Cryopreservation Enables Long-Term Storage of 9-(2-Phosphonylmethoxy-ethyl) Adenine Prodrug-Loaded Reconstituted Lactosylated High-Density Lipoprotein

Chronic hepatitis B is caused by an infection of parenchymal liver cells with hepatitis B virus (HBV). The nucleoside analog 9-(2-phosphonylmethoxyethyl)adenine (PMEA) is a promising candidate drug for the treatment of HBV infections. PMEA effectively inhibits the replication HBV in cell culture (1,2), and the orally available bis(POM) derivative has entered clinical trials (3). Although initial results are promising, the disposition of PMEA remains a matter of concern. Most of the drug is cleared by the kidneys, whereas only a small fraction taken up by parenchymal liver cells (4). To enhance the effectiveness of PMEA against HBV, we developed a carrier-based strategy for selective delivery of PMEA to parenchymal liver cells. PMEA was conjugated to lithocholic acid-3a-oleate (LO), yielding the lipophilic prodrug PMEA-LO (5). The prodrug was incorporated into reconstituted lactosylated high-density lipoprotein (LacNeoHDL), a lipidic carrier that is selectively taken up by parenchymal liver cells via the asialoglycoprotein receptor (6,7). LacNeoHDLassociated PMEA-LO was delivered rapidly to parenchymal liver cells (8). Once internalized, PMEA was released from the PMEA-LO prodrug and phosphorylated to its active metabolite (8). The selective delivery of PMEA to parenchymal liver cells is expected to result in an enhanced therapeutic efficacy against HBV. An important prerequisite for clinical application is that PMEA-LO-loaded LacNeoHDL remains stable during storage and transport. An attractive option for storage of PMEALO-loaded LacNeoHDL is cryopreservation. Freezing may, however, seriously damage the particles. Studies on cryopreservation of lipoproteins focus on low-density lipoprotein (LDL). Freezing and thawing of LDL results in aggregation of the particles (9,10). The aggregation leads to non-specific binding of LDL to cultured human fibroblasts (9), and to increased plasma clearance and enhanced spleen uptake, suggesting phagocytosis by cells of the reticulo-endothelial system (11). However, cryoprotectants like sucrose protect LDL from freezing-induced damage (9,12). In this study we investigated the effect of cryopreservation of PMEA-LO-loaded LacNeoHDL on the stability of the PMEA-LO prodrug and on the physical and biological properties of the prodrug-loaded particles. As sucrose is beneficial for LDL during freezing, we examined the ability of sucrose to stabilize the prodrug-loaded carrier during cryopreservation