Repeated injections of PEGylated liposomal topotecan induces accelerated blood clearance phenomenon in rats

The "accelerated blood clearance (ABC) phenomenon" of PEGylated liposomes following multiple injections has been reported recently. This immunogenicity poses a problem for research into liposomes and hinders their clinical application. However, since doxorubicin liposomes and mitoxantrone liposomes have been reported to fail to induce the ABC phenomenon, some people believe that cytotoxic drugs loaded liposomes will not produce this ABC phenomenon under multiple-dosing regimens. Nevertheless, in the present study, we report that a first injection of the PEGylated liposomal topotecan (a cell cycle-specific drug for the S phase) still produced a strong ABC phenomenon. Likewise, when the first dose of "empty" PEGylated liposomes or topotecan liposomes was increased, the ABC phenomenon of the subsequent dose was accordingly attenuated. Unlike doxorubicin and mitoxantrone, the blood clearance rate of topotecan was dramatically rapid, and the hepatic and splenic accumulations of topotecan liposomes were anomalous because of the ABC phenomenon. These findings may present new challenges to the clinical application of formulations of cytotoxic drugs loaded liposomes that require repeated administrations.     

Accelerated blood clearance of pegylated liposomal topotecan: Influence of polyethylene glycol grafting density and animal species

Upon repeated administration, empty pegylated liposomes lose long‐circulating characteristics, referred to as accelerated blood clearance (ABC) phenomenon. However, pegylated liposomal cytotoxic drug formulations could not elicit the phenomenon. In the study, it was found that repeated injection of pegylated liposomal topotecan could induce ABC phenomenon in Wistar rats, beagle dogs, and mice, which might be associated with the formation of empty liposomes in circulation because of the rapid drug release rate. In rats, the 9% polyethylene glycol (PEG) formulation induced more severe ABC phenomenon than 3% PEG formulation despite the similar anti‐PEG immunoglobulin M (IgM) levels following the first dose. Antibody neutralization experiments revealed that high PEG formulation was easily neutralized by IgM. Repeated administration of 3% PEG formulation in dogs could result in more severe ABC phenomenon. It seems that slow infusion was liable to cause ABC phenomenon. In all animal species, considerable intraindividual variability of IgM levels could be observed. Our observations may have important implications for the development, evaluation, and therapeutic use of pegylated liposomal cytotoxic drug formulations because using the current drug loading technology, most of the cytotoxic drugs could not be stably loaded in liposomes and rapid drug leakage from liposomes might occur in circulation. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 101:3864–3876, 2012     

Time Interval of Two Injections and First-Dose Dependent of Accelerated Blood Clearance Phenomenon Induced by PEGylated Liposomal Gambogenic Acid: The Contribution of PEG-Specific IgM

Repeated injection of PEGylated liposomes can cause the disappearance of long circulating property because of the induction of anti-PEG IgM antibody referred to as “accelerated blood clearance (ABC) phenomenon.” Although ABC phenomenon typically occurs when entrapped drugs are chemotherapeutic agent with low cytotoxic, there is little evidence of accelerated blood clearance of PEGylated herbal-derived compound on repeated injection. Herein, we investigated the blood concentration of PEGylated liposomal gambogenic acid (PEG-GEA-L), a model PEGylated liposomal herbal extract, on its repeated injection to rats. We found time interval between injections had considerable impact on the magnitude of ABC phenomenon induced by PEG-GEA-L. When time interval was prolonged from 3 days to 7 days, ABC phenomenon could be attenuated. Furthermore, its magnitude was enhanced accompanied by a marked rise in the accumulation of PEG-GEA-L in the liver and spleen in a first-dose–dependent manner. Consistently, the level of anti-PEG IgM significantly increased with the first dose of PEG-GEA-L and decreased with the extended time interval between injections, which implies anti-PEG IgM is a major contributor to the ABC phenomenon. Notably, the increased expression of liver anti-PEG IgM was accompanied by an increased expression of efflux transporters in the induction process of the ABC phenomenon.     

Use of polyglycerol (PG), instead of polyethylene glycol (PEG), prevents induction of the accelerated blood clearance phenomenon against long-circulating liposomes upon repeated administration

The accelerated blood clearance (ABC) phenomenon accounts for the rapid systemic clearance of PEGylated nanocarriers upon repeated administrations. IgM production against the polyethylene glycol (PEG) coating in PEGylated liposomes is now known to be responsible for such unexpected pharmacokinetical alterations. The ABC phenomenon poses a remarkable clinical challenge by reducing the therapeutic efficacy of encapsulated drugs and causing harmful effects due to the altered tissue distribution pattern of the drugs. In this study, we investigated the in vivo performance of liposomes modified with polyglycerol (PG) upon repeated injection, and the in vivo therapeutic efficacy of such liposomes when they encapsulated a cytotoxic agent, doxorubicin (DXR). Repeated injection of PEG-coated liposomes in rats induced the ABC phenomenon, while repeated injection of PG-coated liposomes did not. In addition, DXR-containing PG-coated liposomes showed antitumor activity that was superior to that of free DXR and similar to that of DXR-containing PEG-coated liposomes upon repeated administration. These results indicate that polyglycerol (PG) might represent a promising alternative to PEG via enhancing the in vivo performance of liposomes by not eliciting the ABC phenomenon upon repeated administration.     

Pharmacokinetics of pegylated liposomal Doxorubicin: review of animal and human studies

Pegylated liposomal doxorubicin (doxorubicin HCl liposome injection; Doxil or Caelyx) is a liposomal formulation of doxorubicin, reducing uptake by the reticulo-endothelial system due to the attachment of polyethylene glycol polymers to a lipid anchor and stably retaining drug as a result of liposomal entrapment via an ammonium sulfate chemical gradient. These features result in a pharmacokinetic profile characterised by an extended circulation time and a reduced volume of distribution, thereby promoting tumour uptake. Preclinical studies demonstrated one- or two-phase plasma concentration-time profiles. Most of the drug is cleared with an elimination half-life of 20-30 hours. The volume of distribution is close to the blood volume, and the area under the concentration-time curve (AUC) is increased at least 60-fold compared with free doxorubicin. Studies of tissue distribution indicated preferential accumulation into various implanted tumours and human tumour xenografts, with an enhancement of drug concentrations in the tumour when compared with free drug. Clinical studies of pegylated liposomal doxorubicin in humans have included patients with AIDS-related Kaposi's sarcoma (ARKS) and with a variety of solid tumours, including ovarian, breast and prostate carcinomas. The pharmacokinetic profile in humans at doses between 10 and 80 mg/m(2) is similar to that in animals, with one or two distribution phases: an initial phase with a half-life of 1-3 hours and a second phase with a half-life of 30-90 hours. The AUC after a dose of 50 mg/m(2) is approximately 300-fold greater than that with free drug. Clearance and volume of distribution are drastically reduced (at least 250-fold and 60-fold, respectively). Preliminary observations indicate that utilising the distinct pharmacokinetic parameters of pegylated liposomal doxorubicin in dose scheduling is an attractive possibility. In agreement with the preclinical findings, the ability of pegylated liposomes to extravasate through the leaky vasculature of tumours, as well as their extended circulation time, results in enhanced delivery of liposomal drug and/or radiotracers to the tumour site in cancer patients. There is evidence of selective tumour uptake in malignant effusions, ARKS skin lesions and a variety of solid tumours. The toxicity profile of pegylated liposomal doxorubicin is characterised by dose-limiting mucosal and cutaneous toxicities, mild myelosuppression, decreased cardiotoxicity compared with free doxorubicin and minimal alopecia. The mucocutaneous toxicities are dose-limiting per injection; however, the reduced cardiotoxicity allows a larger cumulative dose than that acceptable for free doxorubicin. Thus, pegylated liposomal doxorubicin represents a new class of chemotherapy delivery system that may significantly improve the therapeutic index of doxorubicin.     

Liposomal encapsulated anti-cancer drugs

Among several drug delivery systems, liposomal encapsulated anti-cancer agents represent an advanced and versatile technology. Several formulations of liposomal anthracyclines are approved, e.g. for the treatment of metastatic breast cancer (pegylated and non-pegylated liposomal doxorubicin) or AIDS-related Kaposi's sarcoma (pegylated liposomal doxorubicin and liposomal daunorubicin). Meanwhile, virtually all anti-cancer drugs have been encapsulated in liposomes using different technologies. This review will summarize preclinical and clinical data of approved and exemplary emerging liposomal anti-cancer agents.     

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.     

A pegylated liposomal platform: pharmacokinetics, pharmacodynamics, and toxicity in mice using doxorubicin as a model drug

Aims were to observe pharmacokinetics, pharmacodynamics, and toxicity for constructing a Sino-pegylated liposomal platform. Human hepatocarcinoma cells (Bel7402) and murine hepatocarcinoma cells (H(22)) were used for the cytotoxicity assay and the in vivo solid xenograft tumor model in mice, respectively. Pharmacokinetic results in mice showed that the pegylated liposomal doxorubicin markedly prolonged the blood circulation of doxorubicin. Elimination half-time (T(1/2,gamma)) of pegylated, regular liposomal doxorubicin and free doxorubicin were 46.09 +/- 14.44, 26.04 +/- 3.34, and 23.72 +/- 5.13 h, respectively. The area under the concentration-time curves (AUC(0- infinity )) (h. microg/g) of the pegylated and regular liposomal doxorubicin were 6.8- and 2.6-fold higher than that of free doxorubicin, respectively. Cytotoxicity and antitumor activity in vivo indicated that activity of the pegylated liposomal doxorubicin was higher than that of the regular or the free one, respectively. After two weeks of tail intravenous injection of the pegylated liposomal doxorubicin at a single dose of 10 mg/kg, no significant damage was observed in gastric, intestinal mucosa, and heart muscle, but pronounced damages were found in the control group after dosing free doxorubicin. The results demonstrate that the pegylated liposomes improve the efficacy of toxics and reduce the toxicity, therefore providing favorable evidence for building a pegylated liposomal platform.     

Therapeutic efficacy of the combination of doxorubicin-loaded liposomes with inertial cavitation generated by confocal ultrasound in AT2 Dunning rat tumour model

Abstract

The combination of liposomal doxorubicin (DXR) and confocal ultrasound (US) was investigated for the enhancement of drug delivery in a rat tumour model. The liposomes, based on the unsaturated phospholipid dierucoylphosphocholine, were designed to be stable during blood circulation in order to maximize accumulation in tumour tissue and to release drug content upon US stimulation. A confocal US setup was developed for delivering inertial cavitation to tumours in a well-controlled and reproducible manner. In vitro studies confirm drug release from liposomes as a function of inertial cavitation dose, while in vivo pharmacokinetic studies show long blood circulation times and peak tumour accumulation at 24–48?h post intravenous administration. Animals injected 6?mg kg^?1 liposomal DXR exposed to US treatment 48?h after administration show significant tumour growth delay compared to control groups. A liposomal DXR dose of 3?mg kg^?1, however, did not induce any significant therapeutic response. This study demonstrates that inertial cavitation can be generated in such a fashion as to disrupt drug carrying liposomes which have accumulated in the tumour, and thereby increase therapeutic effect with a minimum direct effect on the tissue. Such an approach is an important step towards a therapeutic application of cavitation-induced drug delivery and reduced chemotherapy toxicity.     

Liposomal drug formulations in the treatment of rheumatoid arthritis

Liposomes have been extensively investigated as drug delivery systems in the treatment of rheumatoid arthritis (RA). Low bioavailability, high clearance rates and limited selectivity of several important drugs used for RA treatment require high and frequent dosing to achieve sufficient therapeutic efficacy. However, high doses also increase the risk for systemic side effects. The use of liposomes as drug carriers may increase the therapeutic index of these antirheumatic drugs. Liposomal physicochemical properties can be changed to optimize penetration through biological barriers and retention at the site of administration, and to prevent premature degradation and toxicity to nontarget tissues. Optimal liposomal properties depend on the administration route: large-sized liposomes show good retention upon local injection, small-sized liposomes are better suited to achieve passive targeting. PEGylation reduces the uptake of the liposomes by liver and spleen, and increases the circulation time, resulting in increased localization at the inflamed site due to the enhanced permeability and retention (EPR) effect. Additionally liposomal surfaces can be modified to achieve selective delivery of the encapsulated drug to specific target cells in RA. This review gives an overview of liposomal drug formulations studied in a preclinical setting as well as in clinical practice. It covers the use of liposomes for existing antirheumatic drugs as well as for new possible treatment strategies for RA. Both local administration of liposomal depot formulations and intravenous administration of passively and actively targeted liposomes are reviewed.     

Absorption and tissue distribution of doxorubicin entrapped in liposomes following intravenous or intraperitoneal administration

Absorption and tissue distribution of free doxorubicin (Dxn) and Dxn entrapped into liposomes have been examined after intravenous (i.v.) or intraperitoneal (i.p.) injection into C57/B1/6 mice. Liposomal encapsulation of Dxn altered its plasma kinetics and tissue distribution. After i.v. administration Dxn in liposomes has a half-life longer than that of free Dxn and it is taken up mostly by tissues rich in reticuloendothelial cells, such as liver and spleen. In the heart and kidney liposomal Dxn reaches a lower concentration than free Dxn. After i.p. injection the tissue distribution of liposomal Dxn is drastically changed. We did not observe the first peak of high concentration in the tissues, the Dxn content in liver and spleen is decreased and its concentration in heart is even more reduced. The results of this study suggest that the route of administration of liposome-entrapped drugs may change both the kinetics of absorption and their tissue distribution and this could result in a different pharmacological effect.     

Doxorubicin liposomal pegylated: new preparation. Breast cancer: not just a question of short-term cardiac effects

(1) There is no reference first-line chemotherapy regimen for metastatic breast cancer. Anthracycline-based combinations are generally used. One of the main problems with anthracyclines is the risk of heart failure, both during and some time after treatment. (2) A liposomal pegylated doxorubicin, an anthracycline, is now available in Europe. The aim of pegylation is supposedly to reduce the cardiotoxicity relative to standard doxorubicin. The marketing licence specifies that liposomal pegylated doxorubicin must not be used in combination with other drugs in people with metastatic breast cancer. This is the second liposomal doxorubicin preparation to be authorised for this use in France; we concluded that the first product, a non-pegylated form, offered no therapeutic advance. (3) According to the only available comparative trial, liposomal pegylated doxorubicin is no more effective than standard doxorubicin in terms of the duration or quality of survival. (4) In this trial, liposomal pegylated doxorubicin was associated with slightly fewer cardioechographic abnormalities than standard doxorubicin. (5) Other adverse events were also less common (hair loss, nausea and vomiting, and neutropenia), while some were more common (palmoplantar erythrodysesthesia, stomatitis and mucitis). Overall, 24% of patients stopped using liposomal pegylated doxorubicin because of adverse events, compared with 11% of patients receiving standard doxorubicin. (6) Unlike liposomal non-pegylated doxorubicin, the liposomal pegylated form is no more difficult than standard doxorubicin to prepare for injection. (7) In practice, when the decision is made to use doxorubicin, the standard form, at an appropriate dose, is suitable for most patients, as long as cardiac function is closely monitored. Differences in the adverse effect profile (especially hair loss) may make liposomal pegylated doxorubicin more attractive to some patients (it costs 20 times more than standard doxorubicin in France).     

Accelerated blood clearance (ABC) phenomenon induced by administration of PEGylated liposome

PEGylated liposomes (approximately 100 nm in diameter) lose their long-circulating characteristic upon repeated injection at certain intervals in the same animal (referred to as the "accelerated blood clearance (ABC) phenomenon"), as described by our group and by researchers in the Netherlands. Recently, it was demonstrated by our group that anti-PEG IgM, induced by the first dose of PEGylated liposomes, is responsible for the ABC phenomenon. The IgM produced in this manner then selectively bound to the surface of subsequently injected PEGylated liposomes, leading to substantial complement activation. It is generally believed that nanocarriers coated with a polymer, such as PEG, have no immunogenicity. However, unexpected immune responses occurred even in response to polymer-coated liposomes. This immunogenicity to PEGylated liposomes presents a serious concern in the development and clinical use of liposomal formulations. In this review, we demonstrate our recent observations regarding with the ABC phenomenon against liposomes.     

Biodistribution of Cyclosporin Encapsulated in Liposomes Modified with Bioadhesive Polymer

The purpose of this investigation was to study the possibility of renewing the immunosuppressive activity of cyclosporin by formulating the compound in liposomes modified with bioadhesive polymers. The liposomes prepared were evaluated both pharmacokinetically and pharmacodynamically. Tissue distribution and plasma pharmacokinetics of cyclosporin and model dye, sudan black, which is as hydrophobic as cyclosporin, were studied in rats after intravenous infusion (10 mg kg^?). The immunosuppressive efficacy of liposomal cyclosporin preparations was studied in the allogenic rat-heart-transplantation model, where cyclosporin therapy (10 mg kg^?) continued for one week. The entrapment of sudan black in liposomes modified with bioadhesive polymers resulted in higher sudan black delivery to the spleen and the liver than with standard sudan-black-loaded liposomes. Among the modified liposomes, those modified with carbopol 941 showed the most remarkable enhancing effect on the delivery of sudan black to these organs and total plasma clearance of sudan black decreased to 38.6 ± 7.8 mL h^? kg^? (standard liposomes, 58.9 ± 64 mL h^? kg^?). Delivery of cyclosporin to the spleen and the liver was increased approximately twofold by modifying the liposomes with carbopol 941. In the preliminary study on the allogenic rat-heart-transplantation model, the mean survival days of the graft were 18.8 ± 2.9 days for the group receiving cyclosporin liposomes modified with carbopol 941, 14.2 ± 4.4 days for the group receiving standard cyclosporin liposomes and 7.6 ± 0.5 days for the group receiving cyclosporin solution. The encapsulation of cyclosporin in liposomes modified with bioadhesive polymer enhanced the residence time of cyclosporin in the systemic circulation, resulting in approximately twofold greater delivery of cyclosporin to the spleen and liver. However, in the allogenic rat-heart-transplantation model no significant difference was detected between the immunosuppressive efficacy of cyclosporin encapsulated in bioadhesive polymer-modified liposomes and that encapsulated in standard liposomes.     

Drug distribution and a pulmonary adverse effect of intraperitoneally administered doxorubicin niosomes in the mouse

Niosomes (non-ionic surfactant vesicles) prepared from C₁₆G₂ (a hexadecyl-diglycerol ether), and loaded with doxorubicin, were administered intraperitoneally to male AKR mice at dose levels of 0, 2.5, 5.0, and 10.0 mg kg^?1. Free drug was given at 10.0 mg kg^?1 by the intraperitoneal route. At a dose level of 10.0 mg kg^?1, peak doxorubicin levels in the central compartment were attained faster with the free drug than with the niosome formulation. However, the peak plasma levels were similar for the free drug and the niosome preparation at the 10 mg kg^?1 dose level. With doxorubicin administered as the niosome preparation by the intraperitoneal route at 2.5, 5.0, and 10.0 mg kg^?1, mean peak plasma concentrations of the drug showed a tendency to be dose-related although the differences were not significant. Over the 24 h period of the experiment, with doxorubicin at 10 mg kg^?1, the niosome formulation delivered significantly more drug to the plasma compartment than the free drug (p <0.05). When doxorubicin was given in niosomes at 2.5, 5.0 and 10.0 mg kg^?1 by the intraperitoneal route, the resulting levels of doxorubicin in cardiac tissue were not dose related and the differences not significant and, although the mean peak cardiac-tissue concentration was higher in animals receiving the free drug at 10.0 mg kg^?1 intraperitoneally than in mice given intaperitoneal doxorubicin niosomes at this dose level, the differences were again not significant. There were clinical signs of toxicity in mice given doxorubicin-containing niosomes intraperitoneally at 5.0 and 10.0 mg kg^?1, and at post-mortem an accumulation of fluid in the pleural cavity was evident. These changes were not seen in mice dosed intraperitoneally with free drug at 10 mg kg^?1, or in animals given doxorubicin niosomes intraperitoneally at 2.5 mg kg^?1. In mice dosed intraperitoneally with doxorubicin niosomes at 12.0 mg kg^?1 and at a dose volume of 0.2–0.4 mL, histological examination of the lungs demonstrated a congestion of the alveolar capillaries, and an increased number of acute inflammatory cells in the alveolar walls. There was no histological evidence of lung toxicity in mice dosed with doxorubicin niosomes at 12.0 mg kg^?1 when the formulation was administered with the higher dose volume of 1.8–2.0 mL. Importantly there was no histological evidence of lung toxicity in mice dosed with empty niosomes intraperitoneally or with doxorubicin niosomes given itravenously at 12.0 mg kg^?1.     

Translocation of liposomes into cancer cells by cell-penetrating peptides penetratin and tat: a kinetic and efficacy study

Unlike conventional liposomes, sterically stabilized liposomes, with their smaller volume of distribution and reduced clearance, preferentially convey encapsulated drugs into tumor sites. Despite these improvements, intracellular delivery is hampered by the stable drug retention of the liposomes, which diminishes the efficacy of the liposomal drug. To facilitate uptake of liposomal drugs into cells, two cell-penetrating peptides, penetratin (PEN) and TAT, derived from the HIV-1 TAT protein, were studied. In contrast to control peptides, both TAT and PEN enhanced the translocation efficiency of liposomes in proportion to the number of peptides attached to the liposomal surface. A peptide number of as few as five could enhance the intracellular delivery of liposomes. The kinetics of uptake was peptide- and cell-type dependent. Intracellular accumulation of TAT-liposomes increased with incubation time, but PEN-liposomes peaked at 1 h and then declined gradually. After treatment with 1 microg/ml doxorubicin equivalents of liposome for 2 h, TAT increased the doxorubicin uptake of A431 cells by 12-fold. However, the improvement of uptake of liposomal doxorubicin was not reflected by cytotoxicity in vitro or tumor control in vivo. Our results demonstrated that merely adding CPP to a liposome encapsulating anticancer drug was inadequate in improving its antitumor activity. An additional approach to enhance the intracellular release of the encapsulated drug is obviously necessary.     

Dose Dependency of Pharmacokinetics and Therapeutic Efficacy of Pegylated Liposomal Doxorubicin (DOXIL) in Murine Models

Stealth (pegylated) liposomal doxorubicin (Doxil) has been extensively studied at the pre-clinical and clinical level in recent years. However, one issue not yet addressed is the effect of dose on tumor localization and therapeutic efficacy of Doxil. Although it has been reported that the pharmacokinetics of drug-free Stealth liposomes is independent of dose within a certain range, clinical pharmacokinetic analysis of Doxil suggests a dose-dependent clearance saturation phenomenon when a broad dose range is examined. In addition, liposome-encapsulated doxorubicin can exert toxic effects on the liver macrophage population in the form of impairment of the phagocytic function and reduced ability of colloid particle clearance. In studies with tumor-bearing mice in which the dose of Doxil was escalated from 2.5 to 20 mg/kg, we demonstrate that dose escalation results in a saturation of Doxil clearance and a disproportional increase of the amount of liposomal drug accumulating in tumor. Experiments with radiolabeled highly negatively-charged liposomes injected into mice previously treated with Doxil are consistent with a partial blockade of the reticulo-endothelial system with relative reduction of liver uptake and greater prolongation of liposome circulation time. The clearance saturation effect is seen after Doxil in a dose-dependent fashion, and not after a similar free doxorubicin dose or similar phospholipid dose in drug-free liposomes. A trend to superior therapeutic efficacy for treatments based on larger doses as compared to smaller split doses, while maintaining an equivalent dose intensity, was also observed. These observations may be relevant to the choice of dose-schedule of Doxil to ensure optimal anti-tumor activity. Therefore, dose-dependent liposomal doxorubicin blockade of the reticulo-endothelial system may prolong liposome circulation time and enhance significantly drug delivery to tumors.     

Dose dependency of pharmacokinetics and therapeutic efficacy of pegylated liposomal doxorubicin (DOXIL) in murine models

Stealth (pegylated) liposomal doxorubicin (Doxil) has been extensively studied at the pre-clinical and clinical level in recent years. However, one issue not yet addressed is the effect of dose on tumor localization and therapeutic efficacy of Doxil. Although it has been reported that the pharmacokinetics of drug-free Stealth liposomes is independent of dose within a certain range, clinical pharmacokinetic analysis of Doxil suggests a dose-dependent clearance saturation phenomenon when a broad dose range is examined. In addition, liposome-encapsulated doxorubicin can exert toxic effects on the liver macrophage population in the form of impairment of the phagocytic function and reduced ability of colloid particle clearance. In studies with tumor-bearing mice in which the dose of Doxil was escalated from 2.5 to 20 mg/kg, we demonstrate that dose escalation results in a saturation of Doxil clearance and a disproportional increase of the amount of liposomal drug accumulating in tumor. Experiments with radiolabeled highly negatively-charged liposomes injected into mice previously treated with Doxil are consistent with a partial blockade of the reticulo-endothelial system with relative reduction of liver uptake and greater prolongation of liposome circulation time. The clearance saturation effect is seen after Doxil in a dose-dependent fashion, and not after a similar free doxorubicin dose or similar phospholipid dose in drug-free liposomes. A trend to superior therapeutic efficacy for treatments based on larger doses as compared to smaller split doses, while maintaining an equivalent dose intensity, was also observed. These observations may be relevant to the choice of dose-schedule of Doxil to ensure optimal anti-tumor activity. Therefore, dose-dependent liposomal doxorubicin blockade of the reticulo-endothelial system may prolong liposome circulation time and enhance significantly drug delivery to tumors.     

Cellular localization of stable solid liposomes in the liver of rats

Small solid liposomes made from distearoylphosphatidyl choline and cholesterol (molar ratio 2 : 1) showed significant stability in plasma, with a half-life of about 24 hr after intravenous injection in rats. The major cellular uptake of intact liposomes was found in the liver and spleen, peaking after 2–4 hr in the liver and after 24 hr in the spleen. Isolation of parenchymal and non-parenchymal cells from rat livers at various intervals after injection of liposomes showed that both cell types adsorbed liposomal membranes and took up the liposomal contents. Our study has shown that most of the liposomal markers found in the liver shortly (< 40 min) after administration stemmed from the liposomes adsorbed to extracellular binding sites, and that uptake into the cells took place subsequently. In non-parenchymal cells, uptake was rapid and the intracellular level remained rather constant after 40 min and for up to 4 hr. The uptake of liposomes by parenchymal cells was slower, it showed a lag-phase of approx. $\text{1}\text{2}$ hr and peaked at 2 hr, whereupon the radioactivity in parenchymal cells dropped. The contents of liposomes behaved in a manner similar to the membranes. It is concluded that, in addition to a rapid uptake of liposomes in non-parenchymal liver cells, there is a significant degree of association with parenchymal cells, provided that the liposomes administered are small (< 100 nm in diameter) and stable.     

Evaluation of a Liposome System for the Delivery of Desferrioxamine to Lungs in Rats

Abstract— Liposomes with various lipid composition and sizes, prepared by two different techniques were evaluated for their potential to deliver desferrioxamine to lungs as a treatment against oxidative lung damage. Multilamellar vesicles (MLV) and reverse evaporation vesicles were prepared out of a lipid mixture containing dipalmitoyl phosphatidylcholine, stearyl amine, cholesterol and vitamin E. The administration of desferrioxamine-encapsulated liposomes to rats by the intravenous route at a dose of 100 mg kg^?1, significantly prolonged the presence of desferrioxamine in all the tested organs when compared with the administration of free desferrioxamine. The injection of reverse evaporation vesicles extruded through a 2 μm polycarbonate membrane exhibited a longer residence time of the desferrioxamine and of liposomal vitamin E in lungs compared with the other types of liposomes tested. The examination of liposome components in the bronchoalveolar lavage fluid (BALF) and the alveolar macrophages recovered from BALF revealed that about 7 × 10^?3% of the administered desferrioxamine dose was recovered by this technique at 3 and 17 h after liposome administration. This high residual concentration in the alveolar space confirms the hypothesis that liposomes can be delivered to the lung tissue when encapsulated in alveolar macrophages.