Stable Incorporation of a Lipophilic Daunorubicin Prodrug into Apolipoprotein E-Exposing Liposomes Induces Uptake of Prodrug via Low-Density Lipoprotein Receptor in Vivo A. JENNY VERSLUIS, ERIK T. RUMP, PATRICK C. N. RENSEN, THEO J. C. VAN BERKEL, and MARTIN K. BIJSTERBOSCH Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research, University of Leiden, Leiden, the Netherlands Accepted for publication October 14, 1998 This paper is available online at http://www.jpet.org ABSTRACT Many tumors express elevated levels of low-density lipoprotein (LDL) receptors. Therefore, native LDL and synthetic LDL-like particles have been proposed as carriers for antineoplastic drugs. We demonstrated earlier that small apolipoprotein E (apoE)-exposing liposomes were specifically recognized by the LDL receptor. In this study, we incorporated a lipophilic deriv- ative of daunorubicin (LAD) into the apoE liposomes. Up to 11 molecules of LAD could be incorporated per particle without significantly changing the size, lipid composition, and ability to bind apoE of the liposomes. The biological fate of the prodrug was largely determined by its carrier (70% of the initially incor- porated LAD was still associated to the liposomes after 4 h of circulation in mice). Compared with free daunorubicin, the cir- culation half-life of the liposome-associated prodrug was sub- stantially prolonged and undesired tissue disposition was re- duced. The role of the LDL receptor in the metabolism of LAD-loaded apoE liposomes was demonstrated in rats with up-regulated hepatic LDL receptors. In these rats, the liver uptake of the prodrug and carrier was increased 5-fold. The addition of apoE was essential for LDL receptor-mediated up- take of the drug-carrier complex. In LDL receptor-deficient mice, the circulation time of both the prodrug and the carrier increased approximately 2-fold compared with wild-type mice. We conclude that LAD-loaded apoE liposomes constitute a stable drug-carrier complex that is well suited for LDL receptor- mediated selective drug delivery to tumors. In the chemotherapy of cancer, the disposition of antineo- plastic drugs in nonmalignant tissues often causes severe side effects. The narrow therapeutic window of these drugs hampers the administration of fully effective doses. A thera- peutic strategy in which antineoplastic drugs are associated with carriers that are selectively taken up by tumor cells may diminish side effects and allow the administration of more effective doses (Tomlinson, 1987). The receptor for low-density lipoprotein (LDL) is an attrac- tive target for the selective delivery of antineoplastic drugs to tumors because it has been found that many tumors of dif- ferent origin express elevated levels of this receptor (Cata- pano, 1987; Vitols, 1991). Especially tumors of gynecological origin and myeloid leukemic cells, but also colon, kidney, lung, and brain tumors, were found to express exceptionally high amounts of LDL receptors (Firestone, 1994). The ele- vated expression of LDL receptors on tumor cells is probably a result of their rapid proliferation. The cells use the choles- terol present in LDL for the synthesis of new membranes. LDL is the predominant cholesterol-transporting lipoprotein in humans. It is a spherical particle of about 23 nm that consists of a polar shell of phospholipids and cholesterol, which surrounds an apolar core of mainly cholesterol esters. A large part of the particle surface is covered with the apo- lipoprotein B (apoB), which is recognized by the LDL recep- tor. After internalization of LDL via its receptor, the particle is degraded in the lysosomes (Brown and Goldstein, 1975, 1986). However, a possible drawback for the use of endogenous LDL for tumor therapy may be its limited availability. Fur- thermore, it has been found that the incorporation of cyto- toxic drugs into native LDL often induces altered physiolog- ical behavior of the particles (Masquelier et al., 1986; De Smidt and Van Berkel, 1990). A wide variety of methods to incorporate antitumor drugs into LDL has been explored, but apparently, the incorporation of drugs into LDL often causes subtle changes in the structure of apoB, which provoke in vivo uptake by mechanisms other than the LDL receptor (De Smidt and Van Berkel, 1990). Received for publication February 20, 1998. ABBREVIATIONS: apoB, apolipoprotein B; apoE, apolipoprotein E; CO, cholesterol oleate; DNR, daunorubicin; 17EE, 17-ethinyl estradiol; EYPC, egg yolk phosphatidylcholine; LAD, conjugate of 3-O-oleoyl-5-cholanic acid and alanine-leucine-alanine-leucine-daunorubicin; LDL, low-density lipoprotein; PBS, 10 mM sodium phosphate buffer (pH 7.4), containing 0.15 M NaCl. 0022-3565/99/2891-0001$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 289, No. 1 Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 289:1–7, 1999 1 at ASPET Journals on August 7, 2016 jpet.aspetjournals.org Downloaded from