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ABSTRACT: The hepatocyte receptor for asialoglycoproteins internalizes galactosyl terminating macromolecules which can be used as hepatotropic drug carriers. Since this receptor is also expressed on the cells of well differentiated human hepatocellular carcinomas (HCCs), we studied whether conjugation of doxorubicin (DOXO) with lactosaminated human albumin (L-HSA) increases the drug efficacy on HCCs induced in rats by diethylnitrosamine (DENA).
DENA was given in the drinking water for 8 weeks. One week after the last day of DENA administration, animals were randomly assigned to three groups. Each group was administered with either saline, free or coupled DOXO (1 microg/g). Rats received 4 weekly intravenous injections. One week after the last administration, rats were killed and HCC development was evaluated by counting the tumor nodules on the surface of hepatic lobes.
In rats treated with L-HSA coupled DOXO the number of neoplastic nodules was significantly lower (P < 0.05) than that counted in animals injected with saline or with free DOXO. Coupled DOXO did not decrease body rat weight, which was markedly reduced by the free drug.
Conjugation with L-HSA increased the antineoplastic efficacy and decreased the systemic toxicity of DOXO administered to rats with HCCs produced by DENA.
Journal of Hepatology 10/2005; 43(4):645-52. · 9.26 Impact Factor
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ABSTRACT: The hepatocyte receptor for asialoglycoproteins (ASGP-R) internalizes macromolecules exposing galactosyl residues (MEGRs) which can be used as liver-addressed drug carriers. This receptor was also found on the cells of the large majority of well differentiated hepatocarcinomas (HCCs). The aim of the present experiments was to ascertain whether ASGP-R of HCCs is functionally active and these tumors can internalize higher quantities of MEGRs than extra-hepatic tissues.
We injected radioactive lactosaminated human albumin (L-HSA) in rats with HCCs produced by nitroso-diethylamine and measured the radioactivity of tumors, surrounding liver, heart, intestine and kidney. L-HSA is a MEGR successfully used in humans as a hepatotropic drug carrier.
The levels of radioactivity of HCCs were two to three times lower than those of surrounding liver, but several times higher than those of extra-hepatic tissues. L-HSA accumulation in the tumors mainly occurred via the ASGP-R, as indicated by the 20 times lower penetration of non-lactosaminated HSA. L-HSA uptake by the well-differentiated tumors were four times higher compared with that by the poorly differentiated forms.
The present results suggest that in the chemotherapy of HCCs expressing the ASGP-R the extra-hepatic toxicity of anticancer agents can be reduced by conjugation to L-HSA.
Liver international: official journal of the International Association for the Study of the Liver 09/2005; 25(4):854-60. · 3.82 Impact Factor
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ABSTRACT: The expression of the asialoglycoprotein receptor on the cells of the large majority of the well differentiated hepatocellular carcinomas can be exploited to improve the chemotherapy of these tumours by coupling anticancer agents to macromolecules taken up by the receptor. In line with this approach, in previous experiments we coupled doxorubicin (DOXO) to lactosaminated human albumin (L-HSA) using the (6-maleimidocaproyl)hydrazone derivative of the drug as an acid sensitive linker. Encouraging results were obtained in laboratory animals using L-HSA-DOXO. This conjugate, however, has the disadvantage of a difficult synthesis, which requires protein thiolation with iminothiolane and can hinder its preparation on a large scale. Here we describe a very simple method of coupling. The HS-groups required for the reaction with the maleimide moiety of DOXO-EMCH are made available in L-HSA by a cleavage of the protein disulphides achieved with tris(2-carboxyethyl) phosphine (TCEP). Contrary to thiolic reducing agents, the use of TCEP eliminates the need of an inert atmosphere and allows a one-step coupling reaction, without purification of the reduced protein before the addition of DOXO-EMCH. As the previous L-HSA-DOXO conjugate, the new conjugate accomplishes a very efficient liver targeting of the drug. This novel method of synthesis should facilitate the preparation of L-HSA-DOXO in the amounts required for clinical studies.
European Journal of Pharmaceutical Sciences 01/2005; 23(4-5):393-7. · 3.21 Impact Factor
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ABSTRACT: 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.
Using saline-injected hepatectomised rats as controls, we studied the effects of the conjugate on the ultrastructure of regenerating hepatocytes and evaluated [3H]thymidine incorporation, mitotic index and rate of DNA recovery in the liver remnant.
L-HSA-DOXO caused a selective drug accumulation in liver remnant, with low DOXO levels in extra-hepatic tissues. It did not change the ultrastructure of hepatocytes and did not increase serum alanine aminotransferase. It decreased [3H]thymidine incorporation and mitotic index, causing a moderate delay in hepatic DNA recovery.
The experiments indicate a substantial resistance of rat regenerating hepatocytes to high intracellular concentrations of DOXO. They support the possibility of using L-HSA-DOXO in an adjuvant chemotherapy after the surgical removal of HCCs which maintain the ASGP-R.
Liver international: official journal of the International Association for the Study of the Liver 07/2004; 24(3):246-52. · 3.82 Impact Factor
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ABSTRACT: In previous experiments fluorodeoxyuridine monophosphate (FUdRMP) was conjugated with lactosaminated human albumin (L-HSA). Fluorodeoxyuridine (FUdR) is an anticancer agent and L-HSA is a hepatotropic carrier of drugs obtained by the covalent linkage of lactose residues to the albumin molecule. The conjugate was synthesised via the imidazolide of FUdRMP at alkaline pH. Peripheral venous administration of L-HSA-FUdRMP produced enhanced FUdR levels in hepatic blood and might accomplish a non-invasive loco-regional chemotherapy of liver micrometastases. In the present paper some physicochemical characteristics of L-HSA-FUdRMP are reported. Polyacrylamide gel electrophoresis indicated that the coupling reaction did not cause covalent aggregation of the L-HSA molecules. 31P NMR spectra of the conjugate showed that FUdRMP was linked to L-HSA by phosphoamide bonds to lysine and histidine residues, and the area of the peak due to the lysine bond represented more than 80% of the spectrum of L-HSA-FUdRMP. MALDI analysis revealed a partial degradation of the peptide backbone of the conjugate which could not be detected using other methods of analysis. The degradation was not caused by the coupling of lactose molecules to albumin, but rather a consequence of FUdRMP conjugation with L-HSA. This fragmentation was dependent on the pH of the medium used for the FUdRMP coupling reaction. By decreasing the pH to 7.5, conjugates were obtained with a lower drug load but with a substantially reduced fragmentation, which should be preferred for a clinical use of L-HSA-FUdRMP.
Rapid Communications in Mass Spectrometry 02/2003; 17(22):2503-7. · 2.79 Impact Factor
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ABSTRACT: Nucleoside analogs (NAs) conjugated with galactosyl terminating peptides selectively enter hepatocytes via the asialoglycoprotein receptor and, after intracellular release from the carrier, partly exit from these cells into the bloodstream, resulting in higher concentrations in liver blood than in systemic circulation. Therefore, conjugates of anticancer NAs can be exploited to accomplish a loco-regional noninvasive treatment of liver micrometastases. In the present experiments we studied whether the enhancement of drug levels in liver blood achieved when NAs are given in the coupled form depends on the rate of drug elimination from the bloodstream. Three NAs, adenine arabinoside (ara-A), 5-fluoro-2'-deoxyuridine (FUdR), and 2',2'-difluorodeoxycytidine, were coupled with lactosaminated human albumin, a galactosyl terminating carrier. In rats that received an intravenous bolus injection of these conjugates, we compared the drug concentrations in liver blood to those in the systemic circulation. We found that enhanced levels of NAs in liver blood were only achieved by administering the conjugates of the drugs (ara-A and FUdR), which are rapidly cleared from the bloodstream. Increased drug levels also were obtained when ara-A and FUdR conjugates were slowly infused (a way of administration often used for anticancer drugs). The experiments also showed that galactosyl terminating conjugates of NAs might have the potential to produce a therapeutic effect only when the coupled drugs are active at low blood concentrations, since the amounts of drugs introduced into hepatocytes and released by these cells in the bloodstream cannot be increased when the receptor for the hepatic uptake of galactosyl terminating peptides is saturated.
Journal of Pharmacology and Experimental Therapeutics 06/2002; 301(2):638-42. · 3.83 Impact Factor
