Felix Kratz

Doxorubicin coupled to lactosaminated human albumin remains confined within mouse liver cells after the intracellular release from the carrier

BACKGROUND The hepatocyte receptor for asialoglycoproteins, which binds and internalises galactosyl terminating peptides, was found to be expressed also on the cells of the majority of hepatocarcinomas. AIMS To verify whether doxorubicin coupling to lactosaminated albumin, a galactosyl terminating neoglycoprotein, produces selective drug accumulation in hepatocytes with reduced concentrations in extra-hepatic tissues, thus facilitating the use of the drug in hepatocarcinoma treatment. METHODS Doxorubicin concentrations were measured in organs of mice injected with the free or coupled drug. RESULTS In mice injected with the coupled drug, the ratios between doxorubicin concentrations in liver and those in heart, intestine, spleen and kidney were 8-14 times higher than in animals that received the same dose of the free drug. CONCLUSIONS Due to the very efficient liver targeting of doxorubicin, the lactosaminated human albumin-doxorubicin conjugate appears to have the potential of improving the chemotherapy of hepatocellular carcinomas through the asialoglycoprotein receptor.

A conjugate of doxorubicin with lactosaminated albumin enhances the drug concentrations in all the forms of rat hepatocellular carcinomas independently of their differentiation grade

Abstract: Background/Aims: Doxorubicin (DOXO) was coupled to lactosaminated human serum albumin (L‐HSA) in order to enhance the drug concentration in the well differentiated hepatocellular carcinomas (HCCs), which can accumulate L‐HSA through the asialoglycoprotein receptor. In the present experiments we compared the DOXO concentrations produced by this conjugate (L‐HSA–DOXO) and by the uncoupled drug in the well, moderately, and poorly differentiated rat HCCs.

Liver-targeted doxorubicin: effects on rat regenerating hepatocytes.

Abstract: Background/Aims: The conjugate of doxorubicin (DOXO) with lactosaminated human albumin (L‐HSA) has the potential of improving DOXO efficacy in the treatment of hepatocellular carcinomas (HCCs) expressing the asialoglycoprotein receptor (ASGP‐R). In view of an adjuvant chemotherapy with L‐HSA–DOXO after the surgical removal of the tumour, in the present experiments we verified whether DOXO accumulation produced by the conjugate can impair the liver regeneration following hepatic resection in non‐cirrhotic liver.

Doxorubicin coupled to lactosaminated albumin: effect of heterogeneity in drug load on conjugate disposition and hepatocellular carcinoma uptake in rats.

Coupling to lactosaminated human albumin (L-HSA) makes doxorubicin (DOXO) an effective drug against chemically induced rat hepatocellular carcinomas (HCCs). In the conjugate there is a large heterogeneity in the number of DOXO molecules bound to one L-HSA molecule. After lyophilization, the molecules with the higher DOXO load form large complexes (C-DOXOL), whereas those with low drug load (C-DOXOS) have the size of the carrier L-HSA. In the present experiments, we demonstrated that in C-DOXOL the molecules are not linked by covalent bonds, but are strongly aggregated probably because of mutual drug-drug interaction between the DOXO residues. In healthy rats and in animals with HCCs which received the same dose (1 microg/g) of DOXO injected in C-DOXOL or in C-DOXOS forms, penetration of the drug in tumors and in tissues was more rapid after administration of the former complex. Three hours after injection of both conjugate forms the intracellular release of DOXO from the carrier was completed. The AUCs from 0.5 to 4h of the levels of the released DOXO in HCCs, surrounding liver and bone marrow of animals injected with C-DOXOL were similar to those calculated in animals given C-DOXOS. This suggests that after administration of the dose of DOXO used in the present experiments the conjugate molecules with lower or higher drug load can exert comparable pharmacological and toxic effects.

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 (13)C 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 (13)C 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.