Pharmaceutical NanotechnologyPharmacokinetics and biodistribution of RGD-targeted doxorubicin-loaded nanoparticles in tumor-bearing mice
Introduction
Doxorubicin is an anthracycline, widely used in the treatment of breast and ovarian cancer (Devita et al., 1989, Martindale, 1996, Vermorken, 2003). However, dose-limiting toxicities occur with doxorubicin therapy and include myelosuppression and cardiotoxicity. Congestive heart failure is of particular concern, and is dosage cumulative such that no more than 550 mg/m2 is recommended as a lifetime total dose (Von Hoff et al., 1979). Reformulation of doxorubicin has been undertaken to improve its pharmacokinetic (PK) and pharmacodynamic profile and minimize its toxicity. Liposomal formulations, including Myocet™ (Elan Pharmaceuticals) and Doxil® (Ortho BioTech) have been approved for clinical use and have similar efficacies and improved toxicity profiles as compared with doxorubicin (Harris et al., 2002). Additional formulations are also being developed to improve drug delivery, including the use of N-(2-hydroxypropyl)methacrylamide (HPMA)–doxorubicin conjugates (Duncan et al., 1998, Kovar et al., 2003, Ulbrich et al., 2003). These conjugates form a hydrogel in which doxorubicin is covalently linked to the water-soluble, biocompatible polymer HPMA via its ketone (forming a hydrazone bond) or amine site (forming an aconityl or amide bond). In addition, peptidic spacers, such as Gly–Phe–Leu–Gly and Gly–Gly, have also been incorporated in an effort to impart a pH-sensitive release profile. Polymeric micelles, most notably comprised of pluronics (polyether block copolymers), PEG-phospholipid and poly(ethyleneglycol)-poly(aminoacid) (PEG-PAA) block copolymers, have also been employed to deliver chemotherapeutics (Kwon, 2003). A formulation comprised of doxorubicin covalently attached to a poly(aspartic acid) backbone, and with additional doxorubicin molecules physically loaded into the core, has advanced to Phase II clinical trials in Japan (Kwon et al., 1994). In this formulation, only the physically entrapped doxorubicin molecules, and not covalently attached doxorubicin, are bioactive and tumorcidal.
The use of peptides capable of targeting tumor is one of the more recent developments for improving the performance and safety of selected chemotherapeutics. A number of RGD peptides and peptidomimetics have been shown to bind preferentially to particular integrins, for instance integrin αvβ3, which is often overexpressed on endothelial cells in tumor neovasculature (Pasqualini et al., 1997). Recently, anti-tumor efficacy was improved by targeting doxorubicin-loaded liposomes to the vasculature of colon cancer using RGD peptides in a xenograft mouse (Schiffelers et al., 2003). The targeting of integrin αvβ3 may lead to endocytosis of the doxorubicin-containing particles, subsequent killing of endothelial cells and tumor vasculature destruction.
We have developed carbohydrate-based NPs incorporating doxorubicin for the treatment of cancer. These NPs are typically between 15 and 30 nm in diameter with a core comprised of a modified, cross-linked carbohydrate: inulin multi-methacrylate (IMMA). Doxorubicin is covalently bound to the NP core via an amide bond. A targeting element consisting of a cyclic peptide with the RGD sequence: cyclo(–Arg–Gly–Asp–d–Phe–Cys–), is also attached using a PEG tether. We report herein our studies using these unique hydrophilic NPs with covalently attached doxorubicin. To define the PK and biodistribution of this formulation a murine mammary tumor model is described.
Section snippets
Chemicals
Doxorubicin HCl, doxorubicinol and doxorubicinone were purchased from Qventas (Branford, CT). Daunorubicin HCl and sodium acrylate (NaA) were obtained from Sigma (St. Louis, MO). The cyclic RGD peptide was supplied by Peptisyntha (Torrance, CA). Inulin was purchased from Carbomer (San Diego, CA). Inulin multi-methacrylate (Mw 1750 approximately), cystine bisacrylamide (CiBA) and poly(ethylene glycol) 400-dibromoacetate (PEG 400 DBA) were prepared in-house. All other reagents were at least
Results
The plasma levels of doxorubicin were determined following a single injection of RGD-targeted doxorubicin-loaded NP (12.5 mg/kg dox equiv.) in female Balb/c mice bearing Cl-66 mammary tumors. The resulting plasma levels over 24 h are shown in Fig. 1 and the PK parameters as determined using the Kinetica v4.2 program are shown in Table 1. The concentration of total doxorubicin in plasma was 213.3 ± 54.6 μg/mL at 2 min following injection and decreased bi-exponentially to essentially undetectable
Discussion
Covalent linkage of doxorubicin to NPs with an IMMA core and i.v. injection (ca. 12.5 mg/kg) resulted in a bi-exponential PK profile. The plasma concentration profile compares favorably with that reported by Seymour et al. (1990). In those studies, the PK of a doxorubicin-HPMA conjugate was studied following i.v. administration of the conjugate at a 5 mg/kg doxorubicin equivalent dose. At 30 min post-injection, Seymour et al. (1990) reported the total drug concentration in plasma to be
Conclusions
In summary, the PK and biodistribution of doxorubicin administered in a carbohydrate NP formulation was determined in plasma, tumor and five selected tissues using a Cl-66-tumor-bearing mouse model. The PK of doxorubicin (total drug content) in plasma followed a two-compartment model following i.v. (bolus) administration of the NPs. The terminal half-life was determined to 5.99 h.
The accumulation of doxorubicin was high in both the liver and spleen. Notably, unconjugated doxorubicin (and
Acknowledgments
The work was supported by a contract from the National Cancer Institute. The authors wish to thank Sowmya Chollate for assistance in PK sample preparation and analysis, and Amy Hsu, Almita Heramia, Anthony Lam and Andrew Goodwin for nanoparticle fabrication.
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