Characterisation of the conjugate of the (6-maleimidocaproyl)hydrazone derivative of doxorubicin with lactosaminated human albumin by 13C NMR spectroscopy

In order to improve the efficacy of doxorubicin (DOXO) in the treatment of hepatocellular carcinomas, the drug was conjugated with lactosaminated human albumin (L-HSA), a hepatotropic drug carrier. Conjugation was performed using the (6-maleimidocaproyl)hydrazone derivative of the drug (DOXO-EMCH). The maleimide group of DOXO-EMCH reacts with the aminoacidic residues of the carrier forming stable bonds, whereas the hydrazone bond is rapidly hydrolysed in the acidic endosomal and lysosomal compartments of the cells allowing the intracellular release of DOXO. To identify the amino acids of L-HSA involved in the bond with DOXO-EMCH, in the present study we synthesized this compound with the 2,3 carbon atoms of the maleimide moiety enriched in the C-13 isotope and used this labelled DOXO-EMCH to prepare two types of L-HSA conjugate. Type I was prepared in analogy to those studied in the anticancer experiments using tris(2-carboxyethyl)phosphine (TCEP) to reduce L-cysteine disulfides and make the sulfhydryl groups available for the reaction with DOXO-EMCH; type II was synthesized omitting TCEP. By C-13 NMR spectroscopy we could demonstrate that in type I conjugate cysteine was the only amino acid residue that reacted with DOXO-EMCH, whereas in type II conjugate lysine was the only amino acid in the reaction with DOXO-EMCH. When hydrolysed in an acidic medium to cleave the hydrazone bond, type I conjugate released only DOXO, whereas type II conjugate also released a derivative of the drug. (C) 2009 Elsevier B.V. All rights reserved.