Receptor-mediated endocytosis of fucosylated neoglycoprotein by macrophages.

The characteristics of the recognition system involved in the receptor mediated endocytosis of the neoglycoprotein, fucose-human serum albumin (HSA) were studied. It was found that (i) fucose-HSA showed strong affinity binding and uptake by various macrophages; (ii) binding was specific for L-fucose and D-mannose; (iii) binding was found to be inhibited by oxidant like H2O2 and swainsonine whereas it was elevated by dexamethasone; (iv) clearance of 125I-fucose-HSA was rapid and strongly inhibited by unlabelled fucose-HSA. Greater than 70% of fucose-HSA was found in liver and more than 60% of this was found in liver lysosomes; (v) uptake of fucose-HSA was thirty-fold more efficient in liver macrophages (Kupffer cells) than in hepatocytes; (vi) moreover, mannose-HSA and ovalbumin which are potent inhibitors of mannose/N-acetylglucosamine receptors inhibited clearance and uptake of fucose-HSA by liver as well as by isolated Kupffer cells suggesting the involvement of both fucose and mannose receptors or a single type of receptor having greater affinity for fucose-HSA than for mannose-HSA. These results emphasize the important role of fucose-terminated glycoproteins in site-specific drug targeting.

Syntheses of several 99mTc and 131I labeled neoglycoalbumins and their differential uptake patterns in animal biodistribution experiments.

Several glycoconjugates, alpha-D-mannopyranosyl, beta-L-fucopyranosyl, alpha-L-rhamnopyranosyl, beta-D-glucopyranosyl and beta-D-galactopyranosyl human serum albumin, were synthesized using C9-tether and radiolabeled with 99mTc and 131I. Both 99mTc and 131I radiolabeled neoglycoalbumins had considerable stability and exhibited similar biodistribution patterns within the experimental limits. The results of biodistribution studies can be explained from the in vitro observations that 99mTc-beta-D-galactopyranosyl albumin binds to hepatic binding protein in liver in a dose-dependent fashion. The radiolabeled glycoalbumins derived from D-mannopyranose and L-fucopyranose also bind in a dose-dependent fashion to the receptors present in the liver sinusoidal cells and spleen macrophages. The beta-D-glucopyranosyl and alpha-L-rhamnopyranosyl neoglycoalbumins accumulate nonspecifically in liver and spleen.

99Tcm-cystine, a renal function and imaging agent: a comparative study in dog with 131I-hippurate and 99Tcm-glucoheptonate to evaluate its functional and imaging characteristics.

99Tcm-cystine, which has been proposed as a renal radiopharmaceutical for evaluating renal morphology and function in a single experiment, is compared with 131I-orthoiodohippurate (OIH) with respect to its renal clearance and extraction parameters and with 99Tcm-glucoheptonate (GHA) regarding its imaging characteristics. In spite of its comparable renal accumulation with 131I-OIH, its clearance (10.1 +/- 1.0 ml min-1 kg-1) was lower than that of 131I-OIH (21.5 +/- 0.9 ml min-1 kg-1) but was higher than that of 125I-iothalamate (5.4 +/- 0.6 ml min-1 kg-1). Extraction efficiencies of 99Tcm-cystine, 131I-OIH and 125I-iothalamate were 39 +/- 5, 64 +/- 4 and 27 +/- 3, respectively. The glomerular filtration components of 99Tcm-cystine and 131I-OIH were 26 and 16% of their respective clearances. In probenecid-treated animals the clearance of both agents was affected to a similar extent and fell to half of their respective control values, whereas tubular secretory components were found to be 19 and 31% of the controls. The kidney images obtained with 99Tcm-cystine were superior to those obtained with 99Tcm-GHA at different time points. Therefore, considering both renal function and imaging properties of 99Tcm-cystine it appears that this radiopharmaceutical offers some definite advantages over the currently available renal agents and commands further study.

Purification and partial characterization of a haemorrhagin (VRH-1) from Vipera russelli russelli venom.

A haemorrhagic toxin (VRH-1) has been purified to homogeneity from Vipera russelli russelli venom by subjecting it to chromatography twice successively on CM-Sephadex C-50. It is a protein of mol. wt 22,000 and contains one mole of Mg2+. Intradermal administration of this haemorrhagin in mice resulted in severe lung haemorrhage but produced little haemorrhage in skin. This apparent organ preference led us to develop a new haemorrhage assay method utilizing dye diffusion from lung in vitro. Proteolytic activity of VRH-1 was demonstrated using dimethylcasein as substrate following quantitation by reaction with trinitrobenzoyl sulfonic acid. Both haemorrhagic and proteolytic activities of VRH-1 were inhibited by serine protease inhibitors like phenylmethyl sulfonyl fluoride and chymostatin, but metal chelators had no effect. Lung haemorrhage is unlikely to be a direct reflection of a high local concentration of VRH-1. The administration of supernatant generated by incubation of chopped liver from untreated mouse and VRH-1 (in subhaemorrhagic dose) results in severe lung haemorrhage. This raises the possibility that VRH-1 leads to the formation of intermediate(s) which causes the haemorrhage.

Pharmacokinetics and biodistribution of methotrexate conjugated to mannosyl human serum albumin.

The superior efficacy of mannosylated neoglycoprotein-conjugated methotrexate, compared with free drug, in eliminating the parasite burden in both the in-vitro macrophage model and in-vivo mouse model of visceral leishmaniasis has been demonstrated previously. In the present study it was found that: (i) methotrexate conjugated to mannosyl human serum albumin (Man-HSA) was taken up rapidly by the liver and spleen, whereas the free drug was taken up by the kidney; (ii) uptake of the conjugate was ten-fold more efficient in liver macrophages (Kupffer cells) than in hepatocytes; (iii) most of the drug conjugate reached the lysosomes of Kupffer cells; and (iv) the active drug was released in the lysosomes of macrophages to act on Leishmania parasites.

Biodistribution of two 99mTc-cardiac glycosides with end glucose unit: effect of lipophilicity on their relative myocardial accumulation.

In biodistribution experiments with tritium labelled cardiac glycoside it was observed that compounds of low lipophilicity showed a considerably higher affinity towards myocardium with respect to other tissues and organs. A similar trend was also observed with 99mTc-cardiac glycosides except for one compound with glucose residue, which in spite of its lower lipophilicity exhibited an unexpectedly low heart to non-target concentration ratio, thereby indicating a possible influence of carbohydrate residue on biodistribution. To confirm this, in this article we radiolabelled two glucose containing cardiac glycosides (K-strophanthin-beta and K-strophanthoside) with 99mTc and, in biodistribution experiments, less lipophilic 99mTc-K-strophanthoside showed a much better heart to non-target ratio over 99mTc-K-strophanthin-beta. It is thus concluded that, in addition to lipophilicity, the affinity of the carbohydrate residue for non-target organs is also an important consideration in determining the structure-distribution relationship of 99mTc-cardiac glycosides.

Preparation and evaluation of technetium-99m labeled cardiac glycoside derivatives as potential myocardial imaging agent.

Three cardiac glycosides, two natural, cymarin and convallotoxin and one synthetic, strophanthidin-beta-D-glucoside were converted to their thiosemicarbazone and subsequently radiolabeled with 99mTc by chelation. The resulting radioactive chelate complexes were evaluated in animals to determine the suitability of this class of compounds for myocardial imaging. It was observed from the animal biodistribution data of the three radioactive compounds, there was a considerable variation in the heart to non-target organ uptake ratio. A possible explanation of this variation was offered in the light of their lipophilic character, protein binding ability and affinity towards non-target receptors. It is anticipated that this study may help to develop a 99mTc-cardiac glycoside complex with better distribution characteristics, and such a compound may offer a suitable alternative to 201Tl, which is at present used for myocardial imaging.