PEG−Doxorubicin Conjugates: Influence of Polymer Structure on Drug Release, in Vitro Cytotoxicity, Biodistribution, and Antitumor Activity

Abstract

Polymer−drug conjugates (polymer therapeutics) are finding increasing use as novel anticancer agents. Here a series of poly(ethylene glycol) PEG−doxorubicin (Dox) conjugates were synthesized using polymers of linear or branched architecture (molecular weight 5000−20000 g/mol) and with different peptidyl linkers (GFLG, GLFG, GLG, GGRR, and RGLG). The resultant conjugates had a drug loading of 2.7−8.0 wt % Dox and contained 50 value = 0.24 μg/mL). Biodistribution in mice bearing sc B16F10 tumors was assessed after administration of PEGs (5000, 10000, or 20000 g/mol) radioiodinated using the Bolton and Hunter reagent or PEG−Dox conjugates by HPLC. The ¹²⁵I-labeled PEGs showed a clear relationship between Mw and blood clearance and tumor accumulation. The highest Mw PEG had the longest plasma residence time and consequently the greatest tumor targeting. The PEG−Dox conjugates showed a markedly prolonged plasma clearance and greater tumor targeting compared to free Dox, but there was no clear molecular weight-dependence on biodistribution. This was consistent with the observation that the PEG−Dox conjugates formed micelles in aqueous solution comprising 2−20 PEG−Dox molecules depending on polymer Mw and architecture. Although PEG−Dox showed greater tumor targeting than free Dox, PEG conjugation led to significantly lower anthracycline levels in heart. Preliminary experiments to assess antitumor activity against sc B16F10 in vivo showed the PEG₅₀₀₀linear (L)−GFLG−Dox and PEG₁₀₀₀₀branched (B)−GLFG−Dox (both 5 mg/kg Dox-equiv) to be the most active with T/C values of 146 and 143%, respectively. Free Dox did not show significant activity in this model (T/C = 121%). Dose escalation of PEG₅₀₀₀(L)−GFLG−Dox to 10 mg/kg Dox-equiv prolonged further animal survival (T/C = 161%). Using the Dox-sensitive model ip L1210 (where Dox displayed a T/C = 150% after single ip dose), the PEG₅₀₀₀(L)−GFLG−Dox displayed a maximum T/C of 141% (10 mg/kg Dox-equiv) using a once a day (×3) schedule. Further studies are warranted with PEG₅₀₀₀(L)−GFLG−Dox to determine its spectrum of antitumor activity and also the optimum dosing schedule before clinical testing.