Carrier-mediated delivery of 9-(2-phosphonylmethoxyethyl)adenine to parenchymal liver cells: a novel therapeutic approach for hepatitis B

Abstract

Our aim is to selectively deliver 9-(2-phosphonylmethoxyethyl)adenine (PMEA) to parenchymal liver cells, the primary site of hepatitis B virus (HBV) infection. Selective delivery is necessary because PMEA, which is effective against HBV in vitro, is hardly taken up by the liver in vivo. Lactosylated reconstituted high-density lipoprotein (LacNeoHDL), a lipid particle that is specifically internalized by parenchymal liver cells via the asialoglycoprotein receptor, was used as the carrier. PMEA could be incorporated into the lipid moiety of LacNeoHDL by attaching, via an acid-labile bond, lithocholic acid-3alpha-oleate to the drug. The uptake of the lipophilic prodrug (PMEA-LO) by the liver was substantially increased after incorporation into LacNeoHDL. Thirty minutes after injection of [(3)H]PMEA-LO-loaded LacNeoHDL into rats, the liver contained 68.9% +/- 7.7% of the dose (free [(3)H]PMEA, <5%). Concomitantly, the uptake by the kidney was reduced to <2% of the dose (free [(3)H]PMEA, >45%). The hepatic uptake of PMEA-LO-loaded LacNeoHDL occurred mainly by parenchymal cells (88.5% +/- 8.2% of the hepatic uptake). Moreover, asialofetuin inhibited the liver association by >75%, indicating uptake via the asialoglycoprotein receptor. The acid-labile linkage in PMEA-LO, designed to release PMEA during lysosomal processing of the prodrug-loaded carrier, was stable at physiological pH but was hydrolyzed at lysosomal pH (half-life, 60 to 70 min). Finally, subcellular fractionation indicates that the released PMEA is translocated to the cytosol, where it is converted into its active diphosphorylated metabolite. In conclusion, lipophilic modification and incorporation of PMEA into LacNeoHDL improves the biological fate of the drug and may lead to an enhanced therapeutic efficacy against chronic hepatitis B.

FIG. 1

Structure of PMEA-LO (the acid-labile phosphonamidate bond is indicated by an arrow).

FIG. 2

Release of [³H]PMEA from [³H]PMEA-LO-loaded LacNeoHDL at lysosomal pH. [³H]PMEA-LO-loaded LacNeoHDL was incubated at pH 4.7 (●) and 7.4 (○) at 37°C. At the indicated times, samples were chromatographically analyzed for released [³H]PMEA as described in Materials and Methods. The amounts of [³H]PMEA released are expressed as a percentage of the total radioactivity analyzed. Values are means ± SEMs for three individual experiments.

FIG. 3

Clearance from plasma and distribution in tissue of [³H]PMEA and [³H]PMEA-LO-loaded LacNeoHDL. Rats were intravenously injected with [³H]PMEA (50 μg/kg of body weight; open circles and open bars) or [³H]PMEA-LO-loaded LacNeoHDL (10 μg of [³H]PMEA/kg of body weight; closed circles and closed bars). At the indicated times, the amounts of radioactivity in plasma (A) and liver (B) were determined. At 30 min ([³H]PMEA-LO-loaded LacNeoHDL) or 40 min ([³H]PMEA) after injection, the amount of radioactivity in the indicated tissues (C) was determined. Values represent means ± SEMs for three rats.

FIG. 4

Effect of asialofetuin on the clearance from plasma and liver association of [³H]PMEA-LO-loaded LacNeoHDL. Rats were intravenously injected with [³H]PMEA-LO-loaded LacNeoHDL (10 μg of [³H]PMEA/kg of body weight). One minute prior to injection of the radiolabeled ligand, the animals received asialofetuin (50 mg/kg of body weight; ●) or PBS (○) by intravenous injection. At the indicated times, the amounts of radioactivity in plasma (A) and liver (B) were determined. Values represent means ± SEMs for three rats.

FIG. 5

Distribution patterns of radioactivity and marker enzymes over subcellular fractions of the liver after injection of [³H]PMEA-LO-loaded LacNeoHDL. Rats were intravenously injected with [³H]PMEA-LO-loaded LacNeoHDL (15 μg of [³H]PMEA/kg of body weight). Five hours later, the liver was perfused with ice-cold 0.25 M sucrose containing 10 mM Tris-HCl buffer (pH 7.5) and was divided into subcellular fractions by differential centrifugation, as described earlier (17). The fractions were assayed for radioactivity, protein content, and the activities of marker enzymes; recoveries were >85%. Blocks from left to right represent nuclear (N), mitochondrial (M), lysosomal (L), microsomal (P), and cytosolic (S) fractions. The relative protein concentration is given on the x axis. The y axis represents the relative specific activity (percentage of total activity recovered divided by percentage of total protein recovered).

FIG. 6

Anion-exchange chromatographic analysis of the radioactivity in a liver extract after injection of [³H]PMEA-LO-loaded LacNeoHDL. Five hours after intravenous injection of [³H]PMEA-LO-loaded LacNeoHDL (15 μg of [³H]PMEA/kg of body weight) into rats, a liver lobule was homogenized in cold (−80°C) methanol. The homogenate was centrifuged, filtered, and analyzed by anion-exchange HPLC as described in Materials and Methods. Fractions of 1 ml were collected and assayed for radioactivity. The elution times of unlabeled PMEA, PMEAp, and PMEApp standards are indicated by arrows.