Vol. 60, Issue 3, 521-527, September 2001

Carrier-Mediated Delivery Improves the Efficacy of 9-(2-Phosphonylmethoxyethyl)Adenine against Hepatitis B Virus

Martin K. Bijsterbosch, Chunxiao Ying, Remco L. A. de Vrueh, Erik de Clercq, Erik A. L. Biessen, Johan Neyts, and Theo J. C. van Berkel

Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research, University of Leiden, Leiden, The Netherlands (M.K.B., R.L.A.D.V., E.A.L.B., T.J.C.V.B.); and Rega Institute for Medical Research, University of Leuven, Leuven, Belgium (C.Y., E.D.C., J.N.)

We recently synthesized a lipophilic prodrug of 9-(2-phosphonyl-methoxyethyl)adenine (PMEA), designated PMEA-LO, and incorporated it into reconstituted lactosylated high-density lipoprotein (LacNeoHDL). In a rat model, LacNeoHDL-associated PMEA-LO was internalized by the asialoglycoprotein receptor on parenchymal liver cells and converted into its active diphosphorylated metabolite. To further evaluate the therapeutic potential of the carrier-associated prodrug, we examined in this study the processing of ¹²⁵I-labeled PMEA-LO-loaded LacNeoHDL by HepG2 cells. Upon incubation with HepG2 cells, PMEA-LO-loaded LacNeoHDL became rapidly cell-associated. The association was saturable and of high-affinity (k_d = 3.8 ± 0.4 nM). Asialofetuin, an established ligand for the asialoglycoprotein receptor, inhibited the association by >75%, which confirms the role of the asialoglycoprotein receptor. Association of the prodrug-loaded particles to HepG2 cells was coupled to degradation. Radiolabeled degradation products appeared in the culture medium with a lag phase of 2 h. Asialofetuin and chloroquine inhibited secretion of degradation products by 75 to 80%, indicating that PMEA-LO-loaded LacNeoHDL is internalized via the asialoglycoprotein receptor and lysosomally processed. The therapeutic potential of LacNeoHDL-associated PMEA-LO was studied by measuring its effects on hepatitis B virus (HBV) replication in Hep AD38 cells (HBV-transfected HepG2 cells). LacNeoHDL-associated PMEA-LO effectively inhibited HBV DNA synthesis. The EC₅₀ value of carrier-associated PMEA-LO was 35 times lower than that of free PMEA (3.4 ± 0.4 and 120 ± 18 ng of PMEA/ml, respectively). We conclude that the present results, combined with our earlier in vivo disposition data, underscore the therapeutic potential and utility of PMEA-LO-loaded LacNeoHDL for treatment of chronic hepatitis B.