Masako Ichihara

Accelerated clearance of PEGylated liposomes in rats after repeated injections

Polyethylene glycol-modified liposomes (PEGylated liposomes) represent promising carrier systems for therapeutic agents. Herein, we report on a study of the effect of repeated injection of PEGylated liposomes on their pharmacokinetics in rats. The first dose resulted in a reduction in the circulation time and an increase in hepatic accumulation of the second dose in a time-interval of injection-dependent manner. No significant increases in the number of Kupffer cells were detectable, although the liver most likely played an important role in the accelerated clearance. Interestingly, the acceleration in clearance became less pronounced, when the third dose was injected at 4, 7 or 14 days after the second injection (the second dose was given 5 weeks after the first injection). An accelerated clearance was evoked in normal rats by the transfusion of serum from rats that had received PEGylated liposomes 5 days earlier, indicating that humoral serum factor(s) are also involved in causing the accelerated clearance. A complement consumption assay indicated that the complement system is not the factor. In summary, multiple injections of PEGylated liposomes clearly altered their pharmacokinetic behavior in rats. It is likely that cellular immunity (Kupffer cells) and humoral immunity are required to cause the phenomenon. The results reported here have a considerable impact in and important implications on the clinical application, design and engineering of PEGylated liposomes for use in repeated intravenous administration.

Accelerated clearance of a second injection of PEGylated liposomes in mice.

We recently reported that the firstly injected PEGylated liposomes dramatically affected the rate of blood clearance of secondly injected PEGylated liposomes in rats in a time interval of injection dependent manner [J. Control. Release (2003)]. Mice are frequently used in evaluations of the therapeutic efficacy of PEGylated liposomal formulations, but the pharmacokinetics of repeatedly injected PEGylated liposomes in mice is not fully understood. In this study, therefore, we examined in mice the effect of the repeated injection of PEGylated liposomes on their pharmacokinetics. An intravenous pretreatment with PEGylated liposomes produced a striking change in the biodistribution of the second dose which was given several days after the first injection. The first dose resulted in a reduction in the circulation half-life of the second dose. The degree of alteration was dependent on the time interval between the injections. The rapid clearance of the second dose was strongly related to hepatic clearance (CLh). This finding suggests that a considerable increase in hepatic accumulation accounts for this phenomenon. But, no liver injury or an increase in the number of Kupffer cells were detected in histopathological evaluations. Collectively, although the multiple injections of the PEGylated liposomes had no obvious physical effects, such as inflammation, their pharmacokinetic behavior was clearly altered in mice. The results obtained here have important implications not only with respect to the design and engineering of liposomes for human use, but for evaluating the therapeutic efficacy of liposomal formulations in experimental animal models as well.

Accelerated blood clearance of PEGylated liposomes containing doxorubicin upon repeated administration to dogs

The accelerated blood clearance phenomenon involving anti-PEG IgM production has been recognized as an important issue for the design and development of PEGylated liposomes. Here, we show that empty PEGylated liposomes and Doxil, PEGylated liposomes containing doxorubicin, both caused anti-PEG IgM production and thereby a rapid clearance of the second and/or third dose of Doxil in Beagle dogs in a lipid-dose, inverse-dependent manner. It appears that the pharmacokinetic profile of the second and third administration of Doxil reflected the presence of anti-PEG IgM circulating in the blood. Doxil plus an excess amount of empty PEGylated liposomes rather enhanced the production of anti-PEG IgM compared to Doxil of the same doxorubicin dose. During sequential administration, increasing the lipid dose of Doxil in each dose by the addition of empty PEGylated liposomes strongly attenuated the magnitude of the ABC phenomenon during the effectuation phase of a second and third dose of Doxil. Our results suggest that the pre-clinical study of anti-cancer drug-containing PEGylated liposomes with dogs must be carefully designed and performed with monitoring of the anti-PEG IgM and liposomal drugs circulating in the blood.

Anti-PEG IgM Response against PEGylated Liposomes in Mice and Rats.

We have reported that PEGylated liposomes lose their long-circulating properties when they are administered repeatedly at certain intervals to the same animal. This unexpected phenomenon is referred to as the accelerated blood clearance (ABC) phenomenon. We recently showed that the ABC phenomenon is triggered via the abundant secretion of anti-PEG IgM in response to the first dose of PEGylated liposomes. However, the details of the underlying mechanism for the induction of anti-PEG IgM production are yet to be elucidated. The present study demonstrated that the spleen is a major organ involved in the secretion of anti-PEG IgM in mice and rats. Anti-PEG IgM production was detected in nude, T-cell deficient mice, but not in SCID mice with B- and T-cell deficiencies. These observations indicate that splenic B-cells secret anti-PEG IgM without help from T-cells. Sequential injections of PEGylated liposomes into the same mice did not promote isotype switching from IgM to IgG. Accordingly, PEGylated liposomes may function as a type-2, T-cell-independent antigen (TI-2 antigen) during anti-PEG IgM production. Although the underlying mechanism that causes an anti-PEG IgM response against PEGylated liposomes is not yet clear, our findings give implications in revealing the anti-PEG IgM response against PEGylated liposome.

Combination therapy of metronomic S‐1 dosing with oxaliplatin‐containing polyethylene glycol‐coated liposome improves antitumor activity in a murine colorectal tumor model

Metronomic chemotherapy has been advocated recently as a novel chemotherapeutic regimen. Polyethylene glycol (PEG)‐coated liposomes are well known to accumulate in solid tumors by virtue of the highly permeable angiogenic blood vessels characteristic for growing tumor tissue, the so‐called “enhanced permeability and retention (EPR) effect”. To expand the range of applications and investigate the clinical value of the combination strategy, the therapeutic benefit of metronomic S‐1 dosing in combination with oxaliplatin (l‐OHP)‐containing PEG‐coated liposomes was evaluated in a murine colon carcinoma‐bearing mice model. S‐1 is an oral fluoropyrimidine formulation and metronomic S‐1 dosing is a promising alternative to infused 5‐FU in colorectal cancer therapy. Therefore, the combination of S‐1 with l‐OHP may be an alternative to FOLFOX (infusional 5‐FU/leucovorin (LV) in combination with l‐OHP), which is a first‐line therapeutic regimen of a colorectal carcinoma. The combination of oral metronomic S‐1 dosing with intravenous administration of liposomal l‐OHP formulation exerted excellent antitumor activity without severe overlapping side‐effects, compared with either metronomic S‐1 dosing, free l‐OHP or liposomal l‐OHP formulation alone or metronomic S‐1 dosing plus free l‐OHP. We confirmed that the synergistic antitumor effect is due to prolonged retention of l‐OHP in the tumor on account of the PEG‐coated liposomes, presumably via alteration of the tumor microenvironment caused by the metronomic S‐1 treatment. The combination regimen proposed here may be a breakthrough in treatment of intractable solid tumors and an alternative to FOLFOX in advanced colorectal cancer therapy with acceptable tolerance and preservation of quality of life (QOL). (Cancer Sci 2010; 101: 2470–2475)

Multiple administration of PEG-coated liposomal oxaliplatin enhances its therapeutic efficacy: a possible mechanism and the potential for clinical application.

We previously developed a PEG-coated cationic liposome that enabled dual targeting delivery of oxaliplatin (l-OHP) to both tumor endothelial cells and tumor cells in a solid tumor. The targeted liposomal l-OHP formulation consequently elicited potent antitumor efficacy in a murine solid tumor model after 3 sequential injections. However, the probable mechanism(s) for this enhanced antitumor activity has not been fully elucidated. In the present study, therefore, the changes in tumor microenvironment induced by sequential administration of liposomal l-OHP were investigated, with emphasis on its impact to the intratumoral localization of the subsequently injected dose. In addition, the potential for anti-PEG IgM production upon repeated administration of liposomal l-OHP-containing PEGylated lipid was clearly revealed. Two sequential injections of liposomal l-OHP induced superior apoptotic activity in tumor tissue and thus resulted in broader intratumor distribution of the subsequent test dose of PEG-coated cationic liposomes, compared with a single injection of liposomal l-OHP. In addition, it was confirmed that repeated administration of liposomal l-OHP did not induce a significant anti-PEG IgM response, indicating that l-OHP encapsulated in PEG-coated liposomes was efficient in abrogating the ABC phenomenon. These results suggest that sequential treatment strategies with liposomal cytotoxic agents might be superior to mono-treatment strategies in achieving alterations in the tumor microenvironment and maintaining/restoring the pharmacokinetics of the formulation, and, therefore, would result in substantial therapeutic efficacy.

Influence of dose and animal species on accelerated blood clearance of PEGylated liposomal doxorubicin.

We recently demonstrated that Doxil loses its long-circulating properties when injected repeatedly at doses below 2 mg/m(2) in dogs. In studies using other animal species, PEGylated liposomal doxorubicin has been reported not to induce the accelerated blood clearance (ABC) phenomenon. We investigated the issue of whether Doxil can elicit the ABC phenomenon in several species. In minipigs, the ABC phenomenon was induced at 2 mg/m(2). In other animal species, the ABC phenomenon was not observed at higher doses (>2 mg/m(2)), but was observed at much lower doses (0.2 mg/m(2)). The pharmacokinetic profile of a second dose of Doxil reflected the circulating anti-PEG IgM level induced by the first dose. The ABC phenomenon was not observed at the clinically recommended DXR dose (20 mg/m(2)) in any animal species. These results indicate that Doxil can cause the ABC phenomenon in all animals tested, the extent of induction was dependent on the first dose of Doxil, and a higher Doxil dose lessened the ABC phenomenon. The current study results suggest that a careful study design including selection of animal species is important for preclinical studies using PEGylated liposomal formulations even if they contain anticancer drugs that suppress the host immune response.

Anti-PEG IgM Production via a PEGylated Nano-Carrier System for Nucleic Acid Delivery

For the systemic application of nucleic acids such as plasmid DNA and small interfering RNA, safe and efficient carriers that overcome the poor pharmacokinetic properties of nucleic acids are required. A cationic liposome that can formulate lipoplexes with nucleic acids has significant promise as an efficient delivery system in gene therapy. To achieve in vivo stability and long circulation, most lipoplexes are modified with PEG (PEGylation). However, we reported that PEGylated liposomes lose their long-circulating properties when they are injected repeatedly at certain intervals in the same animal. This unexpected and undesirable phenomenon is referred to as the accelerated blood clearance (ABC) phenomenon. Anti-PEG IgM produced in response to the first dose of PEGylated liposomes has proven to be a major cause of the ABC phenomenon. Therefore, in a repeated dosing schedule, the detection of anti-PEG IgM in an animal treated with PEGylated lipoplex could be essential to predict the occurrence of the ABC phenomenon. This chapter introduces a method for the evaluation of serum anti-PEG IgM by a simple ELISA procedure, and describes some precautions associated with this method.