Liposome-induced complement activation and related cardiopulmonary distress in pigs: factors promoting reactogenicity of Doxil and AmBisome.

Hypersensitivity reactions to liposomal drugs, often observed with Doxil and AmBisome, can arise from activation of the complement (C) system by phospholipid bilayers. To understand the mechanism of this adverse immune reaction called C activation-related pseudoallergy (CARPA), we analyzed the relationship among liposome features, C activation in human serum in vitro, and liposome-induced cardiovascular distress in pigs, a model for human CARPA. Among the structural variables (surface charge, presence of saturated, unsaturated, and PEGylated phospholipids, and cisplatin vs. doxorubicin inside liposomes), high negative surface charge and the presence of doxorubicin were significant contributors to reactogenicity both in vitro and in vivo. Morphological analysis suggested that the effect of doxorubicin might be indirect, via distorting the sphericity of liposomes and, if leaked, causing aggregation. The parallelism among C activation, cardiopulmonary reactions in pigs, and high rate of hypersensitivity reactions to Doxil and AmBisome in humans strengthens the utility of the applied tests in predicting the risk of CARPA. FROM THE CLINICAL EDITOR: The authors studied complement activation-related pseudoallergy (CARPA) in a porcine model and demonstrate that high negative surface charge and drug effects leading to distortion of liposome sphericity might be the most critical factors leading to CARPA. The applied tests might be used to predict CARPA in humans.

ABCG2 (breast cancer resistance protein/mitoxantrone resistance-associated protein) ATPase assay: a useful tool to detect drug-transporter interactions.

The ATPase assay using membrane preparations from recombinant baculovirus-infected Spodoptera frugiperda ovarian (Sf9) cells is widely used to detect the interaction of compounds with different ATP-binding cassette transporters. However, Sf9 membrane preparations containing the wild-type ABCG2 transporter show an elevated baseline vanadate-sensitive ATPase activity, which cannot be further stimulated by substrates of ABCG2. Therefore, this assay system cannot be used for the detection of ABCG2 substrates. To overcome this difficulty we 1) purified membranes from a selected human cell line expressing wild-type ABCG2, and 2) inhibited the baseline ATPase activity with different inhibitors. In our modified assay, ABCG2 substrates were able to stimulate the baseline ATPase activity of ABCG2 expressed in membranes of human cells. Furthermore, using the specific ABCG2 inhibitors Ko143 or Ko134 allowed us to suppress the baseline vanadate-sensitive ATPase activity. Substrates of ABCG2 could stimulate this suppressed baseline ATPase, resulting in a better signal-to-background ratio and a robust assay to detect substrates of the ABCG2 transporter. The ATPase assay and the direct vesicular transport measurements for estrone-3-sulfate were in good accordance.

[In vitro methods suitable for the prediction of drug and ABC transporter, especially ABCG2 interactions]. / ABC transzporterek-különös tekintettel az ABCG2-re--és gyógyszerjelölt molekulák kölcsönhatásának predikciójára alkalmas in vitro módszerek.

ABCG2, a transporter of the ATP-binding cassette family, is known to play a prominent role in the absorption, distribution, metabolism, and excretion of xenobiotics. Drug-transporter interactions are commonly screened by in vitro systems using transfected insect and/or human cell lines.

Ca2+-dependent control of the permeability properties of the mitochondrial outer membrane and voltage-dependent anion-selective channel (VDAC).

Cell function depends on the distribution of cytosolic and mitochondrial factors across the outer mitochondrial membrane (OMM). Passage of metabolites through the OMM has been attributed to the voltage-dependent anion-selective channel (VDAC), which can form a large conductance and permanently open a channel in lipid bilayers. However, recent data indicate that the transport of metabolites through the OMM is controlled in the cells. Recognizing that the bilayer studies had been commonly conducted at supraphysiological [Ca2+] and [K+], we determined the effect of Ca2+ on VDAC activity. In liposomes, the purified VDAC displays Ca2+-dependent control of the molecular cut-off size and shows Ca2+-regulated Ca2+ permeability in the physiological [Ca2+] range. In bilayer experiments, at submicromolar [Ca2+], the purified VDAC or isolated OMM does not show sustained large conductance but rather exhibits gating between a nonconducting state and various subconductance states. Ca2+ addition causes a reversible increase in the conductance and may evoke channel opening to full conductance. Furthermore, single cell imaging data indicate that Ca2+ may facilitate the cation and ATP transport across the OMM. Thus, the VDAC gating is dependent on the physiological concentrations of cations, allowing the OMM to control the passage of ions and some small molecules. The OMM barrier is likely to decrease during the calcium signal.

Caveolae--an alternative endocytotic pathway for targeted drug delivery.

Caveolae are bottleshape-like invaginations of the plasma membrane. After internalization they are involved in endocytosis, transcytosis, potocytosis, and pinocytosis. Our recently expanded knowledge has made clear that various molecules and macromolecular complexes enter the cells after docking on caveolar receptors, before subsequent internalization of the caveolae. The internalization is initiated by the ligand receptor interaction. Folate, cholesterol, serum lipoproteins, and liposomes are among the most common examples. It is important to point out that, in contrast to the classic clathrin-dependent endocytosis, the caveolar internalization pathway seems to avoid the lysosomes. Internalized caveolae fuse with caveosomes, and the caveosomes deliver their contents into other subcellular (non-lysosomal) compartments. The bypass of the acidic and harmful milieu might be a major advantage for drug delivery via the caveolar pathway. Not all cell types have caveolae. Cells, where the Caveolin I protein is not expressed, do not develop caveolar invaginations. However, these cells have caveolar-equivalent plasma membrane domains, so-called "lipid rafts." Lipid rafts are assembled from the same lipid constituents and proteins as caveolae, but they are flat. Specific ligands may also dock on appropriate receptors of the raft domain. As a complication, certain types of ligand-covered raft receptors can migrate to clathrin-coated pits and get internalized via clathrin-coated vesicles. Nevertheless, suitable ligands or antibodies developed against proteins of the caveolar/raft domains may selectively direct the attached drug carrier to the less harmful caveolar/raft pathway. Thus, understanding of caveolar transport may help in the rational design of more effective drug carriers.

Degranulation and superoxide production depend on cholesterol in PLB-985 cells.

The effect of agents disrupting cholesterol-rich microdomains of the cell membrane was studied on the chemoattractant receptor (FPR and FRPL1) coupled effector responses of promyelocytic PLB-985 cells. Both methyl-beta-cyclodextrin (MbetaCD) and filipin III inhibited exocytosis of primary granules and O(2)(.-) production induced by stimulation of either chemotactic receptor. Alteration of calcium homeostasis of MbetaCD-treated cells does not account for the impairment of the effector responses. Disruption of microfilaments by cytochalasin B (CB) partially reverses the inhibitory effect of cholesterol depletion. Our results provide functional support for the involvement of cholesterol-rich membrane domains in the signaling of chemotactic receptors and call the attention to the possible role of microfilaments in the organization of lipid microdomains.