Polyethylene Glycol Conjugates of Methotrexate Varying in Their Molecular Weight from MW 750 to MW 40000:  Synthesis, Characterization, and Structure−Activity Relationships in Vitro and in Vivo

Poly(ethylene glycol)s (PEGs) are potential drug carriers for improving the therapeutic index of anticancer agents. In this work, the anticancer drug methotrexate (MTX) was activated with N,N‘-dicyclohexylcarbodiimide (DCC) and coupled to amino group bearing PEGs of MW 750, 2000, 5000, 10 000, 20 000, and 40 000. First, the activation process of MTX with DCC in the presence and absence of N-hydroxysuccinimide was analyzed through HPLC. Preincubation of methotrexate with DCC alone at 0 °C proved to be favorable with respect to the amount of activated species and the formation of byproducts. MTX−PEG conjugates were synthesized according to this procedure, isolated through size-exclusion chromatography, and characterized through analytical HPLC, MALDI-TOF spectrometry, and gel permeation chromatography. In a cell-free assay, all of the drug polymer conjugates inhibited the target enzyme of MTX, dihydrofolate reductase (DHFR), to a similar extent, but were not as active as free MTX. Additionally, incubation of the MTX−PEG40000 conjugate for 6 days at 37 °C in phosphate buffered saline (pH 7.4), in cell-conditioned medium, or in human serum revealed no significant release of methotrexate. These results, taken together, indicate that release of MTX from polymer conjugates is not necessary for an effective interaction with the active site of dihydrofolate reductase. Evaluation of the in vitro cytotoxicity of the MTX−PEG conjugates in two adherent and three suspension human tumor cell lines revealed that the IC50 values of the tested compounds increased with the size of the drug−polymer conjugates. The most effective compound tested in these assays was the free drug MTX itself (IC50 value ranging from ∼0.01 to 0.05 μM), while the IC50 values of the polymer conjugates were higher (IC50 value for MTX−PEG750, 2000 and 5000: ∼0.6−3 μM; for MTX-PEG10000 and 20000: ∼2−7 μM; and for MTX−PEG40000: > 6 μM). Subsequently, MTX−PEG5000, MTX−PEG20000, and MTX−PEG40000 were evaluated in a human mesothelioma MSTO-211H xenograft model, and their antitumor effects were compared with free methotrexate and the albumin conjugate MTX−HSA, a conjugate that is currently in phase II clinical trials. In contrast to the in vitro results, the high molecular weight MTX−PEG conjugates exhibited the highest in vivo antitumor activity: At a dose of 40 and 80 mg/kg MTX−PEG5000 was less active than MTX at its optimal dose of 100 mg/kg; MTX−PEG20000 at a dose of 40 mg/kg showed antitumor effic