Carrier-mediated delivery improves the efficacy of 9-(2-phosphonylmethoxyethyl)adenine against hepatitis B virus

We recently synthesized a lipophilic prodrug of 9-(2-phosphonylmethoxyethyl )adenine (PMEA), designated PMEA-LO, and incorporated it into reconstituted lactosylated high-density lipoprotein (LacNeoHDL). In a rat model, LacNeoH DL-associated PMEA-LO was internalized by the asialoglycoprotein receptor o n parenchymal liver cells and converted into its active diphosphorylated me tabolite. To further evaluate the therapeutic potential of the carrier-asso ciated prodrug, we examined in this study the processing of I-125-labeled P MEA-LO-loaded LacNeoHDL by HepG2 cells. Upon incubation with HepG2 cells, P MEA-LO-loaded LacNeoHDL became rapidly cell-associated. The association was saturable and of high-affinity (k(d) = 3.8 +/- 0.4 nM). Asialofetuin, an e stablished ligand for the asialoglycoprotein receptor, inhibited the associ ation by >75%, which confirms the role of the asialoglycoprotein receptor. Association of the prodrug-loaded particles to HepG2 cells was coupled to d egradation. Radiolabeled degradation products appeared in the culture mediu m with a lag phase of 2 h. Asialofetuin and chloroquine inhibited secretion of degradation products by 75 to 80%, indicating that PMEA-LO-loaded LacNe oHDL is internalized via the asialoglycoprotein receptor and lysosomally pr ocessed. The therapeutic potential of LacNeoHDL-associated PMEA-LO was stud ied by measuring its effects on hepatitis B virus (HBV) replication in Hep AD38 cells (HBV-transfected HepG2 cells). LacNeoHDL-associated PMEA-LO effe ctively inhibited HBV DNA synthesis. The EC50 value of carrier-associated P MEA-LO was 35 times lower than that of free PMEA (3.4 +/- 0.4 and 120 +/- 1 8 ng of PMEA/ml, respectively). We conclude that the present results, combi ned with our earlier in vivo disposition data, underscore the therapeutic p otential and utility of PMEA-LO-loaded LacNeoHDL for treatment of chronic h epatitis B.