Conjugation of poly(styrene-co-maleic acid) derivatives to the antitumor protein neocarzinostatin: pronounced improvements in pharmacological properties

Abstract

An anticancer agent of intermediate MW and having both a hydrophilic and hydrophobic nature was developed by utilizing the antitumor protein neocarzinostatin (NCS; MW = 12,000) as a prototype drug. The modification was achieved by reacting the 2 amino groups on NCS with an anhydride group of partially half-esterified (p-E-) or partially hydrolyzed (p-H-) poly(styrene-co-maleic anhydride) (SMA) in 0.8 M NaHCO3. The SMA samples with narrow MW distributions (MW = approximately 2000) were prepared by copolymerizing styrene and maleic anhydride in cumene followed by fractionation by means of a column-elution method. The derivatives p-E- or p-H-SMA were then formed by using the appropriate monoalcohols or H2O, respectively. These SMA derivatives contain .apprx. 2 mol of anhydride residues/mol of SMA. The reaction product, SMA-conjugated NCS (designated as SMANCS), was purified by dialysis followed by gel filtration with Sephadex G-75. The complete reaction yielded essentially a single product, biantennary SMANCS. The MW of the pure SMAMCS was estimated by various methods, including polyacrylamide gel electrophoresis with NaDodSO4 [sodium dodecyl sulfate], HPLC [high-performance liquid chromatography] in the gel permeation mode, fluorescence polarization, and a decrease in both N and protein contents. These results agree with the apparent MW of .apprx. 16,000. Characters of SMANCS was considerably altered from that of parental NCS: solubility characteristics in both organic and aqueous solvents were changed; the biological half-life in blood [rat] was prolonged 10 times; and antitumor activity [mouse S-180 ascitic tumor cells] became more pronounced, but the toxicity was reduced to 1/4 of the parental NCS. The present study has provided a method of improving biologically active substances by polymer conjugation.