Rivka Cohen

Role of complement activation in hypersensitivity reactions to doxil and hynic PEG liposomes: experimental and clinical studies.

ABSTRACT Pegylated liposomal doxorubicin (Doxil) and 99mTc-HYNIC PEG liposomes (HPL) were reported earlier to cause hypersensitivity reactions (HSRs) in a substantial percentage of patients treated i.v. with these formulations. Here we report that (1) Doxil, HPL, pegylated phosphatidylethanolamine (PEG-PE)-containing empty liposomes matched with Doxil and HPL in size and lipid composition, and phosphatidylglycerol (PG)-containing negatively charged vesicles were potent C activators in human serum in vitro, whereas small neutral liposomes caused no C activation. (2) Doxil and other size-matched PEG-PE and/or PG-containing liposomes also caused massive cardiopulmonary distress with anaphylactoid shock in pigs via C activation, whereas equivalent neutral liposomes caused no hemodynamic changes. (3) A clinical study showed more frequent and greater C activation in patients displaying HSR than in non-reactive patients. These data suggest that liposome-induced HSRs in susceptible individuals may be due to C activation, which, in turn, is due to the presence of negatively charged PEG-PE in these vesicles.

Development of liposomal anthracyclines : from basics to clinical applications

The pharmacokinetics of liposome-encapsulated drugs are controlled by the interplay of two variables: the rate of plasma clearance of the liposome carrier, and the stability of the liposome-drug association in the blood stream. The pharmacokinetic properties of the liposomal drug, the vesicle size of the liposome carrier and the vascular permeability of individual tissues will determine the extravasation and biodistribution profile. The pharmacokinetics of polyethylene-glycol-(PEG)-liposomal doxorubicin are characterized by an extremely long circulating half-life, slow plasma clearance and reduced volume of distribution compared to free doxorubicin. These carrier systems show an improved extravasation profile with enhanced localization in tumors and superior therapeutic efficacy in comparison to doxorubicin in free form. These properties are the result of an optimized liposome composition and of a special drug-loading method which produces stable and long-circulating carriers. In clinical studies, doxorubicin encapsulated in PEG-coated liposomes shows a unique pharmacokinetic-toxicity profile and promising antitumor activity.

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

UNLABELLED 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.

Preparation and characterization of well defined D-erythro sphingomyelins.

A simple semisynthetic procedure for the preparation of various D-erythro sphingomyelins (SPMs), differing in their acyl chains, is described. They were prepared by one-step condensation of the desired free fatty acid with sphingosyl phosphorylcholine (SPC) using dicyclohexylcarbodiimide. The D-erythro SPMs were obtained in high purity, high yields and resemble bovine brain SPM in their chromatographic behavior, infrared, circular dichroism (CD) and proton NMR (PMR) spectra as well as in their rate of hydrolysis by Staphylococcus aureus sphingomyelinase. Multilamellar vesicles can be prepared from the semisynthetic SPMs. Their thermotropic behavior is dependent mainly on the acyl chain though it is also affected by the heterogeneity of the sphingosine base composition. Intact sealed small unilamellar vesicles (SUV) cannot be prepared from a single semisynthetic saturated SPM but can be prepared from their mixtures. This acylation procedure can also be applied for preparing simple neutral glycosphingolipids. The sphingolipids prepared by this method can be used to study metabolism, enzymology and physicochemical properties of D-erythro well defined simple sphingolipids.

Characterization of sterically stabilized cisplatin liposomes by nuclear magnetic resonance

Extensive scientific efforts are directed towards finding new and improved platinum anticancer agents. A promising approach is the encapsulation of cisplatin in sterically stabilized, long circulating, PEGylated 100 nm liposomes. This liposomal cisplatin (STEALTH cisplatin, formerly known as SPI-77) shows excellent stability in plasma and has a longer circulation time, greater efficacy and lower toxicity than much free cisplatin. However, so far, the physicochemical characterization of STEALTH cisplatin has been limited to size distribution, drug-to-lipid ratio and stability. Information on the physical state of the drug in the liposome aqueous phases and the drugs interaction with the liposome membrane has been lacking. This study was aimed at filling this gap. We report a multinuclear NMR study in which several techniques have been used to assess the physical nature of cisplatin in liposomal formulations and if and to what extent the drug affects the liposome phospholipids. Since NMR detects only the soluble cisplatin in the liposomes and not the insoluble drug, combining NMR and atomic absorption data enables one to determine how much of the encapsulated drug is soluble in the intraliposomal aqueous phase. Our results indicate that almost all of the cisplatin remains intact during the loading process, and that the entire liposomal drug is present in a soluble form in the internal aqueous phase of the liposomes.

High-dose bupivacaine remotely loaded into multivesicular liposomes demonstrates slow drug release without systemic toxic plasma concentrations after subcutaneous administration in humans.

BACKGROUND:Depot formulations prolong the analgesic effect of local anesthetics and reduce peak plasma drug concentration. This allows for safer administration of larger doses of local anesthetics, which further prolongs the duration of analgesic effect. We previously reported the development of large multivesicular vesicles (LMVVs) remotely loaded with bupivacaine (LMVV liposomal bupivacaine) and demonstrated a >5-fold prolongation of analgesic effect in animals and humans. In this study, we present pharmacokinetic data of LMVV liposomal bupivacaine in humans. METHODS:Healthy volunteers received subcutaneous injections of 20 mL plain 0.5% bupivacaine and, 1 week later, 20 mL of 2% LMVV liposomal bupivacaine in a prospective, open-label, crossover, controlled study. RESULTS:Eight subjects were studied. No subjective side effects of local anesthetics were observed. The maximal plasma concentration and the time to achieve maximal plasma concentration were assessed by modeling plasma concentration–time profiles. Maximal plasma concentration was not significantly different between groups (0.87 ± 0.45 &mgr;g/mL and 0.83 ± 0.34 &mgr;g/mL for plain and liposomal bupivacaine, respectively; P = not significant, 0.83). These values are well below the putative toxic plasma concentration of 2 to 4 &mgr;g/mL. Time to achieve maximal plasma concentration was 7-fold greater for the liposomal preparation (262 ± 149 minutes vs 37.5 ± 16 minutes, P < 0.01). CONCLUSIONS:Peak plasma bupivacaine concentrations were not different in the 2 groups, despite a 4-fold increase in total bupivacaine dose administered in the novel liposomal preparation. The delayed elimination and prolonged redistribution of liposomal bupivacaine to plasma is compatible with the depot-related slow-release effect leading to the prolonged pharmacodynamic effect previously reported.

In vitro tests to predict in vivo performance of liposomal dosage forms.

Design of liposome-based formulations for clinical use can be assisted by employing in vitro assays to predict pharmacokinetics and bioavailability of the drug before employing costly and time-consuming in vivo studies. For such formulations of the anti-cancer drug doxorubicin (DXR) we developed two assays. (A) An assay which determines the dilution-induced DXR release in buffers and plasma. This assay was employed to evaluate two liposomal DXR formulations: (i) membrane-associated liposomal doxorubicin (L-DXR), and (ii) sterically-stabilized liposomes which encapsulate DXR in the aqueous phase of the liposomes (S-DXR). The agreement between the dilution-induced release assay in vitro and the pharmacokinetics of DXR administrated either as L-DXR or as S-DXR in humans suggests that the dilution release assay can be used as a predictor for the pharmacokinetic performance of liposomal formulations. (B) An assay which determines intracellular drug release induced by liposome degradation in the presence of mouse liver lysosome lysate. This assay was used to assess bioavailability of DXR when delivered via L-DXR, which are taken up by the reticuloendothelial system (RES)

Prolonged analgesia from Bupisome and Bupigel formulations: From design and fabrication to improved stability

There is a compelling need for an ultralong-acting local anesthetic. Previously, we demonstrated in mice and humans that encapsulation of bupivacaine into large multivesicular liposomes (Bupisome) prolongs drug residence time and analgesic duration at the injection site while reducing peak plasma concentration. However, we observed considerable leakage of bupivacaine from the liposomes during storage at 4 °C. This deficiency was overcome by modifying the lipid composition of Bupisome and by entrapping them in a Ca-alginate cross-linked hydrogel (Bupigel), forming stable, soft, injectable (3-5 mm) beads. Bupisome are not released from Bupigel, but their encapsulated bupivacaine is released into the bulk solution. Adding 0.5% to 2.0% free bupivacaine to the Bupigel prevented net loss of bupivacaine from the Bupisome after storage at 4 °C for 2 years, and at 37 °C enough bupivacaine was released to prolong analgesia. For injection subcutaneously into mice, the beads are drawn into a syringe, leaving the small amount of free bupivacaine behind. Both Bupisome and Bupigel formulations significantly prolonged analgesia in mice compared to standard bupivacaine, with Bupigel performing better than Bupisome.

Liposomes as a model for the study of the mechanism of fish toxicity of sodium dodecyl sulfate in sea water.

The mechanism underlying the shark repellency of SDS was studied by comparing it with the shark nonrepelling detergent, Triton X-100. The findings can be summarized as follows: (1) The effective concentration of SDS for termination of shark tonic immobility (an immediate and fast response) was close to its critical micellar concentration in sea water (70 microM). The fish lethal concentrations (LD50) were far below the CMC value for SDS, and at CMC level for Triton X-100. (2) In sea water SDS possesses a strong affinity for lipid membranes, expressed in a lipid sea water partition coefficient (Kp) of about 3000. (3) In liposomal systems examined by assays of turbidity, fluorescence resonance energy transfer and kinetics of carboxyfluorescein (CF) release, the pattern of SDS induced changes in the phospholipid bilayer suggests: (a) absence of vesicle-vesicle fusion; (b) occurrence of vesicle size increase, and (c) nonlytic gradual release of CF above and below its CMC values. In contrast, Triton X-100 above its CMC induces membrane solubilization. (4) Assays coupling CF release from liposomes to potassium diffusion potential induced by valinomycin indicate that SDS related CF release can also be attributed to a specific mechanism such as cation pore formation and not only to membrane solubilization. The hypothesis of pore formation by SDS is discussed.

Correlation between the thermotropic behavior of sphingomyelin liposomes and sphingomyelin hydrolysis by sphingomyelinase of Staphylococcus aureus.

The hydrolysis of D-erythro beef brain sphingomyelin and D,L-erythro-N-palmitoylsphingomyelin dispersed as multilamellar liposomes by sphingomyelinase of Staphylococcus aureus is correlated with the thermotropic behavior of the sphingomyelins. In both cases maximal enzymatic hydrolysis was achieved at the beginning of the gel to liquid crystalline phase transition (30 degrees C for beef brain sphingomyelin and 41 degrees C for N-palmitoylsphingosine-phosphorylcholine) with much lower activity both below and above these temperatures. The enzymatic activity was depressed in the presence of cholesterol in the bilayer which also depressed the phase-transition. The profile of the enzymatic activity is explained by the uniqueness of the lipid molecules arrangement at the phase transition.