Bioconjugate Chem. 2005, 16, 775−784
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PEG-Doxorubicin Conjugates: Influence of Polymer Structure on Drug Release, in Vitro Cytotoxicity, Biodistribution, and Antitumor Activity Francesco M Veronese,*,⊥ Oddone Schiavon,⊥ Gianfranco Pasut,⊥ Raniero Mendichi,† Lars Andersson,‡ Anders Tsirk,‡ Jayne Ford,§ Gefei Wu,§ Samantha Kneller,§ John Davies,| and Ruth Duncan*,§ Department of Pharmaceutical Science, University of Padua, Via F. Marzolo 5, 35100 Padua, Italy, Istituto per lo Studio delle Macromolecole (CNR), Via E. Bassini 15, 20133, Milano, Italy, PolyPeptide Laboratories (Sweden) AB, PO Box 30089, SE-200 61 Limhamn, Sweden, Centre for Polymer Therapeutics, Welsh School of Pharmacy, Cardiff University, Redwood Building, King Edward VII Avenue, Cardiff, Wales, U.K., and Polymer Laboratories Ltd, Unit 4/5, The Mynd Industrial Estate Church Stretton, Shropshire, SY6 6EA, U.K . Received November 13, 2004
Polymer-drug conjugates (polymer therapeutics) are finding increasing use as novel anticancer agents. Here a series of poly(ethylene glycol) PEG-doxorubicin (Dox) conjugates were synthesized using polymers of linear or branched architecture (molecular weight 5000-20000 g/mol) and with different peptidyl linkers (GFLG, GLFG, GLG, GGRR, and RGLG). The resultant conjugates had a drug loading of 2.7-8.0 wt % Dox and contained 99%. The 125I-labeled PEGs were each diluted to give 5 × 106 cpm/mL before iv injection (dose ∼ 1.4 mg/kg). The mice were killed after 1 h, 5 h, or 24 h (n ) 3), and a blood sample was taken before the tumor and organs were dissected. Blood and organs (after homogenization) were made up to a known volume and assayed for radioactivity. The biodistribution was expressed as a percentage of dose administered. PEG-Dox Conjugates. Once the sc tumors had become palpable, PEG-Dox conjugates were injected iv at a dose of 5 mg/kg (Dox-equiv). After 1 h, the animals were killed and the organs were dissected as above. In this case an HPLC method (as described above) was used to quantify the total Dox content of blood and tissues. In this case the samples were subjected to acid hydrolysis before extraction and quantitation of Dox aglycone. Antitumor Activity of PEG-Dox in the sc B16F10 and ip L1210 Models. B16F10 Model. C57 black male mice were injected with 1 × 105 B16 F10 murine melanoma cells sc as described above. 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 (ip) with saline, Dox, or PEG-Dox according to the schedules and doses shown in the Results section. L1210 Model. On day 0, 1 × 105 L1210 cells were injected ip into DBA2 mice. On day 1, the mice were randomized into control and treatment groups (n ) 5). They were injected ip (either on day 1 only or on days 1, 2, and 3) with saline, free Dox or the PEG-Dox conju-
Veronese et al. Table 1. Structure and Composition of the PEG-Peptide-Dox Conjugates (a typical conjugate structure is shown)
compd
structure
1 2 3 4 5 6 7 8
linear PEG5000-GFLG-Dox branched PEG10000-GFLG-Dox linear PEG10000-GFLG-Dox branched PEG20000-GFLG-Dox branched PEG10000-GLFG-Dox branched PEG10000-GLG-Dox linear PEG5000-Nle-GGRR-Dox linear PEG5000-Nle-RGLG-Dox
total Dox free Dox contenta contenta (wt %) (% total Dox) 3.0-7.4 5.0 4.3 2.7 5.2 5.8 8.0 6.4
0.53-1.41a 0.99 1.47 0.56 0.24 0.62 1.57 0.89
a Varied in different batches. Total Dox content was evaluated by HPLC on the basis of doxorubicinone released after acid hydrolysis, while free Dox was evaluated on the HPLC of the untreated sample. The amount of unconjugated free PEG remaining in samples was not evaluated.
gate. The precise dose and schedule is 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 measured. Animals were humanely killed when their tumor burden reached the maximum allowable size according to the UKCCCR Guidelines (23). RESULTS AND DISCUSSION
Synthesis and Characterization of PEG-Peptide-Dox Conjugates. Three peptides H-GFLG-OH‚ TFA, H-GLFG-OH‚TFA, and H-GLG-OH‚TFA were synthesized using solid-phase and solution synthesis. The crude products were purified to homogeneity by preparative HPLC using a C18 derivatized silica column, and each peptide was characterized by analytical HPLC, amino acid analysis, and mass spectroscopy (MS). These analyses confirmed the proposed structure and also demonstrated that the two synthetic methods (solid phase and solution) were equally effective in giving pure products (by HPLC purity ranged between 99.3 -100%). The two hydrophilic arginine-containing peptides HGGRR-OH‚ TFA and H-RGLG-OH‚TFA were synthesized using solidphase synthesis only. Again purification was facilitated by reverse phase chromatography as described above. Analysis (HPLC, MS, and amino acid analysis) confirmed peptide structure, and the purity as assessed by analytical HPLC was >97%. Both hydrophobic and the more hydrophilic arginine-containing peptides were prepared in good yield (∼80% yield) using the methods described. Whereas solid-phase synthesis is preferred for rapid
PEG−Peptide−Doxorubicin Conjugates
synthesis of peptides on a small scale, in contrast, for large scale preparation, solution synthesis is frequently used as a less expensive alternative. To prepare PEG-peptide-Dox conjugates, a sequential synthetic procedure was adopted. First the desired peptide linker was bound to PEG, and then Dox was conjugated via the amino glycoside to the peptide carboxylic group using CMC and HOBT as coupling agents (Scheme 1). This method was preferred to conventional PEG-supported liquid-phase synthesis in which the amino acids are added sequentially, as it allows the use of the same stock of peptide for binding to PEGs of different structure. Eight different conjugates were thus synthesized that differ in molecular weight and shape of the polymer (linear or branched PEG) and in composition of the peptide arm (Table 1). In the first group of compounds (PEG-Dox 1-4) the peptide H-GFLG-OH was retained, but PEGs with a linear or a branched form of Mw 5000 g/mol or 10000 g/mol were used (Table 1). In the second group of compounds (PEG-Dox 5, 6) the peptide composition was changed: First by reversal of FL to give LF (compare PEG-Dox 5 and PEG-Dox 1-4) and then by deletion of F completely (PEG-Dox 6). Finally two conjugates containing arginine in the linker were synthesized (PEGDox 7, 8). Arginine was inserted to increase the linker hydrophilicity with the aim of decreasing the tendency of Dox-induced micelle formation in water. Different activation conditions were used when binding the various PEGs to peptides. For linear PEG terminating with an hydroxyl group, chloroformate activation was used to give a stable carbamate linkage between PEG and peptide (PEG-Dox 1 and 3). In this case, aqueousorganic reaction conditions were used to give better dissolution of the peptide. However, it should be noted that a molar excess of peptide was needed to overcome the partial hydrolysis of the nitrophenyl group that takes place simultaneously to the aminolysis. The advantage of this procedure lies in the fact that unreacted PEG is hydrolyzed back to hydroxyl PEG and thus it cannot react with Dox in the following synthetic step. In contrast the branched PEGs had terminal COOH groups due to their unique method of synthesis (24). These PEGs were activated as hydroxysuccinimidyl esters and, since they are easily hydrolyzed by water, the reaction with the peptides was carried out in DMSO. Under these conditions, a large molar excess of peptide was not needed. For the synthesis of the two arginine-containing peptides, a special Nle-containing PEG was used (25). The Nle residue is particularly useful, as it allows an easy analytical characterization of the products by amino acid analysis after acid hydrolysis. Arginine peptides were linked to the terminal COOH group of PEG-Nle-OH previously activated as succinimidyl ester. An aqueousorganic solution was needed since these peptides possess very low solubility in organic solvents. Also in this case a large molar excess of peptide over PEG was used to overcome the hydrolysis of the succinimidyl ester in this solvent mixture. In the PEG-Nle conjugates, the conjugate composition could be confirmed precisely by measuring the Nle:arginine ratio after acid hydrolysis, and the observed ratios were consistent with the theoretical. Dox conjugation to PEG-peptides was carried out in DMF also using CMC in the presence of HOBT (Scheme 1). Excess Dox was used to achieve complete reaction with the PEG-peptides. It is easier to separate the low molecular mass Dox from the large conjugate PEGpeptide-Dox than to remove excess PEG reagent of a similar molecular weight. Free Dox was removed using
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Figure 1. Characterization of PEG-Dox conjugates. Panel a shows an 1H NMR spectrum of the linear PEG5000-GFLG-Dox conjugate. The chemical shifts are shown in Table 2. Panel b shows a C18-HPLC chromatogram of PEG-Dox 1 (UV ) 226 nm). The major peak at tR ) 17.04 min is PEG-Dox 1 and free Dox is at tR ) 12.71 min. The chromatogram indicates the absence of unreacted PEG-AA (no peak at tR ) 16.01 min).
LH-20 Sephadex column chromatography with DMF as eluant. Only occasionally did a small amount of free Dox remain, and in this case a second chromatography run was needed to reduce the percentage free Dox in the product below 2.0% the total Dox content. The purity of the conjugates was assayed by C18HPLC which could resolve conjugated and free Dox and any residual unreacted PEG-peptide. For example, the elution pattern of the PEG-Dox 1 is reported in Figure 1b. A trace of free Dox (∼0.5%) can be seen at tR ) 12.71 min and the major peak at tR ) 17.04 min corresponds to the conjugate. No evidence of unreacted PEG-AA (tR ) 16.01 min) could be seen. The total and free Dox content of all conjugates is shown in Table 1. The peptide composition of each conjugate was confirmed by quantitative amino acid analysis after acid hydrolysis, and the amino acid ratios seen corresponded to the expected within the experimental error of the methods. 1H NMR in DMSO was used to confirm product identity. A typical 1H NMR spectrum (for the linear PEG5000-GFLG-Dox conjugate) is shown in Figure 1a, and the chemical shifts ascribed to the polymer chain, Dox, and the spacer arm protons are reported in Table 2. The integral values of the methoxy group protons of the polymer chain and 2-methyl proton of leucine and 5′-methyl protons of Dox again support the assignment of a 1:1:1 composition of the PEG:peptide:Dox. For in vitro and in vivo experiments, total Dox content of the conjugates was calculated by UV absorption, since PEG has such as a high hydration tendency and it is not possible to remove the strongly bound water by azeotropic distillation due to the instability of Dox under these conditions. The presence of unreacted PEG would also have added to the error if weight % had been used.
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Table 2. Proton Shifts Seen for the Linear PEG5000-GFLG-Dox Conjugate during 1H NMR (see also Figure 1) δ 0.82 1.13 1.47 1.85 2.20 2.75 3.24 3.99 4.20 4.59 4.70-4.90 4.92 5.25 5.92 7.20 7.69 7.92
1H
NMR signals
6H, q, 2CH3 3H, d, CH3-C-5′ 2H, d, CH2 2H, m, H2C-2′ 2H, m, H2C-9 2H, s, CH2 3H, s, OCH3 3H, s, H3C-O-C-1 1H, m, HC-5′ 2H, d, H2C-14 2H, m, OH-C-8 and OH-C-4′ 1H, m, HC-10 1H, m, HC-1′ 1H, t, HO-C-14 5H, m, arom 1H, t, HC-3 2H, d, HC-2 and HC-4
descriptor Leu Dox Phe Dox Dox Dox H-PEG Dox Dox Dox Dox Dox Dox Dox Phe Dox Dox
Table 3. Determination of the Solution Properties of the PEG-Dox Conjugates Using Light Scattering compound
apparent Mw (g/mol × 10-3)
Rga (nm)
aggregation numberb
PEG-Dox-1 PEG-Dox-2 PEG-Dox-3 PEG-Dox-5 PEG-Dox-6 PEG-Dox-7 PEG-Dox-8
120.0 33.6 33.1 28.4 30.6 54.7 35.2
23.3 29.7 15.6 13.0 16.8 45.6 21.3
20.1 3.0 3.0 2.6 2.8 9.1 5.9
Figure 2. Release of Dox from PEG-peptide-Dox conjugates during incubation with isolated rat liver lysosomal enzymes (tritosomes). Each data point represents the mean ( SD of three separate experiments.
a R gives the average radius of the molecule or aggregate. b The g aggregation number indicates the approximate number of PEGDox molecules in the surpramolecular assembly.
Solution Properties of PEG-Dox Conjugates. Evaluation of the properties of the PEG-peptide-Dox conjugates in aqueous solution by light scattering showed clear evidence of conjugate aggregation (Table 3). The extent of aggregation was dependent on conjugate structure, and the linear PEG5000-GFLG-Dox conjugate had by far the greatest tendency to form micelles that had an apparent Mw of 120000 g/mol which is equivalent to an aggregation number of ∼20. In general the other hydrophobic conjugates showed much lower aggregation tendency with an aggregation number of ∼3. Contrary to expectation, however, the more hydrophilic argininecontaining conjugates displayed a relatively high tendency to form micelles. These were composed of ∼6-9 PEG5000-NleRGLG-Dox or PEG5000-NleGGRR-Dox molecules, respectively. The tendency of polymer-drug conjugates to form unimolecular micelles (with bound hydrophobic drug forming the intramolecular core) or multimolecular micellar aggregates is important to note. It has been clearly shown that this phenomenon can limit access of activating enzyme and thus often determines the biological activity of these macromolecular prodrugs (26). Lysosomal Enzyme-Mediated Release of Dox from PEG-Dox Conjugates. Incubation of the PEG-peptide-Dox conjugates 1-6 with tritosomes led to a linear initial release of Dox over 5 h (Figure 2; Table 3). It was evident that the amino acid sequence of the peptide spacer rather than PEG structure had the strongest influence on Dox release since conjugates 1-4, which bore very different PEGs but had identical spacers, released similar quantities of Dox over 5 h. Branched PEG10000-GLFG-Dox (conjugate 5) displayed the fastest initial release rate. The tripeptide-containing linear PEG5000-GLG-Dox (conjugate 6) displayed a slower
Figure 3. In vitro cytotoxicity of the PEG-Dox conjugates assessed using B16 F10 cells (MTT assay) after a 72 h incubation. Error bars represent the mean ( SD and n ) 4-6.
release rate. This pattern of degradation corresponds to the known susceptibilities of these linkers to degradation by lysosomal thiol-dependent proteases when the same sequences were used as side-chains in N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer conjugates containing Dox (27). Interestingly, linear PEG5000-GFLGDox released drug at the same rate as HPMA copolymerGFLG-Dox when incubated with tritosomes, but much more quickly than a PEG-peptide block copolymer bearing GFLG-Dox side chains (28). The arginine-containing linkers displayed the lowest rate of release of free Dox, but this is most likely due to the specificity of the predominant lysosomal proteases rather than the micellerization of these conjugates. In Vitro Cytotoxicity. The in vitro cytotoxicity tests (Figure 3; Table 4) showed the PEG-peptide-Dox conjugates to be 10-100 fold less toxic than free Dox (0.24 µg/mL) against B16F10 cells, an observation consistent with the slower rate of endocytic uptake and lysosomotropic activation as the rate-limiting step. Conjugates 1-4 having the GFLG linker displayed the greatest cytotoxicity (IC50 ) 4.6-8.3 µg/mL). The low cytotoxicity of PEG-GLG-Dox conjugate 6 correlates well with its poor rate of enzymatic activation. However, it is difficult to explain the relatively low cytotoxicity of PEG-GLFGDox conjugate 5 as this contains the linker most susceptible to enzyme attack. Throughout the in vitro testing
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PEG−Peptide−Doxorubicin Conjugates Table 4. Summary of the Biological Properties of the PEG-Dox Conjugates
compound Dox PEG-Dox 1 PEG-Dox 2 PEG-Dox 3 PEG-Dox 4 PEG-Dox 5 PEG-Dox 6 PEG-Dox 7 PEG-Dox 8
release of Dox during incubation with tritosomes (% total Dox liberated) 5h 24 h N/A 30.22 ( 2.19 29.74 ( 3.70 33.07 ( 2.40 29.31 ( 5.48 56.89 ( 8.54 15.50 ( 0.53 3.08 ( 0.90 4.00 ( 1.03
N/Ab 75.9 48.4 51.4 60.6 72.1 37.6 N/A N/A
Table 5. Body Distribution of 125I-Labeled PEGs of Mw 10000 g/mol and 20000 g/mol after iv Injection organ
IC50 (µg/mL)a
B16F10 model T/C (%)
blood
0.2 5.2 8.3 4.6 6.7 72 105 2.4 3.8
121 146 134 113 130 143 N/A N/A N/A
tumor liver heart spleen
a
b
Assessed against B16 F10 cells over 72 h using the MTT assay. N/A; not undertaken. lungs
there seemed to be no clear correlation between PEG structure and cytotoxicity, and this is noteworthy that the free Dox content in each conjugate, even though the levels are low, can influence in vitro cytoxicity. Biodistribution of 125I-Labeled PEGs and PEGDox Conjugates. As would be predicted for watersoluble macromolecules (29, 30), the higher molecular weight 125I-labeled PEGs remained circulating in the blood for longer due to decreased renal clearance after iv injection (Figure 4a; Table 5). This longer plasma residence time led to increased tumor targeting (Figure
kidneys
a
time (h)
PEG 5Ka
PEG 10Ka
PEG 20Ka
1 5 24 1 5 24 1 5 24 1 5 24 1 5 24 1 5 24 1 5 24
0.48 ( 0.05 0.39 ( 0.05 0.26 ( 0.06 0.36 ( 0.02 0.57 ( 0.65 0.11 ( 0.04 5.11 ( 0.50 4.75 ( 0.70 3.75 ( 0.76 0.10 ( 0.03 0.10 ( 0.02 0.03 ( 0.02 0.29 ( 0.03 0.23 ( 0.05 0.08 ( 0.03 0.32 ( 0.16 0.18 ( 0.08 0.09 ( 0.02 0.87 ( 0.09 0.62 ( 0.15 0.20 ( 0.07
2.7 ( 0.54 1.22 ( 0.30 0.57 ( 0.13 1.19 ( 0.20 1.06 ( 0.31 0.26 ( 0.07 4.34 ( 0.38 3.89 ( 0.69 1.79 ( 0.53 0.58 ( 0.12 0.18 ( 0.06 0.08 ( 0.03 0.47 ( 0.12 0.26 ( 0.08 0.16 ( 0.05 0.90 ( 0.05 0.39 ( 0.04 0.19 ( 0.06 1.57 ( 0.40 0.74 ( 0.21 0.20 ( 0.03
10.68 ( 1.02 5.59 ( 0.90 1.45 ( 0.21 1.96 ( 0.77 2.01 ( 0.62 0.81 ( 0.11 4.20 ( 0.55 3.56 ( 0.78 1.60 ( 0.33 1.90 ( 0.30 1.19 ( 0.14 0.23 ( 0.04 1.16 ( 0.15 0.90 ( 0.15 0.37 ( 0.07 2.43. ( 0.33 1.73 ( 0.64 0.54 ( 0.16 3.00 ( 0.30 1.61 ( 0.34 0.44 ( 0.06
Results represent the mean ( SD. n ) 3.
4a; Table 5), but in many cases also gave increased normal tissue exposure (Table 5). Although the highest molecular weight PEG showed greatest tumor levels, comparison of the area under the curve values for each polymer indicated that PEG5000 had the most favorable
Figure 4. Biodistribution of 125I-labeled PEGs and PEG-Dox conjugates after iv administration to mice bearing sc B16F10 tumors. Panel a shows the time-course of radioactivity detected in blood after administration of 125I-labeled PEGs, panel b shows the timecourse of radioactivity detected in tumor tissue and panel c shows the total Dox recovered in all tissues at 1 h after administration of PEG-Dox conjugates. (Analysis by HPLC). The data representmean ( SD where n ) 3-5.
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Table 6. Treatment of Mice Bearing sc B16F10 Murine Melanoma with Various PEG-DOX Conjugates group
mean survival time (days)a
T/C × 100
saline Dox PEG-Dox 1 PEG-Dox 2 PEG-Dox 3 PEG-Dox 4 PEG-Dox 5
5.6 6.8 8.2 7.5 6.3 7.3 8.0
100 121 146a 132a 113 130a 143a
a In this aggressive B16F10 model it is usual to have statistical significance when T/C is greater than 125% and when the group size is n ) 5, as was the case here.
tumor:heart ratio. Considering the known dose-limiting toxicity of Dox, this may be important. The radioactivity associated with 125I-labeled PEGs in the liver were similar for PEG 10000 and 20000 g/mol, but were higher for PEG 5000 g/mol. and did not decrease in the same manner as in other tissues. Nevertheless, for all PEGs investigated, liver uptake was never more than 6% which is only a little higher than other water soluble polymeric drug carriers such as HPMA copolymers, for example. The variability in tumor accumulation of 125I-labeled PEGs may be explained by the tendency of small tumors to accumulate more polymer than large tumors, a phenomenon shown previously with HPMA copolymer-Dox (31). Scatter plots of tumor uptake per gram against tumor weight showed that accumulation at 1 h was higher in smaller tumors (data not shown). As a preliminary study, the biodistribution of the PEG-Dox conjugates, the levels of total Dox in blood, tumor, and the other principal organs, was examined at 1 h after iv injection. All the PEG-Dox conjugates showed a significantly longer plasma half-life than free Dox (Figure 4c). PEG-Dox 4 showed significantly higher blood levels than the other conjugates at 1 h. This is perhaps surprising as light scattering experiments suggested that PEG-Dox 1 had the highest apparent size and would suggest that the micellar aggregate of PEGDox 1 rapidly dissociates in blood. All PEG-Dox conjugates showed higher tumor Dox concentrations than free Dox and importantly lower heart levels (Figure 4c). It is important to note that the blood clearance and tumor targeting of the PEG-Dox conjugates was markedly
different than seen for the corresponding molecular weight 125I-labeled PEGs. This emphasizes the importance of monitoring biodistribution of each polymer-drug conjugate as well as the parent polymeric carrier. Antitumor Activity of PEG-Dox Conjugates. An important factor leading to improved efficacy of polymer conjugates is tumor targeting by the EPR effect. Thus it is important to use solid tumor models to evaluate therapeutic potential of polymer conjugates. As we have used the B16F10 melanoma model to compare the activity of other anticancer conjugates that we have subsequently progressed into clinical trial (32-34), the antitumor activity of PEG-Dox conjugates was assessed using this model. It should be noted that this is a very aggressive tumor, and animals not treated usually die within 6-10 days of the tumor becoming palpable. First studies compared the activity of PEG-Dox conjugates 1-5 at a Dox dose of 5 mg/kg (Dox-equiv). Although Dox showed no significant antitumor activity, both PEG-Dox 1 and PEG-Dox 5 were active although the increase in survival was modest (T/C ∼ 145%) (Table 6). Antitumor activity would appear to correlate with the highest rate of Dox release in the presence of lysosomal enzymes (Table 4). Activity of PEG-Dox 1 was confirmed in a second experiment designed to examine the dose-dependency of activity. Although no clear dose-response was observed antitumor activity was evident, in this case a T/C of ∼ 160% was observed (Table 7). The mouse leukemia L1210, grown as an ip ascites, is classically used to establish the activity of anthracycline derivatives. When PEG-Dox-1 was evaluated in this model, antitumor activity was observed, but survival was reduced in comparison to treatment with Dox alone (Table 8). Considering the differential pharmacokinetics of free Dox and PEG-DOX conjugates at the whole organism and cellular level, this observation is consistent with the need for EPR-mediated targeting to allow polymer conjugates to show therapeutic benefit. Additionally it is noteworthy throughout these experiments that the PEG-Dox conjugates were less toxic as evidenced by their lesser effect on animal weight loss (Figure 5). CONCLUSIONS
Structure of PEG-peptide-Dox conjugates can be tailored to give a 1:1:1 stoichiometry of PEG chain, amino acid spacer, and anthracycline. This offers a unique
Table 7. Treatment of Mice Bearing sc B16F10 Murine Melanoma with PEG5000-GLFG-Dox (PEG-Dox-1). Effect of Dose treatment
dose (mg/kg Dox-equiv)
dosing schedulea
survival (mean ( SD)
T/C (%)b
number of toxic deaths
Saline PEG-Dox-1 PEG-Dox 1 PEG-Dox 1
10 5 1
day 1 day 1 day 1 day 1
8.75 ( 1.9 10.6 ( 2.1 8.6 ( 1.34 8.25 ( 1.71
100 161** 130NS 161NS
0/4 0/5 2/5 0/4
a Single dose given after tumor reaches palpable size. b Statistical significance was calculated using a Student’s t test for small sample sizes. ** ) p > 0.01. NS ) not significant.
Table 8. Effect of ip PEG5000-GLFG-Dox (PEG-Dox 1) Administration on the Survival of DBA2 Mice Bearing ip L1210 Leukaemia. Animals Were Treated on Day 1 or Day 1, 2, and 3 after Tumor Inoculation treatment
dosea
dosing schedule
survival days
survival days (mean ( SD)b
T/C (mean ( SD)
toxic deaths
saline Dox PEG-Dox 1 PEG-Dox 1 PEG-Dox 1 PEG-Dox 1
5 10 1 5 10
1 1 1 1, 2, 3 1, 2, 3 1, 2, 3
18, 18, 18, 18, 18 27, 15, 32, 29, 32 15, 28, 24, 24, 25 22, 24, 21, 22, 23 15, 22, 24, 21, 26 25, 27, 22, 28, 25
18 27 ( 7.0* 23.2 ( 4.87NS 22.4 ( 1.14** 21.6 ( 4.16NS 25.4 ( 2.3**
100 150 ( 39 129 ( 27 124 ( 6 120 ( 23 141 ( 13
0/5 0/5 0/5 0/5 0/5 0/5
a Dose expressed as Dox-equiv (mg/kg). b Statistical significance was calculated using a Student’s t test for small sample sizes. * ) p > 0.05 and ** ) p > 0.001. NS ) not significant.
PEG−Peptide−Doxorubicin Conjugates
Bioconjugate Chem., Vol. 16, No. 4, 2005 783 LITERATURE CITED
Figure 5. Effect of PEG-Dox conjugates on animal weight. The effect of PEG-Dox-1 and free Dox on the weight of mice during antitumor experiment. Panel a shows the weight change in C57 mice bearing a sc B16F10 tumor, and panel b shows the weight change in DBA2 mice bearing ip L1210.
opportunity to prepare anticancer polymeric conjugates of low polydispersity, which is advantageous compared to other pendant polymer conjugates. Solution properties are however complicated by the tendency of PEG-Dox conjugates to form multimolecular aggregates. The aggregation number was dependent on the nature of the PEG used (Mw and architecture), and further study of micellar stability is warranted before further development of such conjugates. The rate of Dox release on exposure to lysosomal enzymes was controlled by the peptidyl linker used, but the nature of the PEG carrier had little influence on this process. In vivo studies confirmed the effect of PEG Mw on the biodistribution of 125I-labeled PEGs. However, the biodistribution of PEG-Dox conjugates was not clearly related to Mw or architecture of the carrier, but was probably governed by the nature and stability of the PEG-Dox aggregates formed. In all cases the PEG-Dox conjugates displayed greater tumor targeting than free Dox and also lower heart levels of the bound anthracycline. This justified evaluation of their antitumor activity in vivo. Experiments in mice bearing either a sc B16F10 tumor or and ip L1210 tumor confirmed the activity of selected PEGDox conjugates and also underlined the importance of the EPR effect as a means to enhance tumor targeting. PEG5000-GFLG-Dox was selected as the lead candidate for further preclinical evaluation. ACKNOWLEDGMENT
This work was supported by BE Project 97- 4133. We would like to thank Dr. Steve Stribbling for technical help with the in vivo experiments.
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