Labeling of Pegylated Liposomal Doxorubicin (Doxil) for Pharmacokinetic and Non-Invasive Imaging Studies

Pharmacokinetic and organ distribution studies of liposomal drugs in humans are a challenge. A direct labeling method using Tc-N,N-bis(2-mercaptoethyl)-N ,N -diethyl-ethylenediamine (BMEDA) complex to label the commercially available pegylated liposomal doxorubicin, Doxil, has been introduced. Biodistributions of Tc-Doxil in normal rats were performed to evaluate the feasibility of using it for monitoring the pharmacokinetics of liposomes encapsulating drugs. Labeling efficiency of Tc-Doxil was 70.6 0.8% (n 3). In vitro incubation of Tc-Doxil in 50% fetal bovine serum or 50% human serum at 37°C showed good labeling stability with 72.3 3.6% or 78.6 1.8% of activity associated with Doxil at 24 h, respectively (n 3). There was a two-phase blood clearance with half-clearance times of 2.2 and 26.2 h after bolus intravenous injection in normal rats. Distribution of Tc-Doxil at 44 h after injection had 19.8 1.3% of injected dose in blood, 14.1 1.7% in liver, 2.6 0.3% in spleen, 9.0 0.8% in bone with marrow, 6.0 0.5% in skin, and 15.3 4.3% in bowel (n 5). Unencapsulated Tc-BMEDA had a very rapid blood clearance with a half-clearance time of only 0.12 h (n 4). By using this Tc labeling method, biodistribution and pharmacokinetics of ammonium gradient liposomes encapsulating drugs can be determined by noninvasive scintigraphic imaging. This labeling method may be extended to Re and Re labeling to combine chemotherapy and radionuclide therapy for tumor treatment. Transmembrane pH or ammonium gradient liposomes are promising drug carriers, because these liposome formulations can achieve high drug encapsulation capacity and high drug entrapment efficiency (Mayer et al., 1986, 1990; Haran et al., 1993). Pegylated liposomes have prolonged residence time in blood, resulting in greater treatment effectiveness and lower treatment complications (Gabizon et al., 2003). Doxil, a commercially available pegylated liposomal doxorubicin, uses an ammonium gradient to load and carry doxorubicin for clinical tumor treatment (Gabizon et al., 2003). Animal and human subject studies have been performed with Doxil to treat various kinds of tumors (Gabizon, 1992; Harrington et al., 2000b). Because it is necessary to know the in vivo behavior of the therapeutic drugs trapped in liposomes as well as the free drugs after liposomal therapeutic drug administration, the pharmacokinetic study of liposomal therapeutic drugs tends to be more complex than routine drugs (Harashima et al., 2002). Several -emitting radionuclides can be used to label liposomes for monitoring the in vivo behavior of liposomes noninvasively (Harrington et al., 2000a,b). By using imaging systems, such as a gamma camera or a positron emission tomography camera, the whole body distribution of a radiolabeled carrier or a radiolabeled compound in each organ or tissue can be measured at different times in a live animal or in a human subject. By using noninvasive imaging, fewer animals are required to study the pharmacokinetics of drugs, making it more efficient and usually cheaper to perform the studies (Contag, 2002). In addition, increased statistical significance can be reached with fewer animals, because the images of each animal acquired at the initial time point can serve as the internal control at subsequent time points. Positron emission radionuclides, such as carbon-11 (C), nitrogen-13 (N), and fluorine-18 (F), may be used to label drug or carrier molecules (Aboagye et al., 2001). The disadvantage of these positron emission tomography radionuclides is that they have relatively short half-lives (C, 20.4 min; N, 10.0 min; and F, 109.8 min), which makes it harder to trace the in vivo behavior of a drug or a carrier for long time