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. 2013:8:1257-68.
doi: 10.2147/IJN.S41701. Epub 2013 Mar 28.

Accelerated drug release and clearance of PEGylated epirubicin liposomes following repeated injections: a new challenge for sequential low-dose chemotherapy

Qiang Yang  1 , Yanling Ma, Yongxue Zhao, Zhennan She, Long Wang, Jie Li, Chunling Wang, Yihui Deng
Affiliations

Accelerated drug release and clearance of PEGylated epirubicin liposomes following repeated injections: a new challenge for sequential low-dose chemotherapy

Qiang Yang et al. Int J Nanomedicine. 2013.

Abstract

Background: Sequential low-dose chemotherapy has received great attention for its unique advantages in attenuating multidrug resistance of tumor cells. Nevertheless, it runs the risk of producing new problems associated with the accelerated blood clearance phenomenon, especially with multiple injections of PEGylated liposomes.

Methods: Liposomes were labeled with fluorescent phospholipids of 1,2-dipalmitoyl-snglycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) and epirubicin (EPI). The pharmacokinetics profile and biodistribution of the drug and liposome carrier following multiple injections were determined. Meanwhile, the antitumor effect of sequential low-dose chemotherapy was tested. To clarify this unexpected phenomenon, the production of polyethylene glycol (PEG)-specific immunoglobulin M (IgM), drug release, and residual complement activity experiments were conducted in serum.

Results: The first or sequential injections of PEGylated liposomes within a certain dose range induced the rapid clearance of subsequently injected PEGylated liposomal EPI. Of note, the clearance of EPI was two- to three-fold faster than the liposome itself, and a large amount of EPI was released from liposomes in the first 30 minutes in a complement-activation, direct-dependent manner. The therapeutic efficacy of liposomal EPI following 10 days of sequential injections in S180 tumor-bearing mice of 0.75 mg EPI/kg body weight was almost completely abolished between the sixth and tenth day of the sequential injections, even although the subsequently injected doses were doubled. The level of PEG-specific IgM in the blood increased rapidly, with a larger amount of complement being activated while the concentration of EPI in blood and tumor tissue was significantly reduced.

Conclusion: Our investigation implied that the accelerated blood clearance phenomenon and its accompanying rapid leakage and clearance of drug following sequential low-dose injections may reverse the unique pharmacokinetic-toxicity profile of liposomes which deserved our attention. Therefore, a more reasonable treatment regime should be selected to lessen or even eliminate this phenomenon.

Keywords: PEGylated liposomes; accelerated blood clearance (ABC) phenomenon; complement; epirubicin; sequential low-dose injections.

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Figures

Figure 1
Figure 1
(A) Blood clearance of epirubicin. From groups A1 to J1 represents pre-dosing with 5% glucose injection, 1, 5, 10, 15, or 50 μmol phospholipids/kg empty liposomes and 1, 5, 10, or 15 μmol phospholipids/kg epirubicin liposomes, respectively. (B) Blood clearance of NBD-PE labeled liposomes. (C) Hepatic and splenic accumulation of epirubicin, 4 hours after intravenous injection of the test dose. (D) Hepatic and splenic accumulation of NBD-PE labeled liposomes. Groups A2 to J2 represents the same protocol of pre-dosing as (A). Notes: Data are shown as mean ± SD, n = 3. P-values apply to differences between the control and treated rats. *P < 0.05; **P < 0.01; ***P < 0.001. Abbreviation: NBD-PE, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl). SD, standard deviation.
Figure 2
Figure 2
Effect of days of sequential injections of epirubicin-encapsulated PEGylated liposomes on the pharmacokinetics and biodistribution of the last dose. (A) Blood clearance. One to 10 represents the number of days of sequential injection with 0.75 mg liposomal epirubicin/kg. (B) Hepatic and splenic accumulation, 4 hours after intravenous injection of the last dose. Notes: Data are shown as mean ± SD, n = 3. P-values apply to differences between the control and treated rats. *P < 0.05. Abbreviation: PEG, polyethylene glycol; SD, standard deviation.
Figure 3
Figure 3
The production of anti-PEG IgM in rats. (A) Groups A to J represent pre-dosing with 5% glucose injection; 1, 5, 10, 15, or 50 μmol phospholipids/kg empty liposomes; and 1, 5, 10, or 15 μmol phospholipids/kg epirubicin liposomes. Anti-PEG IgM in the serum was determined at the seventh day after the first administration and before the last day of repeated injections (B). Notes: Data are shown as mean ± SD, n = 3. P-values apply to difference between the control and treated rats. *P < 0.05; **P < 0.01. Abbreviation: IgM, immunoglobulin M; OD, optical density; PEG, polyethylene glycol; SD, standard deviation.
Figure 4
Figure 4
The leakage of epirubicin from liposomes during incubation with the rat sera. Notes: Groups A to J represent the sera which were collected from rats that had already been pre-dosed with a 5% glucose injection; 1, 5, 10, 15, or 50 μmol phospholipids/kg empty liposomes; and 1, 5, 10, or 15 μmol phospholipids/kg epirubicin liposomes respectively. Data are shown as mean ± SD, n = 3. Abbreviation: SD, standard deviation.
Figure 5
Figure 5
The residual complement activity in rats and mice serum. (A) Groups B to J represent pre-dosing with 1, 5, 10, 15, or 50 μmol phospholipids/kg empty liposomes and 1, 5, 10, or 15 μmol phospholipids/kg epirubicin liposomes. (B) The residual complement activity in mice serum. Notes: Mice were injected via the tail vein with EPI dose of 0.75 mg liposomal EPI/kg (Low EPI-L), 1.5 mg liposomal EPI/kg (High EPI-L), or 0.75 mg free EPI/kg (Free-EPI), once daily for 10 days. In addition, the group of Low plus high EPI-L was injected with 0.75 mg liposomal EPI/kg for the first 5 days, and then the dose was increased to 1.5 mg liposomal EPI/kg. Data are shown as mean ± SD, n = 3 for rats and n = 5 for mice. Abbreviations: EPI, epirubicin; SD, standard deviation.
Figure 6
Figure 6
The anti-tumor activity of sequential low-dose injected epirubicin liposomes on S180 tumor-bearing mice. (A) Tumor volume. (B) The determination of anti-PEG IgM following repeated injections of 5% glucose, free epirubicin, and epirubicin-encapsulated PEGylated liposomes. Notes: S180 tumor-bearing mice were injected via the tail vein with EPI dose of 0.75 mg liposomal EPI/kg (Low EPI-L), 1.5 mg liposomal EPI/kg (High EPI-L), or 0.75 mg free EPI/kg (Free EPI), or the same volumes of 5% glucose (Control), once daily for 10 days. In addition, the group of Low plus high EPI-L was injected with 0.75 mg liposomal EPI/kg for the first 5 days, and then the dose was increased to 1.5 mg liposomal EPI/kg. Data are shown as mean ± SD, n = 5–10. P-values apply to differences between the free epirubicin and liposome-treated rats. *P < 0.05; **P < 0.01; ***P < 0.001. Abbreviations: EPI, epirubicin; OD, optical density; SD, standard deviation.
Figure 7
Figure 7
The tissue distributions of sequentially low-dose injected epirubicin liposomes in S180 tumor-bearing mice. (A) The concentrations of epirubicin in the blood and tissue of mice on the sixth day, 4 hours after injection. (B) The concentrations of epirubicin in the blood and tissue of mice on the tenth day, 4 hours after injection. Notes: Mice were injected via the tail vein with EPI dose of 0.75 mg liposomal EPI/kg (Low EPI-L), 1.5 mg liposomal EPI/kg (High EPI-L), or 0.75 mg free EPI/kg (Free-EPI), once daily for 10 days. In addition, the group of Low plus high EPI-L was injected with 0.75 mg liposomal EPI/kg for the first 5 days, and then the dose was increased to 1.5 mg liposomal EPI/kg. Data are shown as mean ± SD, n = 5. Abbreviations: EPI, epirubicin; SD, standard deviation.
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