Anti-PEG IgM elicited by injection of liposomes is involved in the enhanced blood clearance of a subsequent dose of PEGylated liposomes

https://doi.org/10.1016/j.jconrel.2007.02.010Get rights and content

Abstract

Earlier we reported that PEGylated liposomes lose their long-circulating characteristic when they are administrated twice in the same animal with certain intervals (referred to as the accelerated blood clearance (ABC) phenomenon). We proposed that anti-PEG IgM, induced by the PEGylated liposomes, is responsible for the phenomenon, based on the observation that IgM thus produced selectively binds to the surface of PEGylated liposomes, subsequently leading to substantial complement activation. Interestingly, we found that under certain circumstances administration of conventional liposomes without PEG-coating also caused a strong ABC response upon injection of a second dose of PEGylated liposomes, but not of conventional liposomes. This suggests that also conventional liposomes not modified with PEG can promote an IgM response against PEG. We report here that, irrespective of the presence or absence PEG-coating, a single first dose of liposomes is capable of inducing a strong anti-PEG IgM response and, under certain circumstances, also weak responses against other lipid components. A good correspondence was observed between the amount of IgM associating with both PEGylated and conventional liposomes, concomitant complement activation triggered by those liposomes and the magnitude of the ABC phenomenon against those liposomes. Taken together, our results demonstrate that the ABC phenomenon is fully attributable to production of anti-PEG IgM by the first dose of liposomes and the subsequent complement activation upon a second dose of PEGylated but not conventional liposomes. Although the responsible immunogenic epitopes of the liposomes remain to be determined, the immunogenicity of ‘empty’ liposomes presents a serious concern in the development of liposomal formulations and their use in the clinic. Furthermore, our findings as described here raise important concerns with regard to the safety and efficiency of liposomes currently under development for clinical use.

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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