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Preparation and Antitumor Activity of a Polymeric Derivative of Methotrexate
CLINICAL INVESTIGATION
Preparation and Antitumor Activity of a Polymeric Derivative of Methotrexate Guangxin Zhou, PhD, Xiaoyun Cheng, PhD, Sujia Wu, MD, PhD, Xiqun Jiang, PhD, Xin Shi, MD, PhD, Jiangning Chen, PhD, Junfeng Zhang, PhD and Jianning Zhao, MD, PhD
Abstract: A polymer-drug conjugate was developed by conjugating amino bonds of methotrexate (MTX) to succinoylated a,b-poly[(2hydroxyethyl)-L-aspartamide] (PHEA). The therapeutic efficacy of PHEA-MTX was evaluated in vitro and in vivo. PHEA-MTX showed sustained release properties when incubated in pH 5.5 and pH 7.4 buffering solutions at 37°C. PHEA-MTX induced MG63 cell apoptosis in a time-dependent and concentration-dependent manner in vitro and inhibited the growth of S180 sarcoma in vivo. PHEA-MTX was more potent and, more importantly, displayed less systemic toxicity than free MTX. The enhanced therapeutic effects of PHEA-MTX suggest that the PHEA-MTX conjugate may have a greater potential for chemotherapy of cancers. Key Indexing Terms: PHEA; Methotrexate; Antitumor efficacy; Drug delivery; Polymer prodrug. [Am J Med Sci 2012;344(4):294–299.]
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ethotrexate (MTX) is one of the most commonly used antineoplastic agents for chemotherapy of cancers. However, it has a potent inhibitory effect on the metabolism of folic acid, leading to broad cytotoxicity against many types of proliferative cells, such as bone marrow, mucosal and lymphoid cells.1 To reduce MTX-related adverse effects, a series of drug delivery systems have been attempted, such as encapsulated microspheres,2,3 macromolecule conjugates4,5 and antibody conjugates,6–8 to improve the specificity and selectivity of MTX. However, the efficiency of these strategies is limited. Therefore, development of new derivatives of MTX will be of great significance in effectively and safely treating patients with cancer. Application of a polymer carrier to deliver MTX may be a promising strategy for improving the therapeutic efficacy and safety of MTX. The biological property of a polymer-drug conjugate depends on the types of polymers and the nature of linker between the drug and polymer. The synthetic polymer a,b-poly[(2-hydroxyethyl)-L-aspartamide] (PHEA) has been found to be potentially useful in the delivery of drugs because it is water soluble, biocompatible, easily obtained and modified.7,9,10 However, a PHEA-MTX conjugate formed by directly coupling the carboxylic group of MTX to the hydroxyl group of PHEA did not show any promising activity against HeLa cells in vitro.11 With a similar direct coupling reaction, MTX attached to block or graft derivatives of PHEA could form micelle-like structures and modulate drug From the Department of Orthopedics (GZ, SW, XS, JZ), Jinling Hospital, Nanjing, China; Laboratory of Mesoscopic Chemistry and Department of Polymer Science and Engineering, College of Chemistry and Chemical Engineering (XC, XJ), and State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences (JC, JZ), Nanjing University, Nanjing, China. Submitted May 17, 2011; accepted in revised form November 7, 2011. The first 2 authors contributed equally to this work. Disclosure: The authors report no conflicts of interest. Correspondence: Jianning Zhao, MD, PhD, Department of Orthopedics, Jinling Hospital, Nanjing 210002, China (E-mail: zhaojianning.0207@ 163.com).
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release properties.12–14 However, these polymer-MTX conjugates have limited antitumor activities in vivo. The failure suggests that a suitable spacer between the drug and polymer and/or the presence of free -COOH groups of MTX, especially at the a-position, may be crucial for the activity of the conjugate.5,15 We have successfully developed a derivative of PHEA by modifying PHEA with succinic anhydride (PHEA-suc) in our previous study.16 The redundant hydrophilic succinoyl group in the polymer backbone, together with hydrophobic drugs, may form aggregates, promoting the specific delivery of MTX into tumor cells. We hypothesized that the succinic group could act as a spacer, linking MTX and PHEA, which might facilitate drug release in vitro and in vivo. This study aimed at testing the feasibility of PHEA-suc as the polymerdrug conjugate carrier for MTX by conjugating the -NH2 of MTX with the -COOH of PHEA-suc to form a new derivative, PHEA-MTX, and evaluating its anticancer activity and safety in vitro and in vivo. Our data indicated that the PHEA-MTX had potent antitumor activity against sarcoma in vivo with fewer side effects.
MATERIALS AND METHODS Materials L-Aspartic acid, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, benzotriazol-1-yloxytris(dimethylamino)-phosphonium hexafluorophosphate (BOP reagent), acridine orange and 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical Company (St. Louis, MO). MTX (purity . 98%) was kindly provided by Hengrui Pharmaceuticals (Lianyungang, China). Enzymatic reagent kits for determination of serum alanine aminotransferase (ALT), blood urea nitrogen, creatinine, total protein and albumin were purchased from Jiancheng Biochem Ltd. (Nanjing, China). Succinic anhydride, ethanolamine, N,N-dimethylformamide (DMF), 4-dimethylaminopyridine, sulfo-N-hydroxysuccinimide sodium salt, triethylamine and all other chemicals were of analytical grade and used without further purification. Preparation and Characterization of PHEA-MTX Conjugate PHEA and PHEA-suc were synthesized, as described previously.16 Briefly, PHEA was synthesized by polymerization of L-aspartic acid using 85% phosphoric acid as a catalyst, followed by a ring-opening reaction with ethanolamine. The mean molecular weight of PHEA was 23 kDa (Mw/Mn 5 1.76). PHEA-suc was prepared by esterifying the pendant hydroxyl groups of PHEA with succinic anhydride, using 4-dimethylaminopyridine as a catalyst. The degree of succinoylation of PHEA-suc was determined by titration as 11.8 mol%. The -NH2 groups of MTX were conjugated to PHEA-suc using BOP reagent in the salt-coupling method to protect carboxyl groups in the glutamate moiety of MTX from
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salt formation.17 PHEA-suc (500 mg) was dissolved in 50 mL of DMF and reacted with 350 mg of BOP reagent. A solution of MTX (350 mg) and K2CO3 (220 mg) in 50 mL of DMF was added to the reaction mixture and the temperature elevated to 50°C until a clear solution was obtained. After stirring for 24 hours, the solution was dialyzed against DMF for 2 days and against distilled water for 3 days and then lyophilized. The structure of PHEA-MTX was characterized by a Fourier transform infrared spectrometer (Nicolet 170SX, Thermo Nicolet Corporation, Madison, WI) and by 1H-nuclear magnetic resonance spectroscopy (Bruker DRX-500, Bruker BioSciences Corporation, Fällanden, Switzerland). Based on the assumption that the molar absorptivity of MTX was unchanged by conjugation, the MTX content of PHEA-MTX in 0.1 N HCl was determined by an ultraviolet (UV) spectrophotometer at 307 nm using the calibration curve of MTX.4 Free MTX encapsulated in the PHEA-MTX conjugate was measured by reversed-phase high-performance liquid chromatography with UV detection at 312 nm on an Extend-C18 column (4.6 3 250-mm internal diameter, 5 mm). The mobile phase used for the analysis was phosphate buffer (pH 5.5) and methanol:water (72:28, vol/vol), and the flow rate was 0.5 mL/min. The molecular weight of PHEA-MTX was determined by aqueous size-exclusion chromatography based on polyethylene oxide/polyethylene glycol standards. In Vitro Drug Release Assay MTX release from PHEA-MTX was studied in the buffering solutions at pH 5.5 (HAc-NaAc) and pH 7.4 (Na2HPO4-NaH2PO4), as described previously.16 The sample (2.5 mg) of PHEA-MTX was dissolved in 3 mL of buffering solution and then transferred into a dialysis membrane tube (with a molecular weight cutoff 14,000 Da) against 20 mL of the same buffering solution as releasing medium, with shaking at 37°C in dark for 72 hours. At determined intervals, 1 mL of sample was taken from outside of the tube and measured at 307 nm on a UV spectrophotometer. At the same time, 1 mL medium was supplemented. The cumulative amount of drug released was calculated taking into account the volume of sample solution removed. MTX release ratio was obtained by comparing the released MTX with the initial total MTX of the sample. In Vitro Cytotoxicity Assay Human osteosarcoma cell line MG63 was purchased from American Type Culture Collection (Manassas, VA) and used for in vitro cytotoxicity studies. The cytotoxic activity of PHEA-MTX was quantified by MTT assays. MG63 cells (1.0 3 104 cells/well) were cultured in 96-well plates in RPMI 1640 medium, supplemented with 10% fetal bovine serum for 24 hours. The cells were treated in triplicate with various concentrations of MTX and PHEA-MTX for 72 hours at 37°C. The cells cultured in medium alone were used as negative controls. During the last 4 hours of culture, the cells were exposed to 20 mL of MTT solution (5 mg/mL), and the resultant formazan crystals were dissolved in dimethyl sulfoxide and measured at 550 nm on a microplate reader. The cytotoxicity of individual compounds was expressed as the percentage of the negative controls. The effect of individual compounds on cell apoptosis was observed under a fluorescence microscope. MG63 cells were treated with individual drugs for 72 hours and harvested. The cells (at 107 cells/mL) were stained with 0.1 mg/mL of acridine orange (5 mL) and examined under a fluorescence microscope. Ó 2012 Lippincott Williams & Wilkins
Animal Models Institute of Cancer Research (ICR) mice (18–22 g) were obtained from the Experimental Animal Center of Nanjing Medical University (Nanjing, China) and housed in our specific pathogen-free facility at 21 6 2°C with free access to food pellets and water. Mouse S180 sarcoma cells were obtained from American Type Culture Collection and propagated by intraperitoneal implantation in female ICR mice. ICR mice were inoculated subcutaneously with 2.4 3 106 cells at the axillary region of the animals for solid tumor formation. The experimental protocol was approved by the ethics committee of the university, in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (Publication No. 85-23, revised 1985). The S180 tumor-bearing mice were randomized into 3 groups (n 5 10 per group) on day 5 after inoculation and treated intraperitoneally with saline, MTX (20 mg/kg) or PHEA-MTX (MTX equivalent of 20 mg/kg) on days 5, 7, 9, 11 and 13 after inoculation. The progression of tumors was monitored every other day up to 32 days post-inoculation, and the tumor volume was calculated using the following formula: 1/2 3 a 3 b2, where a is the tumor length and b the width. Body weights were recorded accordingly. At the end of this experiment, the tumors were dissected out, weighted and fixed in 10% neutral buffered formalin solution overnight. Thin paraffin-embedded tumor sections (5 mm) were prepared and stained with hematoxylin and eosin for histological analysis. In Vivo Safety Assessment The S180 tumor-bearing mice (n 5 10 per group) were treated with saline, MTX or PHEA-MTX, as described previously. The mice were carefully observed daily for behavioral changes and survivors. One day after the last drug treatment, blood samples were collected from the mice for routine examination using an autohematology analyzer. Mouse sera were tested for the levels of serum ALT, blood urea nitrogen, creatinine, total protein and albumin using enzymatic reagent kits. Mouse tissues, including the liver, lung, kidney and heart, were dissected out and subjected to histological analysis as mentioned previously. Statistical Analysis All results are expressed as mean 6 standard deviation, unless otherwise noted. The difference among the different groups was analyzed by paired Student’s t test and using oneway analysis of variance for multiple groups. A value of P # 0.05 was considered statistically significant.
RESULTS AND DISCUSSION MTX has been linked with polymers to improve its therapeutic efficacy, which is attributed to its easily modifiable functional groups. For example, both the a- and g-COOH groups in the glutamate moiety and the -NH2 groups at the second and fourth positions in the 2,4-diamino-6-pteridinyl group can be easily modified. However, previous studies have shown that the presence of free -COOH groups, especially at the a-position, is crucial for the maintenance of its cytotoxic activity.5,18 Substitution at the 2-NH2 of the pteridine ring can reduce the inhibitory effects of MTX on dihydrofolate reductase.1 How would modification of MTX improve its activity? In this study, we tested the feasibility of PHEA-suc as a drug carrier and used the PHEAsuc we previously developed to generate the polymer-MTX conjugate by conjugating the -NH2 group in the pteridine of MTX to the PHEA-suc as the succinyl group might facilitate drug release
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FIGURE 1. Structure and characterization of PHEA-MTX. (A) Chemical structure of PHEA-MTX; (B) Fourier transform infrared spectra of MTX (a) and PHEAMTX (b); (C) 1H-nuclear magnetic resonance spectra of MTX (a) and PHEA-MTX (b) dissolved in dimethyl sulfoxide-d6. Data shown are representatives of each compound from 3 independent experiments. MTX, methotrexate; PHEA, a,b-poly[(2-hydroxyethyl)L-aspartamide.
and delivery of the drug into tumors. Subsequently, we evaluated the activity and safety of a novel polymer-MTX conjugate in vitro and in vivo. Preparation and Characterization of PHEA-MTX Conjugate The structure of PHEA-MTX conjugate is outlined in Figure 1A. It did not concern whether the -NH2 at the second or fourth position in the pteridinyl group was used for conjugation. The resultant PHEA-MTX was characterized by infrared spectroscopy (Figure 1B). The PHEA-MTX showed a new signal at 1789.6 cm21, which belonged to the -COOH of MTX, and the
FIGURE 2. In vitro release of MTX from the PHEA-MTX conjugate. PHEA-MTX was dissolved in buffer solutions at pH 5.5 or pH 7.4 at 37˚C, and the released MTX was determined by ultraviolet spectrophotometry. Data are expressed as the mean percent 6 standard deviation of cumulative release in the total amount of MTX from 4 experiments. MTX, methotrexate; PHEA, a,b-poly [(2-hydroxyethyl)-L-aspartamide.
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typical ester bonds of PHEA-suc16 had shifted from 1738.5 to 1716.6 cm21. The structure of PHEA-MTX was further confirmed by 1H-nuclear magnetic resonance spectroscopy (Figure 1C). The specific peaks of MTX were detected at 6.82 ppm (aromatic CH-2’,6’) and 8.53 ppm (aromatic CH-7).18 New peaks appeared at 5.27, 5.0 and 1.91 ppm
FIGURE 3. In vitro cytotoxicity evaluations of MTX and PHEAMTX against MG63 cells. MG63 cells were treated with the indicated concentrations of each drug for varying periods and the cell viability was determined by MTT assays. Data are expressed as the mean percent 6 standard deviation of viable cells in each group from 3 separate experiments. Data shown are representative of individual groups of cells. Unmanipulated cells were used as negative controls. MTT, 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide; MTX, methotrexate; PHEA, a,b-poly[(2-hydroxyethyl)-L-aspartamide. Volume 344, Number 4, October 2012
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ence in releasing rate between pH 5.5 and pH 7.4 buffer solutions was consistent with our previous report.16 Although drug release in vitro is a prerequisite for the biological activity of the conjugate, the dynamics of drug release in vitro might not be the same in vivo. The precise pharmacokinetics of PHEA-MTX in vivo remains to be determined.
FIGURE 4. (A) In vitro cell apoptosis evaluations of MTX and PHEA-MTX against MG63 cells. MG63 cells were treated with 100 mg/mL of MTX and PHEA-MTX for 72 hours and stained with the fluorescent dye acridine orange, followed by an examination under a fluorescence microscope. (B) Histological analysis of tumor sections by hematoxylin and eosin staining after treatment with saline, MTX (20 mg/kg) or PHEA-MTX (MTX equivalent of 20 mg/kg). MTX, methotrexate; PHEA, a,b-poly[(2-hydroxyethyl)-L-aspartamide.
because of the conjugation. MTX content in the PHEA-MTX conjugate was determined to be 5.86 wt%, with no remarkable amount of free MTX (,2 wt% of the total MTX content). The molecular weight of the PHEA-MTX conjugate was determined to be 25 kDa (Mw/Mn 5 1.65). In Vitro Release Studies MTX release behavior from the conjugate was studied in vitro. As shown in Figure 2, MTX was continually released from PHEA-MTX in the buffering solutions tested, and the percentage of MTX released increased with time. The differ-
In Vitro Cytotoxicity Assay To determine the activity of PHEA-MTX, MG63 cells were treated with different concentrations of PHEA-MTX or MTX for varying periods, and the cytotoxicity of PHEA-MTX and MTX was determined by MTT assays (Figure 3). Both MTX and PHEA-MTX showed similar levels of cytotoxicity against human osteosarcoma cells in a dose-dependent and time-dependent manner. PHEA-MTX had a less potent cytotoxic effect than free MTX in the first 2 days but later showed comparable levels of cytotoxicity against human osteosarcoma cells. The difference in pharmacodynamics between PHEAMTX and free MTX may be because of the low penetration of the polymer-drug conjugate as a result of endocytosis, whereas free drug can diffuse freely into the cells.19 Furthermore, MTX was gradually released from the PHEA-MTX conjugate, which would result in lower drug availability compared with free MTX during the early period. However, the endocytosed and released drug would increase the concentration of MTX, which had a potent cytotoxic effect. Alternatively, the tested cells might be resistant to high concentrations of MTX (10 mg/mL).20 The potent cytotoxic effect of PHEA-MTX indicated that the -NH2 group of MTX was feasible for generating a conjugate and did not inactivate MTX activity. MTX can inhibit DNA synthesis and induce apoptosis in chemotherapy.21 We examined whether the PHEA-MTX conjugate could retain the pro-apoptotic activity by staining the drug-treated cells with the fluorescent dye acridine orange.22 As shown in Figure 4A, similar frequency and intensity of
FIGURE 5. In vivo antitumor activity. ICR mice were implanted with S180 cells and randomized into groups that were treated intraperitoneally with saline, MTX (20 mg/kg) or PHEA-MTX (MTX equivalent of 20 mg/kg) on days 5, 7, 9, 11 and 13 after inoculation. The tumor sizes were monitored and body weights measured up to 32 days post-inoculation. At the end of the experiment, the tumors were dissected and imaged. (A) Tumor volume, (B) the net gain in body weights and (C) the representative tumors. Data shown are representative images or expressed as mean 6 standard deviation of each group of mice (n 5 10). MTX, methotrexate; PHEA-suc, succinoylated a,b-poly[(2-hydroxyethyl)-L-aspartamide. Ó 2012 Lippincott Williams & Wilkins
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*Significantly different from control at P , 0.05. ALT, alanine aminotransferase; BUN, blood urea nitrogen; MTX, methotrexate; PHEA, a,b-poly[(2-hydroxyethyl)-L-aspartamide]; RBC, read blood cell; TP, total protein; WBC, white blood cell.
28.5 6 3.2 27.4 6 2.8 30.2 6 1.9 57.4 6 4.6 60.2 6 5.8 59.4 6 5.3 73.5 6 4.2 96.8 6 5.2* 76.2 6 5.2 6.9 6 0.3 8.1 6 0.8* 7.1 6 0.3 28.4 6 2.1 38.7 6 2.2* 28.7 6 1.6 71.4 6 9.2 89.3 6 8.0* 73.5 6 4.2 103 6 8.8 95 6 5.3 99 6 3.8 617 6 45 368 6 31* 587 6 42 6.9 6 0.22 6.3 6 0.35 6.6 6 0.52 2.8 6 0.31 0.7 6 0.21* 2.5 6 0.16 Control MTX PHEA-MTX
TABLE 1. Blood routine and serum biochemical indices of S180 tumor-bearing mice at 14 days after drug administration (mean 6 standard deviation) WBC RBC Platelets Hemoglobin Lymphocytes (%) ALT (U/L) BUN (mmol/L) Creatinine (mmol/L) TP (g/L)
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fluorescent cells were observed in both MTX-treated and PHEA-MTX-treated cells, indicating that PHEA-MTX retained the pro-apoptotic activity of MTX. In Vivo Antitumor Activity To examine the antitumor efficacy of PHEA-MTX, groups of ICR mice were implanted subcutaneously with S180 cells and treated with saline, MTX (20 mg/kg) or PHEA-MTX (MTX equivalent of 20 mg/kg) every other day for 5 days, and the development and progression of solid tumors were monitored up to 32 days post-inoculation (Figure 5). No death was observed, regardless of treatment employed. Whereas the solid tumors in the saline-injected group of mice grew gradually over the observation period, treatment with MTX inhibited the growth of tumors by 85% and treatment with PHEA-MTX further reduced the tumor volume (Figures 5A, B). Histological analysis of the tumor tissues revealed high necrotic levels of cells in the tumors from the PHEA-MTX-treated mice, whereas hypercellular sheets of monomorphic oval cells were observed in the tumors from the salineinjected mice (Figure 4B). In addition, the net gain in body weight in the PHEA-MTX-treated mice was similar to that of the saline-injected mice, although treatment with MTX dramatically reduced the body weight during the first several days (Figure 5B). Apparently, whereas MTX had systemic toxicity, PHEA-MTX may be well tolerated in mice. These data indicate that PHEA-MTX retained its antitumor activity, which was more potent than that of free MTX. The enhanced antitumor activity of PHEA-MTX may be attributed to the accumulation of PHEAMTX in tumor cells by an enhanced permeability and retention effect23 and also to the prolonged gradual release of the drug. Because of the pendant hydrophilic succinic group and hydrophobic MTX residue, PHEA-MTX is likely to form aggregates in biological fluids,24 contributing to the enhanced permeability and retention effect. In Vivo Safety Assessment After drug treatments, we monitored mouse behaviors and found that the PHEA-MTX-treated mice had much alleviated piloerection and adynamia compared with the
FIGURE 6. Histological analysis of the liver, lung, kidney and heart of S180 tumor-bearing mice. Two weeks after inoculation, the liver, lung, kidney and heart from each mouse were dissected and subjected to hematoxylin and eosin staining. Volume 344, Number 4, October 2012
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MTX-treated group. Two weeks after drug treatments, mouse blood cell counts were determined by routine examination (Table 1). Treatment with MTX reduced the number of white blood cells, platelets and lymphocytes but increased the levels of serum ALT, blood urea nitrogen and creatinine. In contrast, treatment with PHEA-MTX did not significantly change blood counts and serum biochemical indices. Further histological examination (Figure 6) revealed that treatment with MTX caused obvious liver lesions with denatured liver cells and histiocytosis of cells surrounding the hepatic sinusoid, whereas PHEA-MTX did not cause significant lesions in the liver. There was no significant difference in the lungs, kidneys and hearts among the different groups of mice. Clearly, in the treated doses, MTX exhibited obvious side effects on the liver and immune cells,25 whereas the conjugated PHEA-MTX had fewer side effects. This phenomenon may stem from PHEA-MTX’s unique pharmacokinetic property, gradual release of MTX and levels of selectivity.26 We are interested in further investigating the mechanisms by which PHEA-MTX exhibits fewer side effects in mice.
CONCLUSIONS A polymer conjugate of PHEA-MTX with a succinic spacer was developed by coupling the amino bonds of MTX to PHEA-suc. This conjugate released MTX gradually in buffer solutions tested. Both PHEA-MTX and MTX showed comparable levels of cytotoxicity and pro-apoptotic activity against human osteosarcoma cells in a concentration-dependent and time-dependent manner. PHEA-MTX showed superior antitumor activity with fewer side effects in mice, as compared with MTX. These data demonstrate that PHEA-suc is suitable for use as a drug carrier and indicate that PHEA-MTX is a promising polymeric drug for chemotherapy. REFERENCES 1. Smal MA, Dong Z, Cheung HTA, et al. Activation and cytotoxicity of 2-a-aminoacyl prodrugs of methotrexate. Biochem Pharmacol 1995;49: 567–74. 2. Zhang Y, Jin T, Zhuo RX. Methotrexate-loaded biodegradable polymeric micelles: preparation, physicochemical properties and in vitro drug release. Colloids Surf B Biointerfaces 2005;44:104–9. 3. Seo DH, Jeong YI, Kim DG, et al. Methotrexate-incorporated polymeric nanoparticles of methoxy poly(ethylene glycol)-grafted chitosan. Colloids Surf B Biointerfaces 2009;69:157–63. 4. Subr V, Strohalm J, Hirano T, et al. Poly [N-(2-hydroxypropyl)methacrylamide] conjugates of methotrexate: synthesis and in vitro drug release. J Control Release 1997;49:123–32. 5. Gurdag S, Khandare J, Stapels S, et al. Activity of dendrimer-methotrexate conjugates on methotrexate-sensitive and -resistant cell lines. Bioconjug Chem 2006;17:275–83. 6. Smith GK, Banks S, Blumenkopf TA, et al. Toward antibody-directed enzyme prodrug therapy with the T268G mutant of human carboxypeptidase a1 and novel in vivo stable prodrugs of methotrexate. J Biol Chem 1997;272:15804–16. 7. Cavallaro G, Licciardi M, Caliceti P, et al. Synthesis, physico-chemical and biological characterization of a paclitaxel macromolecular prodrug. Eur J Pharm Biopharm 2004;58:151–9. 8. Wolfe LA, Mullin RJ, Laethem R, et al. Antibody-directed enzyme prodrug therapy with the T268G mutant of human carboxypeptidase A1: in vitro and in vivo studies with prodrugs of methotrexate and the thymidylate synthase inhibitors GW1031 and GW1843. Bioconjug Chem 1999;10:38–48.
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9. Cavallaro G, Maniscalco L, Campisi M, et al. Synthesis, characterization and in vitro cytotoxicity studies of a macromolecular conjugate of paclitaxel bearing oxytocin as targeting moiety. Eur J Pharm Biopharm 2007;66:182–92. 10. Cavallaro G, Mariano L, Salmaso S, et al. Folate-mediated targeting of polymeric conjugates of gemcitabine. Int J Pharm 2006;307:258–69. 11. Yang SR, Kim SB, Joe CO, et al. Intracellular delivery enhancement of poly(amino acid) drug carriers by oligoarginine conjugation. J Biomed Mater Res A 2008;86:137–48. 12. Kang H, Kim JD, Han SH, et al. Self-aggregates of poly(2-hydroxyethyl aspartamide) copolymers loaded with methotrexate by physical and chemical entrapments. J Control Release 2002;81:135–44. 13. Li Y, Kwon GS. Micelle-like structures of poly(ethylene oxide)-blockpoly(2-hydroxyethyl aspartamide)-methotrexate conjugates. Colloids Surf B Biointerfaces 1999;16:217–26. 14. Li Y, Kwon GS. Methotrexate esters of poly (ethylene oxide)-blockpoly(2-hydroxyethyl-L-aspartamide). Part I: effects of the level of methotrexate conjugation on the stability of micelles and on drug release. Pharm Res 2000;17:607–11. 15. Pignatello R, Spampinato G, Sorrenti V, et al. Lipophilic methotrexate conjugates with antitumor activity. Eur J Pharm Sci 2000;10:237– 45. 16. Cheng XY, Xue WH, Diao HJ, et al. Antitumor activity and toxicological properties of doxorubicin conjugated to a,b-poly[(2-hydroxyethyl)-L-aspartamide] administrated intraperitoneally in mice. Anticancer Drugs 2010;21:362–71. 17. Nagy A, Szoke B, Schally AV. Selective coupling of methotrexate to peptide hormone carriers through a g-carboxamide linkage of its glutamic acid moiety: benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate activation in salt coupling. Proc Natl Acad Sci U S A 1993;90:6373–6. 18. Pignatello R, Guccione S, Forte S, et al. Lipophilic conjugates of methotrexate with short-chain alkylamino acids as DHFR inhibitors. Synthesis, biological evaluation, and molecular modeling. Bioorg Med Chem 2004;12:2951–64. 19. Hovorka O, St’astný M, Etrych T, et al. Differences in the intracellular fate of free and polymer-bound doxorubicin. J Control Release 2002;80:101–17. 20. Diddens H, Niethammer D, Jackson RC. Patterns of cross-resistance to the antifolate drugs trimetrexate, metoprine, homofolate, and CB3717 in human lymphoma and osteosarcoma cells resistant to methotrexate. Cancer Res 1983;43:5286–92. 21. Celtikci B, Lawrance AK, Wu Q, et al. Methotrexate-induced apoptosis is enhanced by altered expression of methylenetetrahydrofolate reductase. Anticancer Drugs 2009;20:787–93. 22. Darzynkiewicz Z, Bruno S, Del BG, et al. Features of apoptotic cells measured by flow cytometry. Cytometry 1992;13:795–808. 23. Iyer AK, Khaled G, Fang J, et al. Exploiting the enhanced permeability and retention effect for tumor targeting. Drug Discov Today 2006;11:812–8. 24. Gautier S, Boustta M, Vert M. Alkylated poly(L-lysine citramide) as models to investigate the ability of amphiphilic macromolecular drug carriers to physically entrap lipophilic compounds in aqueous media. J Control Release 1999;60:235–47. 25. Han JH, Oh YK, Kim DS, et al. Enhanced hepatocyte uptake and liver targeting of methotrexate using galactosylated albumin as a carrier. Int J Pharm 1999;188:39–47. 26. Chau Y, Dang NM, Tan FE, et al. Investigation of targeting mechanism of new dextran-peptide-methotrexate conjugates using biodistribution study in matrix-metalloproteinase-overexpressing tumor xenograft model. J Pharm Sci 2006;95:542–51.
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Preparation and Antitumor Activity of a Polymeric Derivative of Methotrexate Guangxin Zhou, PhD, Xiaoyun Cheng, PhD, Sujia Wu, MD, PhD, Xiqun Jiang, PhD, Xin Shi, MD, PhD, Jiangning Chen, PhD, Junfeng Zhang, PhD and Jianning Zhao, MD, PhD
Abstract: A polymer-drug conjugate was developed by conjugating amino bonds of methotrexate (MTX) to succinoylated a,b-poly[(2hydroxyethyl)-L-aspartamide] (PHEA). The therapeutic efficacy of PHEA-MTX was evaluated in vitro and in vivo. PHEA-MTX showed sustained release properties when incubated in pH 5.5 and pH 7.4 buffering solutions at 37°C. PHEA-MTX induced MG63 cell apoptosis in a time-dependent and concentration-dependent manner in vitro and inhibited the growth of S180 sarcoma in vivo. PHEA-MTX was more potent and, more importantly, displayed less systemic toxicity than free MTX. The enhanced therapeutic effects of PHEA-MTX suggest that the PHEA-MTX conjugate may have a greater potential for chemotherapy of cancers. Key Indexing Terms: PHEA; Methotrexate; Antitumor efficacy; Drug delivery; Polymer prodrug. [Am J Med Sci 2012;344(4):294–299.]
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ethotrexate (MTX) is one of the most commonly used antineoplastic agents for chemotherapy of cancers. However, it has a potent inhibitory effect on the metabolism of folic acid, leading to broad cytotoxicity against many types of proliferative cells, such as bone marrow, mucosal and lymphoid cells.1 To reduce MTX-related adverse effects, a series of drug delivery systems have been attempted, such as encapsulated microspheres,2,3 macromolecule conjugates4,5 and antibody conjugates,6–8 to improve the specificity and selectivity of MTX. However, the efficiency of these strategies is limited. Therefore, development of new derivatives of MTX will be of great significance in effectively and safely treating patients with cancer. Application of a polymer carrier to deliver MTX may be a promising strategy for improving the therapeutic efficacy and safety of MTX. The biological property of a polymer-drug conjugate depends on the types of polymers and the nature of linker between the drug and polymer. The synthetic polymer a,b-poly[(2-hydroxyethyl)-L-aspartamide] (PHEA) has been found to be potentially useful in the delivery of drugs because it is water soluble, biocompatible, easily obtained and modified.7,9,10 However, a PHEA-MTX conjugate formed by directly coupling the carboxylic group of MTX to the hydroxyl group of PHEA did not show any promising activity against HeLa cells in vitro.11 With a similar direct coupling reaction, MTX attached to block or graft derivatives of PHEA could form micelle-like structures and modulate drug From the Department of Orthopedics (GZ, SW, XS, JZ), Jinling Hospital, Nanjing, China; Laboratory of Mesoscopic Chemistry and Department of Polymer Science and Engineering, College of Chemistry and Chemical Engineering (XC, XJ), and State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences (JC, JZ), Nanjing University, Nanjing, China. Submitted May 17, 2011; accepted in revised form November 7, 2011. The first 2 authors contributed equally to this work. Disclosure: The authors report no conflicts of interest. Correspondence: Jianning Zhao, MD, PhD, Department of Orthopedics, Jinling Hospital, Nanjing 210002, China (E-mail: zhaojianning.0207@ 163.com).
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release properties.12–14 However, these polymer-MTX conjugates have limited antitumor activities in vivo. The failure suggests that a suitable spacer between the drug and polymer and/or the presence of free -COOH groups of MTX, especially at the a-position, may be crucial for the activity of the conjugate.5,15 We have successfully developed a derivative of PHEA by modifying PHEA with succinic anhydride (PHEA-suc) in our previous study.16 The redundant hydrophilic succinoyl group in the polymer backbone, together with hydrophobic drugs, may form aggregates, promoting the specific delivery of MTX into tumor cells. We hypothesized that the succinic group could act as a spacer, linking MTX and PHEA, which might facilitate drug release in vitro and in vivo. This study aimed at testing the feasibility of PHEA-suc as the polymerdrug conjugate carrier for MTX by conjugating the -NH2 of MTX with the -COOH of PHEA-suc to form a new derivative, PHEA-MTX, and evaluating its anticancer activity and safety in vitro and in vivo. Our data indicated that the PHEA-MTX had potent antitumor activity against sarcoma in vivo with fewer side effects.
MATERIALS AND METHODS Materials L-Aspartic acid, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, benzotriazol-1-yloxytris(dimethylamino)-phosphonium hexafluorophosphate (BOP reagent), acridine orange and 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical Company (St. Louis, MO). MTX (purity . 98%) was kindly provided by Hengrui Pharmaceuticals (Lianyungang, China). Enzymatic reagent kits for determination of serum alanine aminotransferase (ALT), blood urea nitrogen, creatinine, total protein and albumin were purchased from Jiancheng Biochem Ltd. (Nanjing, China). Succinic anhydride, ethanolamine, N,N-dimethylformamide (DMF), 4-dimethylaminopyridine, sulfo-N-hydroxysuccinimide sodium salt, triethylamine and all other chemicals were of analytical grade and used without further purification. Preparation and Characterization of PHEA-MTX Conjugate PHEA and PHEA-suc were synthesized, as described previously.16 Briefly, PHEA was synthesized by polymerization of L-aspartic acid using 85% phosphoric acid as a catalyst, followed by a ring-opening reaction with ethanolamine. The mean molecular weight of PHEA was 23 kDa (Mw/Mn 5 1.76). PHEA-suc was prepared by esterifying the pendant hydroxyl groups of PHEA with succinic anhydride, using 4-dimethylaminopyridine as a catalyst. The degree of succinoylation of PHEA-suc was determined by titration as 11.8 mol%. The -NH2 groups of MTX were conjugated to PHEA-suc using BOP reagent in the salt-coupling method to protect carboxyl groups in the glutamate moiety of MTX from
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salt formation.17 PHEA-suc (500 mg) was dissolved in 50 mL of DMF and reacted with 350 mg of BOP reagent. A solution of MTX (350 mg) and K2CO3 (220 mg) in 50 mL of DMF was added to the reaction mixture and the temperature elevated to 50°C until a clear solution was obtained. After stirring for 24 hours, the solution was dialyzed against DMF for 2 days and against distilled water for 3 days and then lyophilized. The structure of PHEA-MTX was characterized by a Fourier transform infrared spectrometer (Nicolet 170SX, Thermo Nicolet Corporation, Madison, WI) and by 1H-nuclear magnetic resonance spectroscopy (Bruker DRX-500, Bruker BioSciences Corporation, Fällanden, Switzerland). Based on the assumption that the molar absorptivity of MTX was unchanged by conjugation, the MTX content of PHEA-MTX in 0.1 N HCl was determined by an ultraviolet (UV) spectrophotometer at 307 nm using the calibration curve of MTX.4 Free MTX encapsulated in the PHEA-MTX conjugate was measured by reversed-phase high-performance liquid chromatography with UV detection at 312 nm on an Extend-C18 column (4.6 3 250-mm internal diameter, 5 mm). The mobile phase used for the analysis was phosphate buffer (pH 5.5) and methanol:water (72:28, vol/vol), and the flow rate was 0.5 mL/min. The molecular weight of PHEA-MTX was determined by aqueous size-exclusion chromatography based on polyethylene oxide/polyethylene glycol standards. In Vitro Drug Release Assay MTX release from PHEA-MTX was studied in the buffering solutions at pH 5.5 (HAc-NaAc) and pH 7.4 (Na2HPO4-NaH2PO4), as described previously.16 The sample (2.5 mg) of PHEA-MTX was dissolved in 3 mL of buffering solution and then transferred into a dialysis membrane tube (with a molecular weight cutoff 14,000 Da) against 20 mL of the same buffering solution as releasing medium, with shaking at 37°C in dark for 72 hours. At determined intervals, 1 mL of sample was taken from outside of the tube and measured at 307 nm on a UV spectrophotometer. At the same time, 1 mL medium was supplemented. The cumulative amount of drug released was calculated taking into account the volume of sample solution removed. MTX release ratio was obtained by comparing the released MTX with the initial total MTX of the sample. In Vitro Cytotoxicity Assay Human osteosarcoma cell line MG63 was purchased from American Type Culture Collection (Manassas, VA) and used for in vitro cytotoxicity studies. The cytotoxic activity of PHEA-MTX was quantified by MTT assays. MG63 cells (1.0 3 104 cells/well) were cultured in 96-well plates in RPMI 1640 medium, supplemented with 10% fetal bovine serum for 24 hours. The cells were treated in triplicate with various concentrations of MTX and PHEA-MTX for 72 hours at 37°C. The cells cultured in medium alone were used as negative controls. During the last 4 hours of culture, the cells were exposed to 20 mL of MTT solution (5 mg/mL), and the resultant formazan crystals were dissolved in dimethyl sulfoxide and measured at 550 nm on a microplate reader. The cytotoxicity of individual compounds was expressed as the percentage of the negative controls. The effect of individual compounds on cell apoptosis was observed under a fluorescence microscope. MG63 cells were treated with individual drugs for 72 hours and harvested. The cells (at 107 cells/mL) were stained with 0.1 mg/mL of acridine orange (5 mL) and examined under a fluorescence microscope. Ó 2012 Lippincott Williams & Wilkins
Animal Models Institute of Cancer Research (ICR) mice (18–22 g) were obtained from the Experimental Animal Center of Nanjing Medical University (Nanjing, China) and housed in our specific pathogen-free facility at 21 6 2°C with free access to food pellets and water. Mouse S180 sarcoma cells were obtained from American Type Culture Collection and propagated by intraperitoneal implantation in female ICR mice. ICR mice were inoculated subcutaneously with 2.4 3 106 cells at the axillary region of the animals for solid tumor formation. The experimental protocol was approved by the ethics committee of the university, in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (Publication No. 85-23, revised 1985). The S180 tumor-bearing mice were randomized into 3 groups (n 5 10 per group) on day 5 after inoculation and treated intraperitoneally with saline, MTX (20 mg/kg) or PHEA-MTX (MTX equivalent of 20 mg/kg) on days 5, 7, 9, 11 and 13 after inoculation. The progression of tumors was monitored every other day up to 32 days post-inoculation, and the tumor volume was calculated using the following formula: 1/2 3 a 3 b2, where a is the tumor length and b the width. Body weights were recorded accordingly. At the end of this experiment, the tumors were dissected out, weighted and fixed in 10% neutral buffered formalin solution overnight. Thin paraffin-embedded tumor sections (5 mm) were prepared and stained with hematoxylin and eosin for histological analysis. In Vivo Safety Assessment The S180 tumor-bearing mice (n 5 10 per group) were treated with saline, MTX or PHEA-MTX, as described previously. The mice were carefully observed daily for behavioral changes and survivors. One day after the last drug treatment, blood samples were collected from the mice for routine examination using an autohematology analyzer. Mouse sera were tested for the levels of serum ALT, blood urea nitrogen, creatinine, total protein and albumin using enzymatic reagent kits. Mouse tissues, including the liver, lung, kidney and heart, were dissected out and subjected to histological analysis as mentioned previously. Statistical Analysis All results are expressed as mean 6 standard deviation, unless otherwise noted. The difference among the different groups was analyzed by paired Student’s t test and using oneway analysis of variance for multiple groups. A value of P # 0.05 was considered statistically significant.
RESULTS AND DISCUSSION MTX has been linked with polymers to improve its therapeutic efficacy, which is attributed to its easily modifiable functional groups. For example, both the a- and g-COOH groups in the glutamate moiety and the -NH2 groups at the second and fourth positions in the 2,4-diamino-6-pteridinyl group can be easily modified. However, previous studies have shown that the presence of free -COOH groups, especially at the a-position, is crucial for the maintenance of its cytotoxic activity.5,18 Substitution at the 2-NH2 of the pteridine ring can reduce the inhibitory effects of MTX on dihydrofolate reductase.1 How would modification of MTX improve its activity? In this study, we tested the feasibility of PHEA-suc as a drug carrier and used the PHEAsuc we previously developed to generate the polymer-MTX conjugate by conjugating the -NH2 group in the pteridine of MTX to the PHEA-suc as the succinyl group might facilitate drug release
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FIGURE 1. Structure and characterization of PHEA-MTX. (A) Chemical structure of PHEA-MTX; (B) Fourier transform infrared spectra of MTX (a) and PHEAMTX (b); (C) 1H-nuclear magnetic resonance spectra of MTX (a) and PHEA-MTX (b) dissolved in dimethyl sulfoxide-d6. Data shown are representatives of each compound from 3 independent experiments. MTX, methotrexate; PHEA, a,b-poly[(2-hydroxyethyl)L-aspartamide.
and delivery of the drug into tumors. Subsequently, we evaluated the activity and safety of a novel polymer-MTX conjugate in vitro and in vivo. Preparation and Characterization of PHEA-MTX Conjugate The structure of PHEA-MTX conjugate is outlined in Figure 1A. It did not concern whether the -NH2 at the second or fourth position in the pteridinyl group was used for conjugation. The resultant PHEA-MTX was characterized by infrared spectroscopy (Figure 1B). The PHEA-MTX showed a new signal at 1789.6 cm21, which belonged to the -COOH of MTX, and the
FIGURE 2. In vitro release of MTX from the PHEA-MTX conjugate. PHEA-MTX was dissolved in buffer solutions at pH 5.5 or pH 7.4 at 37˚C, and the released MTX was determined by ultraviolet spectrophotometry. Data are expressed as the mean percent 6 standard deviation of cumulative release in the total amount of MTX from 4 experiments. MTX, methotrexate; PHEA, a,b-poly [(2-hydroxyethyl)-L-aspartamide.
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typical ester bonds of PHEA-suc16 had shifted from 1738.5 to 1716.6 cm21. The structure of PHEA-MTX was further confirmed by 1H-nuclear magnetic resonance spectroscopy (Figure 1C). The specific peaks of MTX were detected at 6.82 ppm (aromatic CH-2’,6’) and 8.53 ppm (aromatic CH-7).18 New peaks appeared at 5.27, 5.0 and 1.91 ppm
FIGURE 3. In vitro cytotoxicity evaluations of MTX and PHEAMTX against MG63 cells. MG63 cells were treated with the indicated concentrations of each drug for varying periods and the cell viability was determined by MTT assays. Data are expressed as the mean percent 6 standard deviation of viable cells in each group from 3 separate experiments. Data shown are representative of individual groups of cells. Unmanipulated cells were used as negative controls. MTT, 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide; MTX, methotrexate; PHEA, a,b-poly[(2-hydroxyethyl)-L-aspartamide. Volume 344, Number 4, October 2012
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ence in releasing rate between pH 5.5 and pH 7.4 buffer solutions was consistent with our previous report.16 Although drug release in vitro is a prerequisite for the biological activity of the conjugate, the dynamics of drug release in vitro might not be the same in vivo. The precise pharmacokinetics of PHEA-MTX in vivo remains to be determined.
FIGURE 4. (A) In vitro cell apoptosis evaluations of MTX and PHEA-MTX against MG63 cells. MG63 cells were treated with 100 mg/mL of MTX and PHEA-MTX for 72 hours and stained with the fluorescent dye acridine orange, followed by an examination under a fluorescence microscope. (B) Histological analysis of tumor sections by hematoxylin and eosin staining after treatment with saline, MTX (20 mg/kg) or PHEA-MTX (MTX equivalent of 20 mg/kg). MTX, methotrexate; PHEA, a,b-poly[(2-hydroxyethyl)-L-aspartamide.
because of the conjugation. MTX content in the PHEA-MTX conjugate was determined to be 5.86 wt%, with no remarkable amount of free MTX (,2 wt% of the total MTX content). The molecular weight of the PHEA-MTX conjugate was determined to be 25 kDa (Mw/Mn 5 1.65). In Vitro Release Studies MTX release behavior from the conjugate was studied in vitro. As shown in Figure 2, MTX was continually released from PHEA-MTX in the buffering solutions tested, and the percentage of MTX released increased with time. The differ-
In Vitro Cytotoxicity Assay To determine the activity of PHEA-MTX, MG63 cells were treated with different concentrations of PHEA-MTX or MTX for varying periods, and the cytotoxicity of PHEA-MTX and MTX was determined by MTT assays (Figure 3). Both MTX and PHEA-MTX showed similar levels of cytotoxicity against human osteosarcoma cells in a dose-dependent and time-dependent manner. PHEA-MTX had a less potent cytotoxic effect than free MTX in the first 2 days but later showed comparable levels of cytotoxicity against human osteosarcoma cells. The difference in pharmacodynamics between PHEAMTX and free MTX may be because of the low penetration of the polymer-drug conjugate as a result of endocytosis, whereas free drug can diffuse freely into the cells.19 Furthermore, MTX was gradually released from the PHEA-MTX conjugate, which would result in lower drug availability compared with free MTX during the early period. However, the endocytosed and released drug would increase the concentration of MTX, which had a potent cytotoxic effect. Alternatively, the tested cells might be resistant to high concentrations of MTX (10 mg/mL).20 The potent cytotoxic effect of PHEA-MTX indicated that the -NH2 group of MTX was feasible for generating a conjugate and did not inactivate MTX activity. MTX can inhibit DNA synthesis and induce apoptosis in chemotherapy.21 We examined whether the PHEA-MTX conjugate could retain the pro-apoptotic activity by staining the drug-treated cells with the fluorescent dye acridine orange.22 As shown in Figure 4A, similar frequency and intensity of
FIGURE 5. In vivo antitumor activity. ICR mice were implanted with S180 cells and randomized into groups that were treated intraperitoneally with saline, MTX (20 mg/kg) or PHEA-MTX (MTX equivalent of 20 mg/kg) on days 5, 7, 9, 11 and 13 after inoculation. The tumor sizes were monitored and body weights measured up to 32 days post-inoculation. At the end of the experiment, the tumors were dissected and imaged. (A) Tumor volume, (B) the net gain in body weights and (C) the representative tumors. Data shown are representative images or expressed as mean 6 standard deviation of each group of mice (n 5 10). MTX, methotrexate; PHEA-suc, succinoylated a,b-poly[(2-hydroxyethyl)-L-aspartamide. Ó 2012 Lippincott Williams & Wilkins
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*Significantly different from control at P , 0.05. ALT, alanine aminotransferase; BUN, blood urea nitrogen; MTX, methotrexate; PHEA, a,b-poly[(2-hydroxyethyl)-L-aspartamide]; RBC, read blood cell; TP, total protein; WBC, white blood cell.
28.5 6 3.2 27.4 6 2.8 30.2 6 1.9 57.4 6 4.6 60.2 6 5.8 59.4 6 5.3 73.5 6 4.2 96.8 6 5.2* 76.2 6 5.2 6.9 6 0.3 8.1 6 0.8* 7.1 6 0.3 28.4 6 2.1 38.7 6 2.2* 28.7 6 1.6 71.4 6 9.2 89.3 6 8.0* 73.5 6 4.2 103 6 8.8 95 6 5.3 99 6 3.8 617 6 45 368 6 31* 587 6 42 6.9 6 0.22 6.3 6 0.35 6.6 6 0.52 2.8 6 0.31 0.7 6 0.21* 2.5 6 0.16 Control MTX PHEA-MTX
TABLE 1. Blood routine and serum biochemical indices of S180 tumor-bearing mice at 14 days after drug administration (mean 6 standard deviation) WBC RBC Platelets Hemoglobin Lymphocytes (%) ALT (U/L) BUN (mmol/L) Creatinine (mmol/L) TP (g/L)
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fluorescent cells were observed in both MTX-treated and PHEA-MTX-treated cells, indicating that PHEA-MTX retained the pro-apoptotic activity of MTX. In Vivo Antitumor Activity To examine the antitumor efficacy of PHEA-MTX, groups of ICR mice were implanted subcutaneously with S180 cells and treated with saline, MTX (20 mg/kg) or PHEA-MTX (MTX equivalent of 20 mg/kg) every other day for 5 days, and the development and progression of solid tumors were monitored up to 32 days post-inoculation (Figure 5). No death was observed, regardless of treatment employed. Whereas the solid tumors in the saline-injected group of mice grew gradually over the observation period, treatment with MTX inhibited the growth of tumors by 85% and treatment with PHEA-MTX further reduced the tumor volume (Figures 5A, B). Histological analysis of the tumor tissues revealed high necrotic levels of cells in the tumors from the PHEA-MTX-treated mice, whereas hypercellular sheets of monomorphic oval cells were observed in the tumors from the salineinjected mice (Figure 4B). In addition, the net gain in body weight in the PHEA-MTX-treated mice was similar to that of the saline-injected mice, although treatment with MTX dramatically reduced the body weight during the first several days (Figure 5B). Apparently, whereas MTX had systemic toxicity, PHEA-MTX may be well tolerated in mice. These data indicate that PHEA-MTX retained its antitumor activity, which was more potent than that of free MTX. The enhanced antitumor activity of PHEA-MTX may be attributed to the accumulation of PHEAMTX in tumor cells by an enhanced permeability and retention effect23 and also to the prolonged gradual release of the drug. Because of the pendant hydrophilic succinic group and hydrophobic MTX residue, PHEA-MTX is likely to form aggregates in biological fluids,24 contributing to the enhanced permeability and retention effect. In Vivo Safety Assessment After drug treatments, we monitored mouse behaviors and found that the PHEA-MTX-treated mice had much alleviated piloerection and adynamia compared with the
FIGURE 6. Histological analysis of the liver, lung, kidney and heart of S180 tumor-bearing mice. Two weeks after inoculation, the liver, lung, kidney and heart from each mouse were dissected and subjected to hematoxylin and eosin staining. Volume 344, Number 4, October 2012
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MTX-treated group. Two weeks after drug treatments, mouse blood cell counts were determined by routine examination (Table 1). Treatment with MTX reduced the number of white blood cells, platelets and lymphocytes but increased the levels of serum ALT, blood urea nitrogen and creatinine. In contrast, treatment with PHEA-MTX did not significantly change blood counts and serum biochemical indices. Further histological examination (Figure 6) revealed that treatment with MTX caused obvious liver lesions with denatured liver cells and histiocytosis of cells surrounding the hepatic sinusoid, whereas PHEA-MTX did not cause significant lesions in the liver. There was no significant difference in the lungs, kidneys and hearts among the different groups of mice. Clearly, in the treated doses, MTX exhibited obvious side effects on the liver and immune cells,25 whereas the conjugated PHEA-MTX had fewer side effects. This phenomenon may stem from PHEA-MTX’s unique pharmacokinetic property, gradual release of MTX and levels of selectivity.26 We are interested in further investigating the mechanisms by which PHEA-MTX exhibits fewer side effects in mice.
CONCLUSIONS A polymer conjugate of PHEA-MTX with a succinic spacer was developed by coupling the amino bonds of MTX to PHEA-suc. This conjugate released MTX gradually in buffer solutions tested. Both PHEA-MTX and MTX showed comparable levels of cytotoxicity and pro-apoptotic activity against human osteosarcoma cells in a concentration-dependent and time-dependent manner. PHEA-MTX showed superior antitumor activity with fewer side effects in mice, as compared with MTX. These data demonstrate that PHEA-suc is suitable for use as a drug carrier and indicate that PHEA-MTX is a promising polymeric drug for chemotherapy. REFERENCES 1. Smal MA, Dong Z, Cheung HTA, et al. Activation and cytotoxicity of 2-a-aminoacyl prodrugs of methotrexate. Biochem Pharmacol 1995;49: 567–74. 2. Zhang Y, Jin T, Zhuo RX. Methotrexate-loaded biodegradable polymeric micelles: preparation, physicochemical properties and in vitro drug release. Colloids Surf B Biointerfaces 2005;44:104–9. 3. Seo DH, Jeong YI, Kim DG, et al. Methotrexate-incorporated polymeric nanoparticles of methoxy poly(ethylene glycol)-grafted chitosan. Colloids Surf B Biointerfaces 2009;69:157–63. 4. Subr V, Strohalm J, Hirano T, et al. Poly [N-(2-hydroxypropyl)methacrylamide] conjugates of methotrexate: synthesis and in vitro drug release. J Control Release 1997;49:123–32. 5. Gurdag S, Khandare J, Stapels S, et al. Activity of dendrimer-methotrexate conjugates on methotrexate-sensitive and -resistant cell lines. Bioconjug Chem 2006;17:275–83. 6. Smith GK, Banks S, Blumenkopf TA, et al. Toward antibody-directed enzyme prodrug therapy with the T268G mutant of human carboxypeptidase a1 and novel in vivo stable prodrugs of methotrexate. J Biol Chem 1997;272:15804–16. 7. Cavallaro G, Licciardi M, Caliceti P, et al. Synthesis, physico-chemical and biological characterization of a paclitaxel macromolecular prodrug. Eur J Pharm Biopharm 2004;58:151–9. 8. Wolfe LA, Mullin RJ, Laethem R, et al. Antibody-directed enzyme prodrug therapy with the T268G mutant of human carboxypeptidase A1: in vitro and in vivo studies with prodrugs of methotrexate and the thymidylate synthase inhibitors GW1031 and GW1843. Bioconjug Chem 1999;10:38–48.
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