Hiroshi Ishihara

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.

Drug delivery to the brain. DOPA prodrugs based on a ring-closure reaction to quaternary thiazolium compounds

In order to explore the possibility of an alternative redox chemical delivery system to a dihydropyridine-pyridinium interconversion system, we prepared DOPA prodrugs coupled with thiazolium precursors, like thiamine disulfides, by forming an ester bond with the amino acid carboxylic moiety while protecting the catechol function with pivalyl groups. The disposition of the prodrugs was evaluated by measuring the concentrations of DOPA regenerated after intravenous administration of the prodrugs and the results were compared with those for DOPA itself. The plasma levels of DOPA demonstrated no significant differences between DOPA and the prodrugs. In contrast, however, brain levels of DOPA were remarkably elevated following administration of the prodrugs. Among the prodrugs examined, ZiPr-DOPA(P)2 was found to most efficiently facilitate delivery of DOPA to brain and this compound showed 30- and 3.7-fold greater increases in the AUC and MRT of DOPA in brain, respectively, than did DOPA itself. These findings suggest that a redox ring-closure system to a quaternary thiazolium can be used as an alternative chemical delivery system to the brain.

Cell-penetrating peptide-conjugated lipid nanoparticles for siRNA delivery.

Lipid nanoparticles (LNP) modified with cell-penetrating peptides (CPP) were prepared for the delivery of small interfering RNA (siRNA) into cells. Lipid derivatives of CPP derived from protamine were newly synthesized and used to prepare CPP-decorated LNP (CPP-LNP). Encapsulation of siRNA into CPP-LNP improved the stability of the siRNA in serum. Fluorescence-labeled siRNA formulated in CPP-LNP was efficiently internalized into B16F10 murine melanoma cells in a time-dependent manner, although that in LNP without CPP was hardly internalized into these cells. In cells transfected with siRNA in CPP-LNP, most of the siRNA was distributed in the cytoplasm of these cells and did not localize in the lysosomes. Analysis of the endocytotic pathway indicated that CPP-LNP were mainly internalized via macropinocytosis and heparan sulfate-mediated endocytosis. CPP-LNP encapsulating siRNA effectively induced RNA interference-mediated silencing of reporter genes in B16F10 cells expressing luciferase and in HT1080 human fibrosarcoma cells expressing enhanced green fluorescent protein. These data suggest that modification of LNP with the protamine-derived CPP was effective to facilitate internalization of siRNA in the cytoplasm and thereby to enhance gene silencing.

Direct, simultaneous measurement of liposome-encapsulated and released drugs in plasma by on-line SPE–SPE–HPLC

A method for the simultaneous measurement of liposome-encapsulated and released drugs in mouse plasma by on-line solid phase extraction (SPE)-SPE-HPLC with direct plasma injection was developed using a doxorubicin (DXR)-containing liposome formulation as the model drug. During SPE, the released DXR was extracted on the 1st restricted-access media (RAM) SPE column, whereas the liposomes were eluted. The eluted liposomes were collapsed on-line, and the released DXR was delivered to the 2nd RAM SPE column for extraction. The retained DXR on the SPE columns was analyzed via HPLC-fluorescent detector by switching the valves. The method was validated and applied to the pharmacokinetic study of DXR in mice after intravenous injection of DXR-containing liposomes.

Specific uptake of asialofetuin-labeled liposomes by isolated hepatocytes

Abstract Interactions between asialofetuin (AF)-labeled liposomes (AF-liposomes) and hepatocytes were investigated in vitro. The hepatocytes were isolated from rat liver by the collagenase perfusion method. Labeling the surface of liposomes with AF enhanced the hepatocyte uptake of liposomes consisting of phosphatidylcholine, phosphatidic acid and cholesterol. The uptake of AF-liposomes (cholesterol-labeled) by hepatocytes decreased on adding AF, and virtual total inhibition was attained with the addition of unlabeled AF-liposomes. The enhancing effect of AF on liposome uptake increased with liposomal cholesterol content. The membrane fluidity of AF-liposomes decreased with increasing cholesterol. From these results, there appears to be the possibility that AF-liposomes are specifically incorporated into hepatocytes through the action of galactose-binding protein.

Preparation of asialofetuin-labeled liposomes with encapsulated human interferon-gamma and their uptake by isolated rat hepatocytes.

The selective delivery of human recombinant interferon (IFN)-γ to isolated rat hepatocytes was studied with asialofetuin (AF)-labeled liposomes. AF-liposomes containing buffer solution were initially prepared by the detergent removal method, and IFN-γ was subsequently encapsulated by the freeze-thawing method without loss of activity. Virtually no free [32P]IFN-γ was internalized into isolated rat hepatocytes, whereas AF-liposomes containing [32P]IFN-γ were taken up to a significant degree. Liposomal binding to the hepatocytes (estimated at 4°C) was one-fifth of the uptake (estimated at 37°C). Since the uptake was inhibited by the addition of free AF, AF-liposomes may be taken up by the action of galactose-binding protein on the hepatocytic cell surface. The liposome preparation method reported in this paper provides a useful means for the encapsulation of unstable macromolecules into AF-liposomes. AF-liposomes were found effectively to carry IFN-γ into hepatocytes in vitro.

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.

Rapid determination of the encapsulation efficiency of a liposome formulation using column-switching HPLC

The feasibility of a rapid automated method for determination of the encapsulation efficiency (EE) of a liposome formulation using a column-switching HPLC system was confirmed by employing several types of liposome formulations containing doxorubicin (DXR). A suspension of DXR liposome was injected directly into an online solid-phase extraction (SPE) system comprising a Diol SPE column and an ODS SPE column connected in series. Free (not encapsulated) DXR was trapped on the Diol SPE column, whereas encapsulated DXR was eluted without interaction. The eluted encapsulated DXR was trapped on the ODS SPE column after being extracted from the inner phase of the liposome by mixing with an organic solvent. Trapped free and encapsulated DXR were eluted sequentially and analyzed separately by gradient HPLC. The time taken by this automated method was only 25min, whereas conventional methods such as ultracentrifugation are time consuming and labor intensive. Validation results and comparison with ultracentrifugation suggested that our method was sufficiently accurate and sensitive to be used to evaluate EE of a liposome formulation without complicated pretreatment.

siRNA-lipid nanoparticles with long-term storage stability facilitate potent gene-silencing in vivo.

Considerable efforts have been directed towards discovering and developing delivery vehicles for RNA therapeutics. While most studies emphasize the efficacy and safety of these delivery vehicles, few reports conduct a comprehensive assessment of their storage stability, a critical property for practical applications. Here, we report a potent and safe lipid nanoparticle with long-term storage stability. Through chemical synthesis and screening of cationic lipids, a formulation has been identified that enables potent knockdown of hepatocyte proteins in mice upon intravenous administration (siRNA ED50 ~0.02 mg/kg). Toxicity studies revealed that a dose of 2mg/kg was well tolerated in rats, the most sensitive rodent model. We identified that a cyclic chemical structure in cationic lipids improved particle stability. The nanoparticles showed over 1.5 year storage stability as a liquid, with over 90% siRNA encapsulation without any changes in particle size. This novel delivery material has promising potential as a drug product that could bring RNA therapeutics to the treatment of liver-related disorders.

Suppression in mice of immunosurveillance against PEGylated liposomes by encapsulated doxorubicin

PEGylated liposomes (PEG-lip) can escape from recognition by immune system and show a longer half-life in the blood than non-PEGylated liposomes. In this study, we investigated the influence of injected PEG-lip encapsulating doxorubicin (PEG-lip-DOX) on the biodistribution of subsequently injected PEG-lip in mice. PEG-lip-DOX, free doxorubicin or empty PEG-lip were initially injected into BALB/c mice via a tail vein, and 3days later [(3)H]-labeled PEG-lip ([(3)H] PEG-lip) were injected into these same mice. At 24h after the injection, the distribution of [(3)H] PEG-lip in the liver and spleen was significantly reduced in the PEG-lip-DOX group compared with that in the free doxorubicin or PEG-lip group. Consequently, the plasma concentration of [(3)H] PEG-lip was significantly elevated by the pretreatment with PEG-lip-DOX. Altered pharmacokinetics was observed at least until 72h after the injection of [(3)H] PEG-lip. The influence of the injected PEG-lip-DOX on the pharmacokinetics of the subsequently injected [(3)H] PEG-lip was clearly observed from 1 to 14days, and slightly observed on days 21 and 28, after the injection of the PEG-lip-DOX. Flow cytometric analysis showed that the number of liver Kupffer cells was significantly reduced after the treatment with PEG-lip-DOX. On the other hand, a similar alteration in the distribution of the subsequently injected [(3)H] PEG-lip was observed in immunodeficient mice such as BALB/c nu/nu and severe combined immunodeficiency (SCID) mice. These findings suggest that immune cells including liver Kupffer cells responsible for recognizing PEG-lip were selectively damaged by the encapsulated doxorubicin in PEG-lip injected initially, which damage led to prolongation of the half-life of subsequently injected [(3)H] PEG-lip in the blood.