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Hepatitis B virus (HBV) infection is the world's most important chronic virus infection. No safe and effective treatment is available at present, and clinical exploration of promising antiviral agents, such as nucleoside analogues is hampered because of significant side-effects due to their aspecific body distribution. We are exploring the possibility of the selective delivery of antiviral active drugs to liver parenchymal cells, the main site of infection and replication of HBV. Chylomicrons, which transport dietary lipids into the liver via apolipoprotein E-specific receptors, could serve as drug carriers. However, their endogenous nature hampers their application as pharmaceutical drug carriers. We report here that incorporation of a derivative of the nucleoside analogue iododeoxyuridine into recombinant chylomicrons leads to selective targeting to liver parenchymal cells. Potentially effective intracellular drug concentrations of 700 nM can be achieved, and we therefore anticipate that these drug carrier complexes represent a conceptual advance in the development of an effective and safe therapy for hepatitis B.

Chylomicrons labelled with [3H]cholesterol/[3H]cholesterol esters in a ratio of 25.5: 74.5, were rapidly removed from rat serum in vivo, and taken up predominantly by the parenchymal liver cells (88.2%) of the hepatic uptake at 15 min after injection). Lactoferrin reduced the liver uptake of chylomicron remnants by 72%, at 20 min after injection. It appeared that the free cholesterol which is present in the chylomicrons is not readily exchanged within the used time period with other cholesterol pools in the animal. Between 10-60 min after injection of 3H-labelled chylomicrons, cholesterol esters are hydrolysed in the liver. Appearance of radioactivity in bile was rapid and at 3, 24 and 72 h after injection, 13.4%, 44.0% and 70.0%, respectively, of the injected dose appeared in bile, mainly as bile acids (> 90%). Lactoferrin reduced the biliary secretion of radioactivity, especially during the first hour after injection. The total amount of radioactivity recovered was 58.0% of the injected dose at 72 h after injection. After injection of beta-migrating very low-density lipoprotein labelled with [3H]cholesterol/[3H]cholesterol esters in a ratio of 23.5:76.5, the maximum amount of radioactivity secreted in bile was much lower than with chylomicrons (2.6% cf. 5.2% at 1 h after injection), although the kinetics of the initial liver association and cholesterol ester hydrolysis were even more rapid. Biliary accumulation of radioactivity was also lower with 50.5% of the injected dose recovered at 72 h after injection. It can be concluded from these studies that the processing of chylomicron remnant cholesterol components in the liver and the subsequent secretion in the bile mainly as bile acids is very efficient. The efficient liver uptake of chylomicron remnants by the liver remnant receptor is thereby essential to achieve this high percentage of removal, thus protecting against extrahepatic cholesterol (ester) deposition.

Upon injection of chylomicrons into rats, chylomicron remnants are predominantly taken up by parenchymal cells, with a limited contribution (8.6% of the injected dose) by Kupffer cells. In vitro storage of partially processed chylomicron remnants for only 24 h leads, after in vivo injection, to an avid recognition by Kupffer cells (uptake up to 80% of the total liver-associated radioactivity). Lactoferrin greatly reduces the liver uptake of chylomicron remnants, which appears to be the consequence of a specific inhibition of the uptake by parenchymal cells. Kupffer-cell uptake is not influenced by lactoferrin. In vitro studies with isolated parenchymal and Kupffer cells show that both contain a specific recognition site for chylomicron remnants. The Kupffer-cell recognition site differs in several ways from the recognition site on parenchymal cells as follows. (a) The maximum level of binding is 3.7-fold higher/mg cell protein than with parenchymal cells. (b) Binding of chylomicron remnants is partially dependent on the presence of calcium, while binding to parenchymal cells is not. (c) beta-Migrating very-low-density lipoprotein is a less effective competitor for chylomicron-remnant binding to Kupffer cells compared to parenchymal cells. (d) Lactoferrin leaves Kupffer-cell binding uninfluenced, while it greatly reduces binding of chylomicron remnants to parenchymal cells. The properties of chylomicron-remnant recognition by parenchymal cells are consistent with apolipoprotein E being the determinant for recognition. It can be concluded that the chylomicron-remnant recognition site on Kupffer cells possesses properties which are distinct from the recognition site on parenchymal cells. It might be suggested that partially processed chylomicron remnants are specifically sensitive to a modification, which induces an avid interaction with the Kupffer cells. The recognition site for (modified) chylomicron remnants on Kupffer cells might function as a protection system against the occurrence of these potential atherogenic chylomicron-remnant particles in the blood.

The uptake in vivo of chylomicrons and beta-migrating very-low-density lipoprotein (beta-VLDL) by rat liver, which is primarily carried out by parenchymal cells, is inhibited, 5 min after injection, to respectively 35 and 8% of the control values after preinjection of lactoferrin. The decrease in the uptake of lipoproteins by the liver caused by lactoferrin is a specific inhibition of uptake by parenchymal cells. Competition studies in vitro demonstrate that chylomicron remnants and beta-VLDL compete for the same recognition site on parenchymal cells. Data obtained in vivo together with the competition studies performed in vitro indicate that chylomicron remnants and beta-VLDL interact specifically with the same remnant receptor. Hepatic uptake of 125I-labelled-alpha 2-macroglobulin in vivo, mediated equally by parenchymal and endothelial cells, is not decreased by preinjection of lactoferrin and no effect on the parenchymal-cell-mediated uptake is found. In vitro, alpha 2-macroglobulin and chylomicron remnants or beta-VLDL show no cross-competition. Culturing of parenchymal cells for 24-48 h leads to a decrease in the cell association of alpha 2-macroglobulin to 26% of the initial value, while the cell association of beta-VLDL with the remnant receptor is not influenced. It is concluded that beta-VLDL and chylomicron remnants are recognized by a specific remnant receptor on parenchymal liver cells, while uptake of alpha 2-macroglobulin by liver is carried out by a specific receptor system (presumably involving the LDL-receptor-related protein) which shows properties that are distinct from those of the remnant receptor.