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Coupling of the antiviral drug ara‐AMP to lactosaminated albumin leads to specific uptake in rat and human hepatocytes
Author(s) -
Jansen Robert W.,
Kruijt Johan K.,
van Berkel THEO J. C.,
Meije Dirk K. F.
Publication year - 1993
Publication title -
hepatology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.488
H-Index - 361
eISSN - 1527-3350
pISSN - 0270-9139
DOI - 10.1002/hep.1840180122
Subject(s) - asialoglycoprotein receptor , endocytosis , conjugate , human serum albumin , scavenger receptor , albumin , in vivo , serum albumin , chemistry , biochemistry , carbodiimide , receptor , hepatocyte , microbiology and biotechnology , in vitro , biology , lipoprotein , cholesterol , mathematical analysis , mathematics
We covalently coupled 9‐β‐D‐arabinofuranosyladenine 5′‐monophosphate (ara‐AMP) to the carrier molecule lactosaminated human serum albumin using a water‐soluble carbodiimide with a two‐step conjugation method (pH 4.5 and pH 7.5) instead of the commonly used single‐step conjugation at pH 7.5. This resulted in a predominantly monomeric conjugate (lac 27 ‐HSA–ara‐AMP 9 ). The conjugate was stable in buffer (pH 7.4) and blood plasma. After in vivo injection, the carrier and the monomeric conjugate were subjected to selective endocytosis in rat hepatocytes, as shown on immunohistochemical study and cellseparation techniques using 125 I‐labeled material. In competition experiments with other ligands for the asialoglycoprotein receptor N‐acetylgalactosamine and asialofetuin, we showed that both lactosaminated human serum albumin and lac 27 ‐HSA–ara‐AMP 9 are subject to endocytosis by this receptor system. Although the coupling of ara‐AMP significantly increased the net negative charge of the conjugate compared with the native carrier, liver uptake was not affected by coadministration of an excess of succinylated human serum albumin (suc‐HSA), a negatively charged ligand for the scavenger receptor. Incubation studies with purified rat liver lysosomes showed that in this acidic and proteolytic environment, mainly ara‐AMP and, to a much lesser extent, ara‐A itself were released from the carrier. After injection into the rat in vivo and in isolated perfused rat liver, no free ara‐AMP or 9‐B‐D‐arabinofuranosyladenine (ara‐A) could be detected in plasma and perfusate, respectively, indicating proper retention of the virally active components in hepatocytes. Uptake experiments with freshly isolated rat and human hepatocytes indicated that human hepatocytes can also carry out endocytosis of lactosaminated human serum albumin and the ara‐AMP conjugate by means of the galactose‐recognizing receptor. (H EPATOLOGY 1993;18:146–152).