Synthesis and characterization of HPMA copolymer-5FU conjugates

Available online at www.sciencedirect.com Chinese Chemical Letters 19 (2008) 137–140 www.elsevier.com/locate/cclet Synthesis and characterization of HPMA copolymer-5-FU conjugates Fang Yuan, Fu Chen, Qing Yu Xiang, Xuan Qin, Zhi Rong Zhang, Yuan Huang * Key Laboratory of Drug Targeting of Ministry of Education, West China School of Pharmacy, Sichuan University, Chengdu 610041, China Received 29 June 2007 Abstract N-(2-Hydroxypropyl) methacrylamide copolymer-5-fluorouracil (PHPMA-FU) conjugates were synthesized by a novel and simplified synthetic route, and characterized by UV, FTIR and HPLC analyses. The conjugated content of 5-fluorouracil (5-FU) was 3.41  0.07 wt%. The stabilities of PHPMA-FU conjugates under different conditions were studied. The results showed that HPMA copolymer was a potential carrier for tumor-targeting delivery of 5-FU. # 2007 Yuan Huang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. Keywords: HPMA copolymer; 5-Fluorouracil; Conjugates; Characterization; Stability The antimetabolite, 5-fluorouracil (5-FU), has a broad spectrum of activities against solid tumors such as cancers of stomach, lung and intestine. Nevertheless, the administration of the drug is always accompanied by systemic toxicities [1,2]. Furthermore, 5-FU has a very short plasma circulation half-life (about 10–20 min) [3]. Therefore, it is necessary to develop a novel tumor-targeting drug delivery system to increase the therapeutic effect, reduce the systemic toxicities and improve the stability of 5-FU. Water-soluble polymer–drug conjugates exhibit selective accumulation in tumor tissue due to ‘EPR’ (enhanced permeability and retention) effect. For more than three decades, researches showed that N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers were excellent carriers for delivery of anticancer agents. These copolymers are well characterized, non-immunogenic and can be tailored to the characteristics of the specific target [4]. In previous study, Putnam and Kopecek synthesized HPMA copolymer-5fluorouracil conjugates based on a-substituted glycine derivatives of 5-FU [5]. However, the synthetic method was complicated and the content of 5-FU in the conjugates was only 1.38 wt%. Therefore, the purposes of our study were: (1) to develop a novel and simplified synthetic route to prepare PHPMA-FU conjugates; (2) to increase conjugated drug content; (3) to study the in vitro stabilities of the conjugates. PHPMA-FU conjugates were synthesized as described in Scheme 1. HPMA 1, methacryloylglycyl-DL-phenylalanylL-leucyl-glycine (MA-GFLG-OH) 2 and 1,3-bis(hydroxymethyl)-5-fluorouracil 4 were prepared and characterized according to literature procedures [6,7]. Precursor 3 was prepared by radical precipitation copolymerization in acetone * Corresponding author. E-mail address: [email protected] (Y. Huang). 1001-8417/$ – see front matter # 2007 Yuan Huang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. doi:10.1016/j.cclet.2007.10.035 138 F. Yuan et al. / Chinese Chemical Letters 19 (2008) 137–140 Scheme 1. Synthesis of HPMA copolymer-5-FU conjugates. Reagents and conditions: (a) AIBN, 50 8C, 24 h and (b) DCC, DMAP, rt, 18 h. (initiator, 2,20 -azobisisobutyronitrile (AIBN), 1.5 wt%; concentration of monomers, 12.5 wt%; molar ratio of HPMA/ MA-GFLG-OH, 9:1). The polymerization mixture was bubbled with argon and polymerized in a sealed ampoule at 50 8C for 24 h. The precipitated polymer was dissolved in methanol and reprecipitated in ether (polymer/methanol/ether 350 mg/2.5 mL/100 mL). The resultant solid was dissolved in distilled water, then dialyzed for 24 h, and finally F. Yuan et al. / Chinese Chemical Letters 19 (2008) 137–140 139 lyophilized. The molecular weight was 1,22,000 g/mol, which was determined viscometrically as previously reported [8]. Conjugate 5 was prepared by the reaction of precursor 3 (1.053 g, contained MA-GFLG-OH 0.6 mmol) with 4 (3.8 g, 12 mmol) in 8.5 mL anhydrous acetonitrile/dimethylformamide (DMF) (5:12, v/v). N,N-dicyclohexylcarbodiimide (DCC) (136.2 mg, 0.66 mmol) and a catalytic amount of 4-dimethylaminopyridine (DMAP) were added under stirring. The reaction was monitored by TLC. The reaction mixture was stirred at room temperature for 18 h, then filtered to remove dicyclohexylurea (DCU) and washed with methanol. Acetonitrile and methanol were removed by vacuum evaporation. The residue was dissolved in methanol and reprecipitated in ether. The resultant solid was purified by dialysis at 4 8C for 12 h and finally lyophilized. The structure of 5 was confirmed by UV, FTIR and HPLC analyses as reported [3,6]. UV lmax: 4, 266 nm; 5, 264 nm; no absorption was observed for 3 from 200 to 400 nm. IR (KBr, cm1): 3384, 2971, 2933, 1642, 1536, 1378, 1261, 1204, 1138, 952, 702. The HPLC profiles were shown in Fig. 1, from which, we can see that the retention time of 5-FU was 8.908 min, while there was no chromatographic Fig. 1. HPLC profiles of conjugate 5, precursor 3 and 5-FU. Conditions: ALLTECH-ODS column (250 mm  4.6 mm, 5 mm,); mobile phase, purified water; flow rate, 1 mL/min; detection wavelength, 266 nm; column temperature, 30 8C. Fig. 2. Stabilities of conjugates in different PBS buffers at 37  1 8C. Fig. 3. Stability of conjugates in plasma at 37  1 8C. 140 F. Yuan et al. / Chinese Chemical Letters 19 (2008) 137–140 Table 1 Half-life values for the degradation of conjugate 5 in PBS and plasma Medium Degradation kinetics t1/2 (h) PBS pH 7.02 PBS pH 8.01 Plasma First-order kinetics First-order kinetics Zero-order kinetics 40.5 20.2 32.4 peak for unhydrolyzed conjugates. As for the hydrolyzed conjugates (the conjugates were hydrolyzed with 3 mol/L NaOH to release 5-FU), there was a chromatographic peak at 8.849 min. The conjugated and free 5-FU contents in the conjugates were 3.41  0.07 and 0.04  0.01 wt%, respectively. All these results indicated that 5-FU was successfully attached to the polymer carrier covalently. In the preliminary study, we used the method, in which 1,3bis(hydroxymethyl)-5-FU reacted with MA-GFLG-OH at first, then copolymerized with HPMA. But this method led to a very low 5-FU content (only 0.28 wt%). It may own to the unstability of MA-GFLG-5-FU during the copolymerization. So, the synthesized route in Scheme 1 was used finally and the conjugated 5-FU content was two times higher compared with the method reported previously [5]. Stabilities of the conjugates were investigated in PBS buffers with various pH values (Fig. 2) and in plasma in vitro (Fig. 3) at 37  1 8C. The percentages of the residual conjugates were determined by HPLC and calculated using the following equation:   1  Ct conjugate ð%Þ ¼  100% Co where Ct is the concentration of free 5-FU at different time t, Co is the concentration of 5-FU after complete hydrolysis. The data resulting from the degradation of the conjugates in slightly basic environment (pH 7.02, 8.01) and plasma were modeled to estimate the kinetic parameters. From Table 1, we can see that the release profiles at pH 7.02, 8.01 were both fitted to first-order kinetics while that of plasma could be modeled to zero-order kinetics. It can be concluded that PHPMA-FU conjugates are relatively stable in acidic environment while in slightly basic environment (pH 7.02, 8.01), most of the drugs were released within 60 h and 96 h, respectively. As for the stability test in plasma, the conjugates were stable during the first 12 h, only less than 25% of the drugs were released, and the release continued to 60 h. PHPMA-FU conjugates exhibited longer half-life in plasma (32.4 h), which demonstrated that the polymer drug conjugates may remain more intact in the systemic circulation for site-specific therapy. Additionally, the results of stability study also proved that the conjugates were successfully synthesized. In summary, PHPMA-FU conjugates were successfully synthesized using a novel synthetic route and characterized. And the conjugates could be used as a potential tumor-targeting delivery system for 5-FU. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 30500636) and Ministry of Education (NCET-06-0786). Reference [1] [2] [3] [4] [5] [6] [7] [8] R.W. Brockman, E.P. Anderson, Annu. Rev. Biochem. 32 (1963) 463. M. Nichifo, E.H. Schach, L.W. Seyrnou, J. Control. Release 48 (1997) 165. T. Ouchi, Y. Hagihara, K. Takahashi, Y. Takano, I. Igarashi, Drug Des. Discov. 9 (1992) 93. Y. Huang, H. Ghandehari, Y.R. Duan, A. Nan, Z.R. Zhang, J. Drug Deliv. Sci. Technol. 14 (2004) 187. D. Putnam, J. Kopecek, Bioconjug. Chem. 6 (1995) 483. P. Rejmanova, J. Pohl, M. Baudys, V. Kostka, J. Kopecek, Makromol. Chem. 184 (1983) 2009. S. Ahmad, S. Ozaki, T. Nagase, et al. Chem. Pharm. Bull. 35 (1987) 4137. A.M. Sookne, M. Harris, Ind. Eng. Chem. 37 (1945) 475.