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Studies of Poly(ethylene glycol) Modification of HM-3 Polypeptides Kang Zhou,† Xiaoqing Zheng,† Han-Mei Xu,* Jie Zhang, Yaqing Chen, Tao Xi, and Tingting Feng Department of Life Science and Technology, China Pharmaceutical University, Nanjing, 210009, P. R. China. Received December 4, 2008; Revised Manuscript Received April 7, 2009
An RGD modified endostatin-derived synthetic peptide, named HM-3, is a polypeptide angiogenesis inhibitor previously synthesized in our laboratory. Its robust inhibitory effects on endothelial cell migration and tumor growth have been demonstrated by in vivo and in vitro activity assays. The RGD integrin recognition sequence enables the selective binding of HM-3 and its specific targeting to tumor cells that express high levels of integrin. However, the drug has relatively short half-life in vivo, thus requiring administration twice a day to achieve its optimal in vivo antitumor efficacy. In the current study designed to prolong HM-3 half-life, we used methoxypoly(ethylene glycol)-aldehyde (mPEG-ALD) to specifically modify its N terminus and optimized the reaction condition via monitoring the modification by reverse-phase high-performance liquid chromatograph (RP-HPLC) under varying stoichiometric ratios (nmPEG10k-ALD:nHM-3), reaction times, and pH values. The maximal modification rate was achieved in a reaction when substrates mPEG10k-ALD and HM-3 were mixed at the molar ratio of 2:1 in a pH 6 phosphate buffer after 4 h incubation at room temperature. The reaction product of this optimal reaction was purified to 96% purity by RP-HPLC. Compared with HM-3, the newly modified PEG10k-HM-3 was shown to be more active in the inhibition of angiogenesis in the chorioallantoic membrane of chick embryos (CAM), its rate of in vitro degradation in serum was markedly reduced, and its in vivo half-life was prolonged by 5.86-fold relative to unmodified HM-3 after intravenous injection into male SD rats.
INTRODUCTION Endostatin, the C-terminal fragment of collagen XVIII, with a molecular weight of 20-kDa, can specifically inhibits endothelial cell proliferation and migration, and reduces vascularization and blood flow in gliosarcoma (1). It also dramatically inhibits growth of the various primary tumors in mice. Interaction of endostatin with integrin implicates its potential targets for function (2, 3). Endostatin binds one atom of zinc (Zn) per monomer via the three histidines in the NH2 terminus of the molecule (histidines 1, 3, and 11) and aspartic acid 76 (4-6). Numerous attempts have been made to produce biologically active endostatin via recombinant DNA technology (7). However, when expressed in E. coli, recombinant endostatin accumulated as inclusion bodies in the cytoplasm. During in vitro renaturation or the refolding process, the recovery of soluble and active endostatin was very low and most of the protein precipitated again during the refolding process owing to the renaturation of proteins containing multiple disulfide bonds (8-10). A highly efficient production of biologically active endostatin, with large quantity and low cost, urgently needs both further structure-function relationship investigation and its potential application as antitumor remedy. To achieve this goal, several groups have synthesized and studied the peptides that correspond to the partial sequence of endostatin (11-16). Two groups have reported the activity of a peptide containing an 11 amino acid peptide (ES-2, IVRRADRAAVP) derived from the amino terminus of endostatin (11, 12). In our previous study, we found that the 11 amino acid peptide dramatically inhibited angiogenesis on the chorioallantoic membrane of chick * Correspondence to: Center of Bitechnology, Department of Life Science and Technology, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, 210009, P. R. China; Tel: 86-25-83271007; Fax: 8625-85438355; E-mail:
[email protected]. † Kang Zhou and Xiaoqing Zheng are the first coauthor.
embryo (CAM) in vivo but had no antitumor effect in tumorbearing mice. We speculate that this difference might be caused by lesser distribution of the drug to tumor tissue in mouse models than that in chick embryos. In order to validate this hypothesis, a new strategy that increases the target delivery of the peptide to tumor is needed. In the past decade, many molecules with RGD peptide have been described as efficiently delivering drugs to tumor vasculature endothelium (17, 18). Therefore, in our previous study, we get a RGD-modified endostatin-derived synthetic peptide HM-3 (IRRADRAAVPGGGGRGD); it displayed higher antitumor effects (19). Safety experiments were conducted to study the possible toxic effects of HM-3; there no evidence of toxicity in the treated animals. These results show that HM-3 is an effective and safe peptide as an anticancer drug. However, in the in vivo pharmacokinetic studies, the halflife of HM-3 in male SD rats was measured at 27.66 ( 7.37 min. This indicates that, in any future clinical applications, higher doses or more frequent administration is required to maintain sufficient drug concentration in the blood, which in turn will exacerbate toxic effects on the patients. Protein modification is an effective method to improve efficacy and prolong drug half-lives. PEG modification of drugs can enhance protein solubility and stability, slow down drug metabilization within the body, reduce or eliminate immunogenicity, increase circulation time in the bloodstream, and lengthen administration intervals (20, 21). Lee et al. (16) and Na et al. (23, 24) have successfully modified polypeptide drugs using PEG, prolonged in vivo clearance half-lives of glucagonlike peptide-1 and salmon calcitonin (sCT), and enhanced their pharmacokinetic properties and efficacy. This current study was aimed at identifying the appropriate PEG modifying reagents suitable for HM-3, to achieve the optimal combination of biological and pharmacokinetic properties following the specific
10.1021/bc900070r CCC: $40.75 2009 American Chemical Society Published on Web 05/04/2009
PEG Modification of HM-3 Polypeptides
modification, ultimately to provide a promising lead for development of new antitumor drugs.
MATERIALS AND METHODS 1. Materials. HM-3 (99% purity) was chemically synthesized. mPEG10k-ALD (80% purity) was purchased from Beijing Kaizheng Biotech Development Co. Ltd., batch number 080540. Trifluoroacetate (TFA), acetonitrile (ACN), and sodium cyanoborohydride were purchased from TEDIA, ROE Scientific Inc., and Shanghai Darui Fine Chemicals Co. Ltd., respectively. White leghorn chicken embryos and male SD rats were purchased from Nanjing Medical Instrument Factory and New Drug Screening Center at China Pharmaceutical University, respectively. 2. Methods. 2.1. PEG Modification of HM-3 and Purification. A series of reactions were set up using varying stoichiometric ratios (nmPEG10k-ALD:nHM-3)of mPEG10k-ALD and HM-3 (1 mg, 1 mg/mL) in phosphate buffers (0.1 mol/mL) at pH 5, 6, and 7. The final concentration of sodium cyanoborohydride was 0.05 mol/mL. Sample aliquots were taken after 2, 3, and 4 h; then, glycine was added to terminate the reactions. The reaction mixtures were first analyzed for the modification rate by RP-HPLC on a C18 column (150 × 4.6 mm, 5 µm, YMC). The mobile phases contained 0.1% TFA in ACN (solution B) and 0.1% TFA in water (solution A), and was applied as a gradient of 10-100% solution B (or 90-10% solution A). Detection wavelength was set at 220 nm. The flow rate was at 1 mL/min, and the running time was 15 min. The reaction products were further purified by a semipreparative RP-HPLC with a C18 column. The mobile phases were 0.1% TFA in ACN (solution B) and 0.1% TFA in water (solution A), and was applied as a gradient of 35-50% solution B (or 65-50% solution A). The detection wavelength was set at 220 nm; the flow rate was at 3 mL/min, and the running time was 20 min. 2.2. Validation of Reaction Products. Modification products were analyzed by RP-HPLC and SDS-PAGE electrophoresis. For the SDS-PAGE identification, reaction mixtures were separated on a SDS-PAGE gel comprising 5% stacking gel and 16% separation gel. The gel was stained with Coomassie brilliant blue, and destained until the background was washed off. For the HPLC identification, the polypeptide substrate and the reaction products were analyzed by RP-HPLC as described above in section 2.1, and their HPLC chromatograms were compared. 2.3. In Vitro Stability of PEG10k-HM-3 in Serum. In aliquots of 0.9 mL SD rat serum, 0.1 mL of HM-3 (6 mol/mL), PEG10kHM-3 (6 mol/mL), and saline buffer were added into separate tubes and incubated in a 37 °C water bath. At 0, 2, 6, 10, 15, 20, 25, 50, and 60 min of incubation, 0.1 mL aliquots were taken and precipitated with perchloric acid. After centrifugation to obtain the supernatants, RP-HPLC analysis was carried out to measure the drug concentration in the supernatants, and retention percentages were calculated. 2.4. In ViVo Pharmacokinetics of PEG10k-HM-3. Male SD rats weighing roughly 200 g were used, and blood samples were taken the day before the experiment as control. PEG10k-HM-3 (35 mg/kg) was injected via tail vein, and blood samples were collected through the canthus at different time points and stored at -70 °C before use. Blood samples were precipitated with perchloric acid and centrifuged. The supernatants were analyzed by RP-HPLC to measure the drug concentration. The pharmacokinetic parameters in SD rats were calculated using the DAS v1.0 (Drug and Statistics for Windows) software and then subjected to statistical analyses.
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2.5. CAM Angiogenesis Assays. In 7 day chick embryos, after the air chamber was opened, the shell membrane was removed and sealed by a transparent tape to form a pseudoair chamber which also serves as transparent observation windows. After a 1 day stabilization period, the transparent tape was cut open, and a drug carrier plate with a 5 mm diameter was placed in the chamber. Ten microliters of each test reagent was added onto the plate. The physiological saline and endostatin (8 mg/ mL) were used as negative and positive controls, respectively. In the experimental groups, HM-3 (1 mg/mL) and PEG10k-HM-3 (6.6 mg/mL) were used (1 mg/mL HM-3 and 6.6 mg/mL PEGHM-3 yield equal molar concentrations). After 48 h incubation at 37 °C, the pseudoair chambers of chick embryos were opened and fixed in the fixative (formaldehyde-acetone, 1:1) for 5 min. The chick embryo was subsequently removed, and the CAM was carefully peeled off from the eggshell and laid out flat for photography. The medium-small blood vessels within a radius of 5 mm from the center of the carrier plate were counted and subjected to statistical analysis.
3. RESULTS 3.1. PEG Modification of HM-3 and Purification of the Modification Product. A series of modification reactions were set up by mixing mPEG-ALD and HM-3 at different molar ratios under conditions with varying buffer pH and reaction time. The reaction mixtures were analyzed by RP-HPLC to assess effects on the reactions by different conditions. The results (Figure 1A) showed that there was only one single peak for the modification product, and the maximal modification rate of 88.7% was achieved when the substrate molar ratio (nmPEG10kALD:nHM-3) was set at 2, and the reactions were incubated for 4 h at RT in a pH 6 phosphate buffer. The reaction time and substrate molar ratio have the most profound effects on the modification rate. Reaction products were purified through semipreparative RPHPLC. Analyses showed that the purity of the products was greater than 96% (Figure 1B). 3.2. Product Validation. After HM-3 modification by mPEGALD, the product was shown as a single band by electrophoresis analysis (Figure 2), and RP-HPLC chromatograms (Figure 1) also revealed the presence of a single peak, confirming that only one modification product was generated from the modification reaction. 3.3. In Vitro Serum Stability of PEG10k-HM-3. Multiple enzymes are present in the blood that can degrade polypeptides and proteins. After the drug was mixed with serum and incubated at 37 °C, RP-HPLC analysis was performed to measure drug retention percentage in the serum at different time points. The degradation rate of HM-3 in serum was very high, as HM-3 became undetectable after only 15 min incubation. By contrast, 48.8% of PEG10k-HM-3 remained intact after 15 min (Figure 3), and 16.5% could still be detected after 60 min. These results demonstrate that, after modification, the polypeptide became less susceptible to the degradative enzymes in the serum, and its stability was markedly enhanced. 3.4. In Vivo Pharmacokinetic Behaviors of PEG10k-HM-3. After intravenous injection into male SD rats, the drugs before and after modification were tested for a panel of pharmacokinetic parameters. The results (Table 1 and Figure 4) showed significantly improved in vivo pharmacokinetic behavior of the drug after PEG modification. The half-life of HM-3 in the blood of these rats was 27.66 ( 7.37 min. After the modification, the half-life of PEG10k-HM-3 was 162.08 ( 36.57 min, which was 5.86-fold longer than that of the unmodified drug, and the clearance rate was measured at 566.34 ( 56.68 L/min/kg, 141.59-fold slower relative to the unmodified HM-3. The area under the curve (AUC) was increased by 123.47-fold to reach
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Figure 1. RP-HPLC analysis of reaction mixtures obtained by reaction of mPEG-ALD with HM-3 at different pHs, molar ratios (nmPEG-ALD:nHM-3), and reaction times as indicated. Analysis was performed on a C18 column with water/acetonitrile/TFA as eluent and a flow rate of 1 mL/min, and peaks were monitored at 220 nm (A). RP-HPLC analysis of the PEGylated HM-3 Purification (B). Table 1. Pharmacokinetic Parameters after PEGylated HM-3 Administration to SD Ratsa T1/2 (min)
Cl (L/min/kg) MRT (min)
AUC (ug/L min)
PEG10k-HM-3 162.08 ( 36.57 566.34 ( 56.68 69.39 ( 6.21 61.62 ( 4.56 HM-3 27.66 ( 7.37 4.00 ( 0.80 0.49 ( 0.13 a Data are means ( SD (n ) 3). T1/2, half life; AUC, area under the curve; MRT, mean residence time; Cl, clearance; Vd, apparent distribution volume.
Figure 2. SDS-PAGE analysis of the PEGylated HM-3 purification and HM-3.
Figure 4. Plasma concentration of PEG10k-HM-3(35 mg/kg) after injection via tail vein to SD rates. Values are mean ( SD (n ) 3).
Figure 3. Residual amounts of equal molar HM-3 and PEG10k-HM-3 in plasma in vitro at 37 °C. Values are mean ( SD (n ) 3).
61 623.73 ( 4564.47 ng/L min. Finally, the average residence time of the modified PEG10k-HM-3 was 69.39 ( 6.21 min. 3.5. Inhibitory Effects on Angiogenesis in the CAM Model. 48 h after drug administration into the chick embryos, medium and small blood vessels were counted within the 5 mm radius from the center of the drug carrier plate. The results demonstrated significant inhibitory effects of PEG10k-HM-3 on angiogenesis. Compared with the negative control, both vessel number and branching were significantly reduced after treatment
Figure 5. Chick chorioallantonic membranes treated with endostain (8 mg/mL), HM-3 (1 mg/mL), and PEG10k-HM-3 (6.6 mg/mL) for 48 h.
with either PEG10k-HM-3 or HM-3 (Figures 5 and 6). The blood vessels were as follows: endostatin 30 ( 5.0, HM-3 34.7 ( 7.3, and PEG10K-HM-3, 25.7 ( 5.6. Moreover, PEG10k-HM-3-treated CAMs showed much lower density of medium-small blood vessels and fewer to almost
PEG Modification of HM-3 Polypeptides
Figure 6. Blood vessels of chick chorioallantonic membrane treated with endostain (8 mg/mL), HM-3 (1 mg/mL), and PEG10k-HM-3 (6.6 mg/mL) for 48 h. Values are represented as mean ( SD (n ) 8). *p < 0.05 vs control group, p < 0.05 (HM-3 group vs PEG10k-HM-3 group).
no small vessels adjacent to the drug carrier plate compared with the control samples (Figure 6). The diameter of blood vessels in PEG10k-HM-3-treated sample also seemed smaller. Together, these results indicate a more potent antiangiogenesis activity after HM-3 was modified.
4. DISCUSSION HM-3 is an 18-amino-acid polypeptide synthesized in our laboratory. Previous studies found that it has robust antitumor activities, especially against liver and stomach tumors. HM-3 exhibits no overt toxic side effects and therefore may have great potential in cancer therapy. However, most protein and peptides have short half-lives in vivo, and the plasma half-life of HM-3 is only 10-27 min. In animal experiments, we had to administer the drug twice daily via tail intravenous injection to achieve significant antitumor activity. To improve HM-3 therapeutic potentials for future applications, this current study sought to increase its in vivo half-life using PEG modification. Due to its nontoxic and nonimmunogenic properties, PEG has been widely used in drug modification to increase drug halflife without compromising activity. For polypeptides, many factors may affect modification reactions, including reaction time, temperature, substrate molar ratio, and so on. The current study reports a systematic optimization of these parameters. mPEG-ALD is one group of modifying agents which can react with free amino groups, and the modifications typically occur at the N termini and lysine residues of proteins (25). The HM-3 polypeptide contains no lysine residue (19), therefore it could only be modified at its N terminus. The RP-HPLC chromatogram (Figure 1) showed only one peak for the modification product, and protein gel electrophoresis also confirmed the single identity. We have also tried to determine the precise molecular weight by MODI-TOF. However, this was not successful, possibly because the modification product was difficult to ionize. A series of substrate molar ratios were tested. The maximal modification rate was obtained when the ratio of mPEG10kALD:HM-3 was 2 (Figure 1). The modification rate increased over time and reached the peak at 4 h. Different pH values were also tested and did not show significant effects on the reaction (Figure 1). Previously, Kinstler et al. documented an important characteristic of mPEG-ALD when they tried to modify G-CSF with PEG under acidic conditions (pH ) 5); coupling of aldehyde and R-amino group was highly selective. This is because, under low pH, the N termini of proteins are susceptible to protonation (25-27), therefore conducive to site-specific modification. Given that HM-3 is a polypeptide with an isoelectric point greater than 11, optimal reaction conditions for mPEG-ALD should be a buffer system with a working range pH 6-9.5. For this reason, we selected pH 6 phosphate buffer as the final reaction system.
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Previous in vivo studies have shown that HM-3 is mainly distributed in liver and kidney after administration (data not shown). Concerted action of various enzyme systems in the blood, liver, and kidney, as well as glomerular filtration, leads to in vivo fast degradation of HM-3 and short retention time in the body. However, after HM-3 was modified by PEG, its metabolic rate in the blood was reduced, and the half-life became longer. This could be due to several reasons. First, PEG may occupy the surface area of the drug, consequently blocking binding of degradative enzymes to the drug. Second, the size of the drug became larger after modification, which may reduce the loss due to glomerular filtration. As a result, the metabolic rate was decreased, and the circulation time in the body was increased. Our studies provide preliminary evidence for improved pharmacodynamic and pharmacokinetic behaviors of the drug. Future experiments are needed to validate these results. After HM-3 modification and purification, the antiangiogenesis activity was shown to be significantly enhanced in the CAM experiment. CAM models are widely used models for angiogenesis study. In this isolated in vivo system, the drug can be directly delivered to the target site; since the chick embryos remain viable during the entire experiment, the drug metabolism is active. However, such metabolic activities are still largely different from the metabolic flux in live animals. Therefore, functional analysis of PEG10kHM-3 requires more detailed animal experiments under in vivo conditions. If PEG10k-HM-3 can be administered only once a day and still achieve or exceed the antitumor activities we previously observed with HM-3 by twice-daily administration, our experimental approach would be considered a success. In addition, the immunogenicity of PEG10k-HM-3 needs to be tested. Previously, we have attempted to generate mono- and polyclonal antibodies of HM-3 for radioimmunassays. The molecular weight of HM-3 is only 1788, which makes it difficult to generate antibodies. After it was coupled with a macromolecule (BSA), antibodies of high titer and specificity were obtained. It remains to be further investigated whether modification of HM-3 mPEG10k-ALD might trigger immunogenic reaction in animals for antibody production. Furthermore, our previous experiments demonstrated that HM-3 had no overt toxic side effects on animals. Acute toxicity tests performed in mice showed that the maximum tolerable dose of HM-3 is 1926 mg/kg, more than 600-fold higher than its effective dose (3 mg/kg). Toxicological studies should also be carried out for mPEG10k-ALD modified HM3. Future therapeutic applications require that the modified product displays high antitumor activity while maintaining low immunogenicity and toxicity.
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