Biomacromolecules 2005, 6, 914-926
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Poly(ethylene glycol)-Poly(ester-carbonate) Block Copolymers Carrying PEG-Peptidyl-Doxorubicin Pendant Side Chains: Synthesis and Evaluation as Anticancer Conjugates Lars Andersson,† John Davies,‡ Ruth Duncan,*,§ Paolo Ferruti,*,| Jayne Ford,§ Samantha Kneller,§ Raniero Mendichi,⊥ Gianfranco Pasut,# Oddone Schiavon,# Clive Summerford,‡ Anders Tirk,† Francesco M. Veronese,*,# Veronica Vincenzi,| and Gefei Wu§ PolyPeptide Laboratories (Sweden) AB, PO Box 30089, SE 20061 Limhamn, Sweden, Polymer Laboratories Ltd., Essex Road, Church Stretton, Shropshire, SY6 6AX United Kingdom, Centre for Polymer Therapeutics, Welsh School of Pharmacy, Cardiff University Redwood Building, King Edward VII Avenue, Cardiff, Wales, United Kingdom, Dipartimento di Chimica Organica e Industriale, Universita` di Milano, Via Venezian 21, 20133 Milano, Italy, Istituto di Chimica delle Macromolecole del CNR, Via E. Bassini 15, 20133 Milano, Italy, and Department of Pharmaceutical Science, University of Padua, Via F. Marzolo 5, 35100 Padua, Italy Received October 1, 2004; Revised Manuscript Received December 2, 2004
Water soluble polymer anticancer conjugates can improve the pharmacokinetics of covalently bound drugs by limiting cellular uptake to the endocytic route, thus prolonging plasma circulation time and consequently facilitating tumor targeting by the enhanced permeability and retention (EPR) effect. Many of the first generation antitumor polymer conjugates used nonbiodegradable polymeric carriers which limits the molecular weight that can be safely used to 95% yield was obtained. The solubility range of the product was in the range of 1 mg in 27-72 µL of distilled water. Determination of Bound and Free Dox. Total Dox. A stock solution was prepared by dissolving 5.00 mg of Dox in 10.0 mL of 1 M HCl, and it was warmed at 50 °C for 3 h to generate the aglycone. Samples containing the equivalent of 25-200 µg of Dox were prepared by diluting the stock solution (1:3) with DMSO. These samples were analyzed using the HPLC method described above to generate a calibration curve. The amount of bound Dox in the final product could then be determined by HPLC analysis following incubation of DBM2-PEG4000-S-PEG3000-GFLG-Dox (5 mg) with 1 M HCl at 50 °C for 3 h. Free Dox. A calibration curve was obtained using samples of Dox subjected to serial dilution and then subjected to HPLC using the method described above. The amount of
PEG-Poly(ester-carbonate)-Dox Conjugates
Figure 5.
1H
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NMR of heterobifunctional (-S-PEG3000-OSu)2.
Scheme 3. Synthesis of HS-PEG3000-GFLG-Dox
free Dox in the conjugates was determined by HPLC analysis of 7 mg of the conjugates. Evaluation of Dox Release from DBM2-PEG4000-SPEG3000-GFLG-Dox by Isolated Lysosomal Enzymes. Experiments were carried out as described previously.15,16 Briefly, rat liver lysosomal enzymes (Tritosomes) were isolated according to the method of Trouet17 and were standardized for activity by measuring their ability to degrade the substrate Bz-PVR-p-nitroanilide (NAp). Protein content
of the Tritosomes was also measured. The batch used here contained 0.96 mg protein/mL and had an activity 24.7 nmole NAp release/min/mg protein. DBM2-PEG4000-S-PEG3000-GFLG-Dox conjugates were dissolved in 0.2 M citrate-phosphate buffer pH 5.5 containing Triton x-100 (0.2%), reduced glutathione (5 mM) and EDTA (1 mM) to give a final concentration of 100 µg dox-equiv./ mL. Tritosomes (400 µL per mL of incubation mixture) were added to start the assay, and the sample was thoroughly mixed. All reagents were maintained at 37 °C throughout. Samples (100 µL) were taken over 5 h and immediately frozen in liquid nitrogen and stored frozen until analysis by HPLC. A method previously described18,19 was used as an initial step to extract free Dox from the samples containing the internal standard daunomycin (Dnm). The analysis carried out by HPLC. In this case 2-propanol (29% in water) adjusted to pH 3.2 was used as a mobile phase, flow rate (1 mL/min) with a C18 Bondapack column (3.9 × 150 mm) using fluorescence detection. Evaluation of in Vitro Cytotoxicity. The cytotoxicity of the DBM2-PEG4000 and the DBM2-PEG4000-S-PEG3000GFLG-Dox conjugates was assessed using the adherent
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Scheme 4. Synthesis of DBM2-PEG4000-S-PEG3000-GFLG-Dox
Andersson et al.
injected i.p. (either on day 1 only or on days 1, 2, and 3) with saline, free Dox, or the conjugate. The precise dose and schedule is also shown in the Results section. In both cases, animals were monitored daily for weight change, and for the B16F10 tumor model, the tumor size was also measured. Animals were humanely killed when their tumor burden reached the maximum allowable size according to the UKCCCR Guidelines.21 3. Results and Discussion
murine B16F10 melanoma cell line as previously described.20 Cells were grown in RPMI medium supplemented with 10% fetal calf serum and seeded at a density of 104 cells per well in 96-well tissue culture plates. After 24 h, the medium was replaced with serial dilutions of either Dox or the conjugate added in fresh medium (100 µL). All stock solutions were filtered through a 0.22 µm membrane filter to ensure sterility. The cells were then incubated for 67 h at 37 °C. Then 20 µL of sterile MTT solution (0.5% w/v in PBS) was added to each well. After additional 5 h incubation, the medium was removed and optical grade DMSO added to dissolve the insoluble formazan dye formed. Absorbance at 550 nm was measured using a microtiter plate reader, and the cell viability was expressed as a percentage viability of cells incubated with medium alone. Antitumor Activity of DBM2-PEG4000-S-PEG3000-GFLGDox in the s.c. B16F10 and i.p. L1210 Tumor Models. All animal experiments were conducted according to the United Kingdom Coordinating Committee on Cancer Research (UKCCCR) Guidelines.21 B16F10 Model. C57 black male mice were injected with 1 × 105 B16 F10 murine melanoma cells s.c. They were then left for 10-12 days until tumor size reached between 9 and 25 mm2. Animals were randomized into control and treatment groups (n ) 5) and injected intraperitoneally (i.p.) with saline, Dox, or conjugates according to the schedules and doses shown in the Results section. L1210 Model. On day 0, 1 × 105 L1210 cells were injected i.p. into DBA2 mice. On day 1, the mice were randomized into control (C) and treatment (T) groups (n ) 5). They were
The synthetic strategy adopted to assemble this high molecular weight carrier (Figure 1, Table 1) was to first synthesize separately the polymer backbone and the drugcarrying sidearms and then to assemble them as the final step via a Michael addition reaction. The challenge was, on one hand, the preparation of a polymeric precursor (DBM2PEG) containing biodegradable maleate or fumarate units with activated double bonds in the main backbone and, on the other hand, the synthesis of a heterobifunctional PEG, carrying at one end the drug moiety attached through a peptide spacer and at the opposite end a function suitable for the Michael addition to the main chain. The overall aim was to create a high molecular weight, biodegradable carrier with the ability to carry a sufficiently high Dox payload to make the conjugate suitable for therapeutic development. Synthesis and Characterization of DBM2-PEG Block Copolymers. The polymeric precursor was prepared by chain extension reaction between PEG4000 and bis-(4-hydroxytetramethylene)maleate (DBM) blocks connected by carbonate bonds. The synthesis chosen was suitable for large-scale synthesis and involved diphenyl carbonate as the coupling agent, and the chain-extension reaction was performed in the melt at 120 °C and under oil-pump vacuum to eliminate the phenol byproduct. Potassium carbonate (1%) was preferred as the catalyst due to its low toxicity. 1 H NMR confirmed the structure of the final product (DBM2-PEG4000; Figure 2) but revealed quantitative rearrangement of the maleate units induced by the high reaction temperature into fumarate units. Moreover, PEG and DBM segments were randomly distributed along the polymer backbone. This contrasts with DBM2-PEG block copolymers reported earlier,9 prepared by the phosgene method, in which all unsaturated units were in cis configuration. It is noteworthy that these new products exhibited a considerably higher shelf-stability. No evidence of cross-linking was found in the “diphenyl carbonate-produced” block copolymers. It is postulated that the cis products became insoluble on storage due to cross-linking by intermolecular cycloaddition, a reaction probably less favored by a trans configuration. The 1H NMR analysis of DBM2-PEG copolymers revealed that the number of double bonds (more specifically the number of DBM units present in the products) was approximately 20% lower than expected from the initial composition of the reaction mixture. Probably, the combination of high temperature and vacuum causes some loss of DBM during polymerization. However, allowance should be made for the inherent approximation of 1H NMR as the quantitative analytical method.
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PEG-Poly(ester-carbonate)-Dox Conjugates
Table 1. Characteristics of the Polymers Used DBM2-PEG4000 Batch 1 and 2 DBM2-PEG4000-S-PEG3000-GFLG-Dox Batch 1 and 2
product name
total Dox (wt %)
free Dox (% total Dox)
Mw (g/mol)
Mn (g/mol)
Mw/Mn
[η] (dL/g)
double bonds per PEG unita
DBM2-PEG4000 batch 1 DBM2-PEG4000 batch 2 DBM2-PEG4000-S-PEG3000-GFLG-Dox batch 1 DBM2-PEG4000-S-PEG3000-GFLG-Dox batch 2
0 0 3.4 4.0
0 0 0.6 0.7
100 000 190 000 NDa NDa
36 800 53 000 NDa NDa
2.72 3.58 NDa NDa
1.07 1.39 NDa NDa
1.5 1.5 NDa NDa
a
The molar ratio DBM/PEG in the polycondensation mixture was 2:1. b ND ) not determined.
GPC analysis was conducted on several batches of DBM2PEG4000 (Figure 3). Depending on the scale of production, the Mw of different batches varied between 100 000 and 190 000 g/mol and all preparations were very polydisperse. The Mw/Mn was between 3.5 and 5.0. Three batches prepared on a large scale displayed similar molecular weight characteristics, as assessed using poly(methyl methacrylate) (PMMA) standards (Figure 3) and their molecular weights were batch 1 Mw ) 183 000 g/mol, Mn ) 37 900 g/mol and Mw/Mn ) 4.82; batch 2 Mw ) 167 000, Mn ) 167 000, Mw/Mn ) 4.69; and batch 3 Mw ) 179 000, Mn ) 35 600, Mw/Mn ) 4.69. By varying the relative amounts of the two diol-terminating blocks, block copolymers of DBM2-PEG can be prepared with a wide range of compositions. However, it should be noted that as the DBM content was increased the final products showed decreased aqueous solubility and most biological studies were conducted using intermediates prepared from the DBM2-PEG4000. Model Reactions to Study Addition to the Double Bond of DBM-PEG Block Copolymers. It should be noted that the cis and trans DBM-PEG block copolymers described above showed no gross variation in reactivity toward the Michael addition. Before proceeding to conjugate the PEGGFLG-Dox sidearms, it was necessary to establish the
optimum chemistry for conjugation. The kinetics of reaction of the PEG-SH and PEG-NH2 (as model compounds) with DBM2-PEG4000 was investigated using N-ethylmaleimide (NEM) since this molecule possesses a double bond similar to that in DBM2-PEG4000 (Figure 4). The addition was monitored by decrement of NEM absorbance at 305 nm due to disappearance of the molecule double bond. PEG-SH showed higher reactivity in terms of kinetic rate and yield of reaction. Therefore, the thiol derivative was chosen as lead compound for the preparation of the pendant conjugate, HS-PEG3000-GFLG-Dox. Synthesis and Characterization of the Heterobifuctional PEG and HS-PEG-GFLG-Dox. To create the HS-PEGGFLG-Dox intermediate, used to graft the DBM2-PEG, it was first necessary prepare a heterobifuctional PEG having one -COOH terminal and one -SH terminal group. Succinimidyl carbonate activated, protected thiol-containing, heterobifunctional PEG, (-S-PEG3000-OSu)2, was synthesized using a 5-step synthesis from commonly available starting materials. The method used was suitable for scale-up with no chromatographic separations required. The derivative was characterized by 1H NMR (Figure 5). The (-S-PEG3000-OSu)2 was coupled to H-GFLG-OH leading to (-S-PEG3000-GFLG-OH)2 that was purified and
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Figure 6.
1H
Andersson et al.
NMR of DBM2-PEG4000-S-PEG3000-GFLG-Dox.
also characterized by reverse phase in HPLC, demonstrating the absence of free peptide. Doxorubicin was conjugated to the carboxylic groups of (-S-PEG3000-GFLG-OH)2, using EDC/HOBt activation. The obtained (-S-PEG3000-GFLG-Dox)2 was purified by preparative RPC. The chemical route and the purification procedure allowed the preparation of the conjugate in quantitative yield in terms of drug loading as verified by the determination of the bound doxorubicin. The final step consisted in the reduction of the disulfide double bond by DTT, and the product was purified by RPC. The reduction was quantitative in 30 min, and DTT does not degrade the drug. The purification was carefully chosen since it is important to avoid the reoxidation of the free thiol group during the elimination of the reductive agent. The overall yield was 63%. Synthesis and Characterization of DBM2-PEG4000-SPEG3000-GFLG-Dox. To prepare DBM2-PEG4000-S-PEG3000GFLG-Dox, HS-PEG2000-GFLG-Dox and DBM2-PEG4000 were prepared separately, and their thiol or double bond reactivity was tested toward model molecules. The addition reaction of HS-PEG3000-GFLG-Dox thiol group to DBM2PEG4000 double bonds was conducted in degassed solvent to avoid the formation of a dimer due to disulfide bond linkage. To obtain high purity DBM2-PEG4000-S-PEG3000GFLG-Dox, a double preparative GPC using LH-60 resin was preformed, which allows the use of an organic solvent
such as methanol since water hydrolyses the ester bonds in the DBM2-PEG4000. Characterization was carried out by analytical GPC, 1H NMR (Figure 6), and UV-vis to ensure that the drug was not degraded during the synthesis. Because of the high complexity of the final grafted block copolymer loaded with the drug, the 1H NMR investigation was performed to ensure the addition of graft polymer thiol group to the DBM2-PEG double bonds. This has been verified by the decrease of double bond H signals, both the cis (6.24 ppm singlet) and the trans ones (6.24 ppm singlet), of the block copolymer in the final pure construct. Biological Properties of DBM2-PEG4000-S-PEG3000GFLG-Dox. Although the DBM-PEG-Dox conjugates were relatively soluble in water, it became clear at the outset of biological testing that they do not possess the desired solubility in physiological buffers and tissue culture medium. In fact, they were difficult to dissolve in these media, even after prolonged sonication. Polymer-drug conjugates are usually inert prodrugs, and it is essential that they liberate Dox to display antitumor activity in vivo.22 The GFLG linker chosen to bind Dox to the PEG sidearm here was originally designed to facilitate Dox conjugation to HPMA copolymer conjugates. This linker allows specific cleavage by the lysosomal thiol-dependent proteases.23 The two HPMA copolymer conjugates containing -GFLG-Dox have shown promise in Phase I/II clinical trials with reduced Dox-related toxicity and antitumor activity in
PEG-Poly(ester-carbonate)-Dox Conjugates
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Figure 7. Release of Dox from PEG5000-GFLG-Dox and DBM2PEG4000-S-PEG3000-GFLG-Dox during incubation with isolated rat liver lysosomal enzymes. Date show mean ( s.d., n ) 3.
Figure 9. Weight loss in animals treated with Dox and Dox conjugates. Panel a shows the weight loss in C57 Mice bearing B16F10 tumors and panel b the weight loss in DBA2 mice bearing i.p. L1210.
Figure 8. Cytotoxicity of the polymers (panel a) and Dox conjugates (panel b) toward B16 F10 cells grown in vitro. Data represent mean ( s.d.; n ) 6.
chemotherapy-refractive disease.24,25 HPMA copolymerGFLG-Dox (26) and PEG5000-GFLG-Dox13 release >20% of the bound Dox when incubated for 5 h with isolated lysosomal enzymes in vitro. In contrast, the DBM2-PEG4000S-PEG3000-GFLG-Dox conjugate showed a much slower rate of degradation (Figure 7), and
