic drugs or radiosensitizer tested so far. Our results support the idea that the increased tumor uptake of PG–TXL and the sustained release of TXL in the tumor can enhance the radiopotentiation activity of TXL. To determine whether prior irradiation affects tumor uptake of PG–TXL, [ 3 H]PG–TXL was injected into mice with OCa-1 tumors 24 h after 15 Gy local irradiation. The uptake of [ 3 H]PG–TXL in irradiated tumors was 28–38% higher than that in nonirradiated tumors at different times after [ 3 H]PG– TXL injection, indicating that tumor irradiation increased accumulation of PG–TXL in the tumors. The enhancement factors, as measured by incremental tumor growth delay compared with PG–TXL alone, ranged from 1.36 to 4.44, the values depending on the doses of PG–TXL and the radiation delivered. Complete tumor regression was observed at higher radiation doses (.10 Gy) and higher PG– TXL doses (.80 mg eq. TXL / kg) [79]. A similar observation was made in a mammary MCa-4 carcinoma model [80]. Interestingly, combined radiotherapy and TXL yielded an enhancement factor of less than 1.0 in MCa-4 tumors, indicating that only additive or even subadditive interactions were produced when irradiation preceded TXL treatment [80]. Thus, conjugation of TXL to PG is necessary for an improved response and the supra-additive effect of combined radiation and PG–TXL therapy is in part due to modulation of the enhanced permeability and retention effect of macromolecules by radiation.
6. Novel poly( L-glutamic acid)-based polymers The performance of PG may be further improved by modifying the architectural structure and composition of the polymer while maintaining its useful characters, i.e. water solubility, carboxyl functionality, biocompatibility, and biodegradability. To reduce the nonspecific uptake of PG–drug conjugates by the cells of the reticuloendothelial system and prolong their blood circulation time, other polymers, such as PEG, can be introduced to PG either in the
709
form of block or graft copolymers. We have prepared a diblock copolymer of PEG and PG that is capable of further modification with a homing moiety at the terminus of the PEG block of the copolymer [76]. Pechar et al. [81] prepared a PEG multiblock copolymer containing Glu–Lys–Glu diamine tripeptide in its backbone via interfacial polymerization. The polymer had a weight-average molecular weight of about 50,000 and a very broad molecular weight distribution. Dox was conjugated to the carboxyl groups of the polymer via an enzymatically degradable Gly–Phe–Leu–Gly spacer. In a preliminary animal study, significantly greater antitumor activity and lower systemic toxicity were observed with the block copolymer–Dox conjugate than with free Dox? HCl after intravenous injection into mice bearing subcutaneously inoculated colorectal C26 tumors [81]. One potential caveat of a targeted drug delivery system is that receptor density, ligand-binding affinity, and nonspecific cellular uptake can limit the accumulation of drugs at the target sites. Coupling multiple ligands to the surface of polymeric carriers can potentially lead to significant enhancement in binding affinity through a multivalent cluster interaction. To develop polymeric drug carriers that possess multiple functional groups on the surface of the polymers with functionality different from those residing along the polymer’s side-chains, we synthesized star-shaped poly( L-glutamic acid) (S-PG n , where ‘S’ stands for star and ‘n’ indicates the number of PG arms) [82]. S-PGn polymers were obtained by the ring-opening polymerization of the N-carboxyanhydride of g-benzyl-glutamic acid using poly(amidoamine) dendrimers as the initiators, followed by treatment with HBr to remove the benzyl protecting groups. Fig. 5 shows the structure of S-PG 8 with eight PG arms. The molecular weight of S-PG n , and hence the spacing between the dendrimer core and the terminal functional groups, was accurately controlled by varying the molar ratio of monomer to initiator. The number of PG arms and the number of amino functional groups at the chain termini were controlled by simple variations in initiator structure. Gel permeation chromatography revealed that these polymers had a narrow molecular weight distribution with a polydispersity of 1.1–1.7. The Mark–Houwink ‘a’ values of S-PG 8 , particu-
710
C. Li / Advanced Drug Delivery Reviews 54 (2002) 695 – 713
Fig. 5. Structure of star-shaped PG with eight PG arms. Note the multiple amino groups at the ends of each PG chain.
larly that of S-PG 16 , were much smaller than that of linear PG, suggesting that S-PG n polymers assume a long chain branched structure. To evaluate the ability of S-PG n to carry water-insoluble drugs, TXL was conjugated to S-PG 8 using a carbodiimide-mediated reaction. The resulting conjugates contained 20–25% TXL (w / w) and were highly water-soluble (.20 mg eq. TXL / ml). The antitumor effects of S-PG 8 –TXL and TXL were determined by their ability to delay tumor growth in C3Hf / kam mice bearing a syngeneic murine ovarian OCa-1 tumor following a single intravenous injection of each drug. S-PG 8 – TXL, at an equivalent TXL dose of 80 mg / kg, caused an absolute growth delay of 24 days, whereas TXL at a dose of 60 mg / kg caused a growth delay of only 14 days. At their respective dose levels, S-PG n – TXL caused a maximum of 4.5% body weight loss,
whereas TXL caused a 10% body weight loss [82]. Our data indicate that S-PG n –TXL is efficacious against OCa-1 tumor with reduced toxicity and that S-PG n may be a promising new type of polymer suitable for targeted drug delivery.
7. Conclusions PG polymer possesses appropriate physicochemical properties suitable for the development of polymer–drug conjugates in order to achieve selective delivery of drugs to solid tumors. Earlier efforts have used animal models that may not necessarily reflect the importance of the EPR effect on the enhanced antitumor activity of polymeric chemotherapeutics. The bonds that link drug molecules and PG together
C. Li / Advanced Drug Delivery Reviews 54 (2002) 695 – 713
are essential in order for PG–drug conjugates to exhibit significant in vivo efficacy. PG–TXL (CT2103) is the first PG-based agent to enter clinical trials. Preliminary clinical experience with this conjugate has demonstrated several promising features. Other PG-based drug conjugates are expected to be introduced into clinical studies in the near future. Future emphasis will focus on the synthesis and characterization of novel PG-based polymers to improve their pharmacological properties and on the development of targeted drug delivery systems.
References [1] H. Ringsdorf, Structure and properties of pharmacologically active polymers, J. Polym. Sci. Symp. 51 (1975) 135–153. [2] H. Maeda, Y. Matsumura, Tumoritropic and lymphotropic principles of macromolecular drugs, Crit. Rev. Ther. Drug Carrier Syst. 6 (1989) 193–210. [3] L.E. Gerlowski, R.K. Jain, Microvascular permeability of normal and neoplastic tissues, Microvasc. Res. 31 (1986) 288–305. [4] R.K. Jain, The next frontier of molecular medicine: delivery of therapeutics, Nature Med. 4 (1998) 655–657. [5] W.G. Roberts, G. Palade, Neovasculature induced by vascular endothelial growth factor is fenestrated, Cancer Res. 57 (1997) 765–772. [6] F. Fuertges, A. Abuchowski, The clinical efficacy of poly(ethylene glycol)-modified proteins, J. Control. Release 11 (1990) 139–148. [7] H. Maeda, SMANCS and polymer-conjugated macromolecular drugs: advantages in cancer chemotherapy, Adv. Drug Deliv. Rev. 6 (1991) 181–193. [8] D. Putnam, J. Kopecek, Polymer conjugates with anticancer activity, Adv. Polym. Sci. 122 (1995) 55–123. [9] R. Duncan, F. Spreafico, Polymer conjugates. Pharmacokinetic considerations for design and development, Clin. Pharmacokinet. 27 (1994) 290–306. [10] J. Drobnik, F. Rypacek, Soluble synthetic polymers in biological systems, Adv. Polym. Sci. 57 (1984) 2–50. [11] M. Idelson, E.R. Blout, High molecular weight poly(a-Lglutamic acid): preparation and physical rotation changes, J. Am. Chem. Soc. 80 (1958) 4631–4634. [12] W.E. Hanby, S.G. Waley, J. Watson, Synthetic polypeptides. Part II. Polyglutamic acid, J. Chem. Soc. (1950) 3239–3249. [13] P. Palacios, P. Bussat, D. Bichon, Novel solid-phase synthesis of thio-terminated-poly(a-amino acid)–drug conjugate, J. Biochem. Biophys. Met. 23 (1991) 67–72. [14] A. Brack, G. Spach, New example of helical polymerization, Nature (Phys. Sci.) 229 (1971) 124–125. [15] H. Block, Poly(g-benzyl-L-glutamate) and Other Glutamic Acid Containing Polymers, Gordon and Breach, New York, 1983.
711
[16] E. Peggion, E. Scoffone, A. Cosani, A. Portolan, Polymerization of g-benzyl-L-glutamate N-carboxyanhydride: effects of conditions of polymer precipitation on the molecular weight distribution, Biopolymers 4 (1966) 695–704. [17] W.D. Fuller, M.S. Verlander, M. Goodman, A procedure for the facile synthesis of amino acid N-carboxyanhydrides, Biopolymers 15 (1976) 1869–1871. [18] R. Katakai, Y. Iisuka, An improved method for the synthesis of N-carboxy a-amino acid anhydrides using trichloromethyl chloroformate, J. Org. Chem. 50 (1985) 715–716. [19] T. Deming, Facile synthesis of block copolypeptides of defined architecture, Nature 390 (1997) 386–389. [20] G. Zhang, M.J. Fournier, T.L. Mason, D.A. Tirrell, Biological synthesis of monodisperse derivatives of poly(a,Lglutamic acid): model rodlike polymers, Macromolecules 25 (1992) 3601–3603. [21] S.M. Yu, V.P. Conticello, G. Zhang, C. Kayser, M.J. Fournier, T.L. Mason, D.A. Tirrell, Smectic ordering in solutions and films of a rod-like polymer owing to monodispersity of chain length, Nature 389 (1997) 167–170. [22] D.W. Leung, G. Pietz, C. Tompkins, P. Bergman, Expression of fusion proteins composed of polyglutamic acid linked to interferon or granulocyte colony stimulating factors, in: 5th International Symposium on Polymer Therapeutics: From Laboratory to Clinical Practice, Cardiff, UK, Jan. 3–5, 2002, p. 9. [23] G.A. Birrer, A.-M. Cromwick, R.A. Gross, g-Poly(glutamic acid) formation by Bacillus licheniformis 9945a: physiological and biochemical studies, Int. J. Biol. Macromol. 16 (1994) 265–275. [24] J. Weber, Poly(g-glutamic acid)s are the major constituents of nematocysts in Hydra (Hydrozoa, Cnidaria), J. Biol. Chem. 265 (1990) 9664–9669. [25] M. Morcellet, C. Loucheux, Viscosity / molecular weight relationship of poly(a-L-glutamic acid) in water and in water / dioxane mixtures, Biopolymers 15 (1976) 1857–1862. [26] S.P. Rao, W.G. Miller, Effect of residual side chain blocking groups and side chain charge on polypeptide conformation in aqueous solution, Biopolymers 12 (1973) 835–843. [27] P.K. Dupta, D. Gillespie, K. Hammons, M.A. Haney, Characterization of polyamino acids by use of GPC-viscometry technology, J. Pharm. Biomed. Anal. 9 (1991) 865–870. [28] A. Tsutsumi, B. Perly, A. Forchioni, C. Chachaty, A magnetic resonance study of the segmental motion and local conformations of poly( L-glutamic acid) in aqueous solutions, Macromolecules 11 (1978) 977–986. [29] V. Dolnik, M. Novotny, J. Chmelik, Electronmigration behavior of poly-( L-glutamate) conformers in concentrate polyacrylamide gels, Biopolymers 33 (1993) 1299–1306. [30] W.G. Miller, Degradation of poly(a-L-glutamic acid). I. Degradation of high molecular weight PGA by papain, J. Am. Chem. Soc. 83 (1961) 259–265. [31] W.G. Miller, Degradation of synthetic polypeptides. II. Degradation of poly-a-L-glutamic acid by proteolytic enzymes in 0.20 M sodium chloride, J. Am. Chem. Soc. 86 (1964) 3913–3918. [32] J. Pytela, R. Kotva, M. Metalova, F. Rypacek, Degradation
712
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
C. Li / Advanced Drug Delivery Reviews 54 (2002) 695 – 713 of N 5 -(2-hydroxylethyl)-L-glutamine and L-glutamic acid homopolymers and copolymers by papain, Int. J. Biol. Macromol. 12 (1990) 241–246. T. Hayashi, M. Iwatsuki, Biodegradation of copoly( L-aspartic acid / L-glutamic acid) in vitro, Biopolymer 29 (1990) 549– 557. H.-C. Chiu, P. Kopeckova, S.S. Deshmane, J. Kopecek, Lysosomal degradability of poly(alpha-amino acids), J. Biomed. Mater. Res. 34 (1997) 381–392. C.J.T. Hoes, W. Potman, W.A.R. van Heeswijk, J. Mud, B.G. de Grooth, J. Greve, J. Feijen, Optimization of macromolecular prodrugs of the antitumor antibiotic Adriamycin, J. Control. Release 2 (1985) 205–213. L.A. McCormick-Thomson, R. Duncan, Poly(amino acid) copolymers as a potential soluble drug delivery system. 1. Pinocytotic uptake and lysosomal degradation measured in vitro, J. Bioact. Compat. Polym. 4 (1989) 242–251. B.K. Kishore, P. Lambricht, G. Laurent, P. Maldague, R. Wagner, P.M. Tulkens, Mechanism of protection afforded by polyaspartic acid against gentamicin-induced phospholipidosis. II. Comparative in vitro and in vivo studies with poly-L-aspartic, poly-L-glutamic and poly-D-glutamic acids, J. Pharmacol. Exp. Ther. 255 (1990) 875–885. J.W. Singer, E. Nudelman, P. de Vries, S. Shaffer, Metabolism of poly-L-glutamic acid (PG) paclitaxel (CT-2103): intratumoral peptide cleavage followed by esterolysis, in: 5th International Symposium on Polymer Therapeutics: From Laboratory to Clinical Practice, Cardiff, UK, Jan. 3–5, 2002, p. 52. K. Akamatsu, Y. Yamasaki, M. Nishikawa, Y. Takakura, M. Hashida, Development of a hepatocyte-specific prostaglandin E1 polymeric prodrug and its potential for preventing carbon tetrachloride-induced fulminant hepatitis in mice, J. Pharmacol. Exp. Ther. 290 (1999) 1242–1249. L.A. McCormick-Thomson, D. Sgouras, R. Duncan, Poly(amino acid) copolymers as a potential soluble drug delivery system. 2. Body distribution and preliminary biocompatibility testing in vitro and in vivo, J. Bioact. Compat. Polym. 4 (1989) 252–268. D. Bayley, M.-R. Sancho, J. Brown, L. Brookman, K. Petrak, P. Goodard, A. Steward, Soluble polymeric carriers for drug delivery. Part 6. Preparation and biodistribution of N 5 -hydroxyethyl-L-glutamine-co-L-glutamic acid copolymers in rats, J. Bioact. Compat. Polym. 8 (1993) 51–68. H. Sumi, K. Kawabe, N. Nakajima, Effect of various polyamino acids and D- and L-amino acids on the blood fibrinolytic system, Comp. Biochem. Physiol. 102B (1992) 159–162. C. Li, D.F. Yu, R.A. Newman, F. Cabral, L.C. Stephens, N. Hunter, L. Milas, S. Wallace, Complete regression of wellestablished tumors using novel water-soluble poly( L-glutamic acid)–paclitaxel conjugates, Cancer Res. 58 (1998) 2404– 2409. C. Li, J.E. Price, L. Milas, N.R. Hunter, S. Ke, D.F. Yu, C. Charnsagavej, S. Wallace, Antitumor activity of poly( Lglutamic acid)–paclitaxel on syngeneic and xenografted tumors, Clin. Cancer Res. 5 (1999) 891–897.
[45] A.D. Kenny, Evaluation of sodium poly(a-L-glutamic acid) as a plasma expander, Proc. Soc. Exp. Biol. Med. 100 (1959) 778–780. [46] C.J.T. Hoes, J. Grootoonk, R. Duncan, I.C. Hume, M. Bhakoo, J.M.W. Bouma, J. Feijen, Biological properties of Adriamycin bound to biodegradable polymeric carriers, J. Control. Release 23 (1993) 37–54. [47] Y. Kato, M. Saito, H. Fukushima, Y. Takeda, T. Hara, Antitumor activity of 1-b-D-arabinofuranosylcytosine conjugated with polyglutamic acid and its derivative, Cancer Res. 44 (1984) 25–30. [48] E. Hurwitz, M. Wilchek, J. Pitha, Soluble macromolecules as carriers for daunorubicin, J. Appl. Biochem. 2 (1980) 25–35. [49] A.D. Marre, H. Soyez, E. Schacht, Synthesis of macromolecular mitomycin C derivatives, J. Control. Release 32 (1994) 129–137. [50] M. Nichifor, E.H. Schacht, L.W. Seymour, Macromolecular prodrug of 5-fluorouracil. 2. Enzymatic degradation, J. Control. Release 39 (1996) 79–92. [51] W.A.R. van Heeswijk, C.J.T. Hoes, T. Stoffer, M.J.D. Eenink, W. Potman, J. Feijen, The synthesis and characterization of polypeptide–adriamycin conjugates and its complexes with Adriamycin. Part 1, J. Control. Release 1 (1985) 301– 315. [52] F. Zunino, G. Pratesi, A. Micheloni, Poly(carboxylic acid) polymers as carriers for anthracyclines, J. Control. Release 10 (1989) 65–73. [53] E. Mochizuki, Y. Inaki, K. Takemoto, Synthesis of poly-Lglutamates containing 5-substituted uracil moieties, Nucleic Acids Res. Symp. 16 (1985) 121–124. [54] H.G. Batz, H. Ringsdorf, H. Ritter, Pharmacologically active polymers. 7. Cyclophosphamide- and steroid hormone-containing polymers as potential anticancer compounds, Makromol. Chem. 175 (1974) 2229–2239. [55] Y. Moromoto, K. Sugibayashi, S. Sugihara, K.-I. Hosoya, S. Nozake, Y. Ogawa, Antitumor agent poly(amino acid) conjugates as a drug carrier in chemotherapy, J. Pharm. Dyn. 7 (1984) 688–698. [56] C.F. Roos, S. Matsumoto, Y. Takarura, M. Hashida, H. Sezaki, Physicochemical and antitumor characteristics of some polyamino acid prodrugs of mitomycin C, Int. J. Pharm. 22 (1984) 75–87. [57] E.A. Oldham, C. Li, S. Ke, S. Wallace, P. Huang, Comparison of action of paclitaxel and poly( L-glutamic acid)– paclitaxel conjugate in human breast cancer cells, Int. J. Oncol. 16 (2000) 125–132. [58] A. Multani, C. Li, M. Ozen, A.S. Imam, S. Wallace, S. Pathak, Cell-killing by paclitaxel in a metastatic murine melanoma cell line is mediated by extensive telomere erosion with no decrease in telomerase activity, Oncol. Rep. 6 (1999) 39–44. [59] C. Li, R.A. Newman, Q.-P. Wu, S. Ke, W. Chen, T. Hutto, C. Charnsangavej, S. Wallace, Biodistribution of paclitaxel and poly( L-glutamic acid)–paclitaxel conjugates in mice with ovarian OCa-1 tumor, Cancer Chemother. Pharmacol. 46 (2000) 416–422. [60] Y.N. Sat, A.M. Burger, H.H. Fiebig, E.A. Sausville, R.
C. Li / Advanced Drug Delivery Reviews 54 (2002) 695 – 713
[61]
[62]
[63]
[64]
[65]
[66]
[67]
[68]
[69]
[70]
Duncan, Comparison of vascular permeability and enzymatic activation of the polymeric prodrug HPMA copolymer– doxorubicin (PK1) in human tumor xenografts, Proc. Am. Assoc. Cancer Res. 40 (1999) 419. R. Todd, J. Sludden, A.V. Boddy, M.J. Griffin, L. Robson, J. Cassidy, D. Bissett, M. Main, M.D. Brannan, S. Elliott, K. Fishwick, M. Verrill, H. Calvert, Phase I and pharmacological study of CT-2103, a poly( L-glutamic acid)–paclitaxel conjugate, ASCO, 2001 (abstract 439). P. Sabbatini, C. Aghajanian, M. Hensley, S. Pezzulli, C. Oflaherty, S. Soignet, M. Lovegren, J. Esch, S. Funt, O. Odujinrin, M. Warner, M.G. Bolton, D. Spriggs, Early findings in a Phase I study of PG–paclitaxel (CT-2103) in recurrent ovarian or primary peritoneal cancer, in: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics, 2001, p. 96, abstract 470. A.P. Kudelka, C.F. Verschraegen, E.M. Loyer, S. Wallace, D.M. Gershenson, M.C. Vincent, C. Alden, L. Ho, M.G. Bolton, M. Warner, P. Garzone, J.J. Kavanagh, Preliminary report of a Phase I study of escalating dose PG–paclitaxel (CT-2103) and fixed dose cisplatin in patients with various solid tumors, in: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics, 2001, p. 59, abstract 287. Y. Zou, Q.-P. Wu, W. Tansey, D. Chow, M.-C. Hung, C. Charnsangavej, S. Wallace, C. Li, Effectiveness of water soluble poly( L-glutamic acid)–camptothecin conjugate against resistant human lung cancer xenografted in nude mice, Int. J. Oncol. 18 (2001) 331–336. J.W. Singer, P. Devries, R. Bhatt, J. Tulinsky, P. Klein, C. Li, L. Milas, R.A. Lewis, S. Wallace, Conjugation of camptothecins to poly( L-glutamic acid), Ann. NY Acad. Sci. 922 (2000) 136–150. J.W. Singer, R. Bhatt, J. Tulinsky, K.R. Buhler, E. Heasley, P. Klein, P. de Vries, Water-soluble poly( L-glutamic acid)– Gly–camptothecin conjugates enhance camptothecin stability and efficacy in vivo, J. Control. Release 74 (2001) 243–247. P. de Vries, R. Bhatt, I. Stone, P. Klein, J. Singer, Optimization of CT-2106: a water-soluble poly-L-glutamic acid (PG)–camptothecin conjugate with enhanced in vivo antitumor efficacy, in: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics, 2001, p. 21, abstract 100. G.F. Rowland, G.J. O’Neill, D.A.L. Davies, Suppression of tumor growth in mice by drug–antibody conjugate using a novel approach to linkage, Nature 255 (1975) 487–488. R.S. Greenfield, P.D. Senter, A.T. Daues, K.A. Fitzgerald, S. Gawlak, W. Manger, G.R. Braslawsky, In vitro evaluation of immunoconjugates prepared by linking mitomycin C to monoclonal antibodies via polyglutamic acid carriers, Antibody Immunoconjug. Radiopharm. 2 (1989) 201–216. F. Xu, X.T. Jiang, Preparation and in vitro activity of monoclonal antibody–pharmorubicin immunoconjugates, Acta Pharm. Sin. 31 (1996) 632–636.
713
[71] E. Galun, D. Shouval, R. Adler, M. Shahaar, M. Wilchek, E. Hurwitz, M. Sela, The effect of anti-a-fetoprotein–Adriamycin conjugate on a human hepatoma, Hepatology 11 (1990) 578–584. [72] B.A. Goff, M. Bamberg, T. Hasan, Photoimmunotherapy of human ovarian carcinoma cells ex vivo, Cancer Res. 51 (1991) 4762–4767. [73] Y. Kato, N. Umemoto, Y. Kayama, H. Fukushima, Y. Takeda, T. Hara, Y. Tsukada, A novel method of conjugation of daunomycin with antibody with a poly-L-glutamic acid derivative as intermediate drug carrier. An anti-a-fetoprotein antibody–daunomycin conjugate, J. Med. Chem. 27 (1984) 1602–1607. [74] Y. Tsukada, Y. Kato, N. Umemoto, Y. Takeda, T. Hara, H. Hirai, An anti-alpha-fetoprotein antibody–daunorubicin conjugate with a novel poly-L-glutamic acid derivative as intermediate drug carrier, J. Natl. Cancer Inst. 73 (1984) 721–729. [75] P. Palacios, P. Bussat, D. Bichon, Novel solid-phase synthesis of thio-terminated-poly(a-amino acid)–drug conjugate, J. Biochem. Biophys. Met. 23 (1991) 67–72. [76] J. Vega, S. Ke, Z. Fan, S. Wallace, C. Charsangavej, C. Li, Targeting Adriamycin to EGF receptors by site-specific conjugation of monoclonal antibody to poly( L-glutamic acid), in: 5th International Symposium on Polymer Therapeutics: From Laboratory to Clinical Practice, Cardiff, UK, Jan. 3–5, 2002, p. 63. [77] C. Li, S. Ke, Q.-P. Wu, W. Tansey, N. Hunter, L.M. Buchmiller, L. Milas, C. Charnsangavej, S. Wallace, Potentiation of ovarian OCA-1 tumor radioresponse by poly( Lglutamic acid)–paclitaxel conjugate, Int. J. Radiat. Oncol. Biol. Phys. 48 (2000) 1119–1126. [78] L. Milas, N. Hunter, K. Mason, C. Li, S. Wallace, Poly( Lglutamic acid)–paclitaxel conjugate is a potent enhancer of tumor radiocurability, J. Natl. Cancer Inst. (submitted for publication). [79] C. Li, S. Ke, Q.-P. Wu, W. Tansey, N. Hunter, L.M. Buchmiller, L. Milas, C. Charnsangavej, S. Wallace, Tumor irradiation enhances the tumor-specific distribution of poly(lglutamic acid) conjugated paclitaxel and its antitumor efficacy, Clin. Cancer Res. 6 (2000) 2829–2834. [80] S. Ke, L. Milas, C. Charnsangavej, S. Wallace, C. Li, Potentiation of radioresponse by polymer–drug conjugates, J. Control. Release 74 (2001) 237–242. [81] M. Pechar, K. Ulbrich, V. Subr, Poly(ethylene glycol) multiblock copolymer as a carrier of the anti-cancer drug doxorubicin, Bioconjug. Chem. 11 (2000) 131–139. [82] C. Li, W. Tansey, J. Vega, S. Wallace, C. Charsangavej, Synthesis and characterization of dendritic polyamino acids as potential carriers for targeted drug delivery, in: 5th International Symposium on Polymer Therapeutics: From Laboratory to Clinical Practice, Cardiff, UK, Jan. 3–5, 2002, p. 31.
