Anti-PEG IgM elicited by injection of liposomes is involved in the enhanced blood clearance of a subsequent dose of PEGylated liposomes
Introduction
Liposomes which are sterically stabilized with surface-coupled polyethylene glycol (PEG) can enhance their lifetime and that of entrapped therapeutic agents in the blood circulation [1], [2], [3]. It is hypothesized that the presence of PEG on the liposome attracts a water shell to the liposomal surface, providing a steric barrier against opsonins and/or recognition by cells of mononuclear phagocyte system (MPS) [4], [5], [6]. This, in turn, results in a decrease in the elimination rate of liposomes from the blood stream.
We and others have found that an intravenous injection of PEGylated liposomes causes a second dose of PEGylated liposomes, injected a few days later, to lose their long-circulating characteristics and accumulate extensively in liver, despite the presence of PEG on the surface of the liposomes [7], [8], [9], [10]. This phenomenon is referred to as the “accelerated blood clearance (ABC) phenomenon”. Although the details of underlying mechanism are not yet elucidated, we proposed the following tentative mechanism for the induction of the ABC phenomenon on the basis of our earlier results [11], [12], [13]: anti-PEG IgM, produced in the spleen in response to an injected dose of PEGylated liposomes, selectively binds to the PEG on a second dose of these liposomes, injected several days later, and subsequently activates the complement system. This, in turn, leads to opsonization of the liposomes by C3 fragments and, as a consequence, to enhanced uptake of the liposomes by the Kupffer cells in liver.
Earlier reports from our laboratory [11], [12], [13], [14] indicate that ‘empty’ PEGylated liposomes are immunogenic and promote antibody, especially IgM, responses against a second dose of such liposomes. This suggests that any PEGylated liposomal formulation, even if containing non-immunostimulatory payloads such as cytotoxic agents [14], may display unexpected pharmacokinetic behavior upon repeated injection and, as a consequence, may show less therapeutic efficacy or even cause undesirable side effects. Therefore, a strategy to abrogate the immunogenicity of PEGylated liposomes without significantly compromising their in vivo performance would be highly desirable for the further development of this otherwise promising drug delivery system. Therefore, studies providing further insight in the mechanisms underlying the ABC phenomenon are of great importance.
We have shown that anti-PEG IgM is responsible for the induction of the ABC phenomenon in rats [12], [13]. Similar results had been reported. Judge et al. [15] showed that anti-PEG antibodies were induced after injection of plasmid DNA-containing PEGylated liposome in mice. Srode et al. [16] showed that anti-PEG IgG was induced in response to the first injection of empty PEGylated liposome in rabbits. Both reports demonstrated that the induction of anti-PEG antibodies causes accelerated blood clearance of subsequently injected PEGylated liposomes. However, a clear-cut relationship between the amount of anti-PEG antibodies associated with PEGylated liposomes and the degree of induced ABC phenomenon has not been established. In addition, our earlier studies revealed that, at higher lipid dose (5 μmol phospholipid (PL)/kg), even conventional liposomes (without PEG-coating) can induce enhanced clearance of a subsequently injected dose of PEGylated, but not conventional, liposomes [17]. This raised the question if anti-PEG IgM is also produced following injection of conventional liposomes, causing accelerated clearance of a subsequent dose of PEGylated liposomes. In the present study we therefore addressed these issues. Our findings raise important concerns regarding the safety and efficiency of PEGylated liposomes currently in use or under consideration for clinical application.
Section snippets
Materials and animals
Hydrogenated egg phosphatidylcholine (HEPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-n-[methoxy(polyethylene glycol)-2000] (mPEG2000-DSPE) and DSPE were generously donated by Nippon Oil and Fat (Tokyo, Japan). Cholesterol (CHOL) was of analytical grade (Wako Pure Chemical, Osaka, Japan). All lipids were used without further purification. Sepharose 4 Fast Flow was purchased from Amersham-Pharmacia Biotech (Upsala, Sweden). Rhodamine-derivatized dihexadecanoylglycerophosphoethanolamine
Hepatic clearance of a second dose of PEGylated or conventional liposomes
After induction of the ABC phenomenon, a subsequent dose of PEGylated liposomes is rapidly cleared by Kupffer cells in the liver [7], [11]. Hepatic clearance reflects the liposome uptake activity of the Kupffer cells and hence is a good parameter of the magnitude of the induced ABC phenomenon [9], [10]. As shown in Fig. 1 (open bars, pre-injection with saline), hepatic clearance of a single dose of test PEGylated or conventional liposomes is very low. Upon a pre-injection with low- or high-dose
Discussion
The results described here clearly indicate that pre-administration of liposomes promotes the production of anti-liposome IgM regardless of the presence or absence of PEG-coating on their surface (Fig. 2). We demonstrated that the major liposomal component responsible for the recognition of the PEGylated liposomes by the anti-liposome IgM is the PEG moiety, in line with the observation that also the enhanced clearance phenomenon observed with test-dose liposomes is specifically seen with
Conclusion
The results presented here demonstrate that i.v. injected liposomes, regardless of the presence of a PEG-coating, are capable of inducing strong IgM responses against the PEG moiety of PEGylated liposomes and weak responses against the other lipid components. Although the exact mechanism of the induction the ABC phenomenon is not yet clear, our results clearly indicate that the production of such IgM is a major trigger transforming a subsequently injected dose of PEGylated liposomes from a less
Acknowledgements
We thank Dr. G.L. Scherphof for his helpful advice in writing the manuscript. This study was supported, in part, by research grant from The Kao Foundation for Arts and Sciences.
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