A Polymeric Prodrug of 5-Fluorouracil-1-Acetic Acid Using a Multi-Hydroxyl Polyethylene Glycol Derivative as the Drug Carrier

<div><p>Purpose</p><p>Macromolecular prodrugs obtained by covalently conjugating small molecular drugs with polymeric carriers were proven to accomplish controlled and sustained release of the therapeutic agents <i>in vitro</i> and <i>in vivo</i>. Polyethylene glycol (PEG) has been extensively used due to its low toxicity, low immunogenicity and high biocompatibility. However, for linear PEG macromolecules, the number of available hydroxyl groups for drug coupling does not change with the length of polymeric chain, which limits the application of PEG for drug conjugation purposes. To increase the drug loading and prolong the retention time of 5-fluorouracil (5-Fu), a macromolecular prodrug of 5-Fu, 5-fluorouracil-1 acid-PAE derivative (5-FA-PAE) was synthesized and tested for the antitumor activity <i>in vivo</i>.</p><p>Methods</p><p>PEG with a molecular weight of 38 kDa was selected to synthesize the <i>multi-hydroxyl polyethylene glycol</i> derivative (PAE) through an addition reaction. 5-fluorouracil-1 acetic acid (5-FA), a 5-Fu derivative was coupled with PEG derivatives via ester bond to form a macromolecular prodrug, 5-FA-PAE. The <i>in vitro</i> drug release, pharmacokinetics, <i>in vivo</i> distribution and antitumor effect of the prodrug were investigated, respectively.</p><p>Results</p><p>The PEG-based prodrug obtained in this study possessed an exceedingly high 5-FA loading efficiency of 10.58%, much higher than the maximum drug loading efficiency of unmodified PEG with the same molecular weight, which was 0.98% theoretically. Furthermore, 5-FA-PAE exhibited suitable sustained release in tumors.</p><p>Conclusion</p><p>This study provides a new approach for the development of the delivery to tumors of anticancer agents with PEG derivatives.</p></div

<i>In vitro</i> drug release of 5-FA-PAE.

<p>(A) Drug release profiles of 5-FA-PAE in PBS and saline. 100 µl 5-PA-PAE was added to preheated release media (PBS of different pH values or saline) and incubated at 37°C with stirring. Samples were collected at fixed time intervals, acidified by hydrochloric acid (1 M) and analyzed by HPLC. (B) Drug release profiles of 5-FA-PAE in murine tumor homogenate and plasma. The samples from mouse plasma and tumor homogenate were obtained in duplicate at each time point (100 µl each). For hydrolysis, 100 µl sodium hydroxide (1 M) were added to samples followed by 100 µl hydrochloric acid (1 M). The other group was not subjected to hydrolysis by substituting sodium hydroxide solution with saline and acidifying with 50 µl hydrochloric acid. The differences of 5-FA in the two groups at the same time point was the unreleased 5-FA in each sample. Each value represents the mean ± SD (n = 3).</p

Tumor weight and tumor control rate of mice administrated with saline, 5-Fu, 5-FA or 5-FA-PAE.

<p>*<i>p</i><0.05 vs. saline group.</p><p>**<i>p</i><0.01 vs. saline group.</p>Δ<p><i>p</i><0.05 vs. 5-Fu group.</p><p>Each value represents the mean ± SD (n = 6).</p><p>Tumor weight and tumor control rate of mice administrated with saline, 5-Fu, 5-FA or 5-FA-PAE.</p

Biodistribution of 5-FA (A) and 5-FA-PAE (B) after <i>i.v.</i> injection.

<p>The tumor-bearing animal model was established by subcutaneous injection of H22 cells into Kunming mice. The control group and the test groups were administered intravenously with 20 mg/kg of 5-FA or 5-FA-PAE (equivalent to 20 mg/kg of 5-FA), respectively. The mice were exsanguinated and sacrificed at predetermined time points. Tissues (heart, liver, spleen, lung, kidney, brain and tumor) were collected, weighed and homogenized with two fold concentrated physiological saline. The samples of the test group were treated as hydrolyzed samples, whereas those of the control group were treated as unhydrolyzed samples. All data are presented as the concentration of 5-FA. Each value represents the mean ± standard deviation (n = 6).</p

Pharmacokinetic parameters of 5-FA and 5-FA-PAE after i.v. injection in rats.

<p>AUC, area under the plasma concentration−time curve; MRT, mean residence time; VRT, variance of mean residence time; t_1/2: elimination half life; T_max, time of maximum concentration; V, apparent volume of distribution; CL, clearance; Cmax, the maximum of 5-FA concentration in plasma.</p><p>Each value represents the mean ± SD (n = 5).</p><p>Pharmacokinetic parameters of 5-FA and 5-FA-PAE after i.v. injection in rats.</p

The antitumor effects on tumor-bearing mice.

<p>Mice were i.v injected with saline (20 mg/kg, 0.160 mmol/kg), 5-FA-PAE (284 mg/kg, 0.160 mmol/kg), 5-FA (30 mg/kg, 0.160 mmol/kg) or 5-Fu (20.47 mg/kg, 0.160 mmol/kg) on day 3, 5, 7, 9, 11, 13 and 15 after inoculation of H22 cells. On day 20, mice were sacrificed. Tumors and organs were removed and weighed. (A) The tumor volumes after inoculation (n = 6–12). * <i>p</i><0.05, ** <i>p</i><0.01. (B) Images of tumors in tumor-bearing mice on day 20 after inoculation of tumor cells (n = 6).</p

Synthesis routes of PAE (A) and 5-FA-PAE conjugates (B).

<p>(A) PA was synthesized by adding NaH to PEG and the mixture was stirred for 4 h at 120°C. Then PAE was obtained by adding AGE to the mixture. (B) 5-FA was added dropwise to PAE in dimethylformamide, then NHS and EDC·HCl were added. After incubation, the mixture was precipitated with isopropanol. The obtained residue was recrystallized by isopropanol several times and dried in vacuum at 40°C overnight to produce 5-FA-PAE.</p

Drug concentration in tumor and plasma.

<p>The tumor-bearing mice model was described in the “<i>In vivo</i> biodistribution” section. The control group and the test groups were administered intravenously with 20 mg/kg of 5-FA and 5-FA-PAE (equivalent to 20 mg/kg of 5-FA), respectively. (A) Drug concentration of 5-FA and conjugated 5-FA-PAE in tumor at different time points. (B) Ratio of drug concentration in tumor vs. that in plasma of 5-FA and conjugated 5-FA-PAE.</p

Pharmacokinetics of 5-FA and 5-FA-PAE after <i>i.v.</i> injection.

<p>The control group and the test groups were administered intravenously with 20 mg/kg of 5-FA or 189 mg/kg of 5-FA-PAE (equivalent to 20 mg/kg of 5-FA) dissolved in physiological saline, respectively. Each plasma sample of the 5-FA-PAE group was divided into two portions (treated as hydrolyzed and unhydrolyzed), which were analyzed by HPLC to determine the plasma concentrations of released 5-FA and total 5-FA of the conjugate whereas the plasma samples of the control group were treated as unhydrolyzed samples. Each value represents the mean ± standard deviation (n = 6).</p

The molecular weight of the polymeric carrier (PEG, PA, PAE) and the prodrug (5-FA-PAE).

<p>PDI: polydispersity.</p><p>The molecular weight of the polymeric carrier (PEG, PA, PAE) and the prodrug (5-FA-PAE).</p