A comparison of liposomal formulations of doxorubicin with drug administered in free form: changing toxicity profiles.

The anthracycline antibiotic doxorubicin has wide activity against a number of human neoplasms and is used extensively both as a single agent and in combination regimens. In addition to the use of free, unencapsulated doxorubicin, there are two US Food and Drug Administration approved liposomal formulations of doxorubicin currently available, with several additional liposomal formulations being researched either in the laboratory or in clinical trials. The two approved liposomal formulations of doxorubicin have significantly different lipid compositions and loading techniques, which lead to both unique pharmacokinetic and toxicity profiles, distinct from those of the unencapsulated form. This article discusses the toxicities associated with the free form of doxorubicin, as well as those associated with the two most common liposomal formulations, namely Doxil and Myocet. One of the key toxicity issues linked to the use of free doxorubicin is that of both an acute and a chronic form of cardiomyopathy. This is circumvented by the use of liposomal formulations, as these systems tend to sequester the drug away from organs such as the heart, with greater accumulation in liver, spleen and tumours. However, as will be discussed, the liposomal formulations of doxorubicin are not without their own related toxicities, and, in the case of Doxil, may be associated with the unique toxicity of palmar-plantar erythrodysaesthesia. Overall, the use of liposomal doxorubicin allows for a greater lifetime cumulative dose of doxorubicin to be administered, however acute maximal tolerated doses differ significantly, with that of Myocet being essentially equivalent to free doxorubicin, while higher doses of Doxil may be safely administered. This review highlights the differences in both toxicity and pharmacokinetic properties between free doxorubicin and the different liposomal formulations, as have been determined in pre-clinical and clinical testing against a number of different human neoplasms. The need for further testing of the liposomal formulations prior to the replacement of free doxorubicin with liposomal doxorubicin in any established combination therapy regimens, as well as in combination with the newer therapeutics such as monoclonal antibodies is also discussed.

Antibody conjugation methods for active targeting of liposomes.

Liposomes are useful drug delivery vehicles since they may protect encapsulated drugs from enzymatic degradation and rapid clearance in vivo, or alter biodistribution, potentially leading to reduced toxicities (1,2). A major limitation to the development of many specialized applications is the problem of directing liposomes to tissues where they would not normally accumulate. Consequently, a great deal of effort has been made over the years to develop liposomes that have targeting vectors attached to the bilayer surface. These vectors have included ligands such as oligosaccharides (3,4), peptides (5,6), proteins (7,8) and vitamins (9). Most studies have focused on antibody conjugates since procedures for producing highly specific monoclonal antibodies (MAbs) are well established. In principle it should be possible to deliver liposomes to any cell type as long as the cells are accessible to the carrier. In practice it is usually not this simple since access to tissue, competition, and rapid clearance are formidable obstacles. It has also been shown that antibodies become immunogenic when coupled to liposomes (10,11), although in similar experiments with ovalbumin we have demonstrated that immunogenicity can be suppressed by formulating the liposomes with the cytotoxic drug doxorubi-cin (12). Such issues as these suggest that the development of antibody-targeted liposomes for in vivo applications will present difficult challenges.

An immune response to ovalbumin covalently coupled to liposomes is prevented when the liposomes used contain doxorubicin.

It is now well established that liposomes with surface associated proteins are immunogenic. Repeated administration of protein coated liposomes elicits the generation of antibodies and the elimination of proteoliposome increases markedly in animals 'immunized' with such liposomes. This immune response compromises the therapeutic potential of liposomal formulations that rely on the use of protein- or peptide-based targeting ligands to enhance cell specificity. Strategies to suppress or inhibit such immune responses must be developed if this technology is going to prove therapeutically viable. This study evaluates whether an immune response to a protein, covalently attached to liposomes by a thioether bond between N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP)-modified-protein and N-(4-(P-maleimidophenyl)butyryl) (MPB)-activated lipids, can be suppressed when the liposomes used contain the anti-cancer drug doxorubicin. To assess this, the highly immunogenic protein ovalbumin was conjugated onto liposomes composed of distearoylphosphatidylcholine/cholesterol (DSPC/Chol) with sufficient poly(ethylene glycol)-modified distearoyl phosphatidylethanolamine (PEG-DSPE) (2 mol%) to prevent liposome aggregation during protein coupling and to engender increased circulation lifetimes. The immune response to these liposomes with and without encapsulated doxorubicin was measured by: (1) monitoring liposome elimination after 3 weekly i.v. injections in C3H/HeJ mice and (2) measuring the anti-ovalbumin antibody levels by an ELISA assay. One week after a single dose of ovalbumin-coated PEG liposomes (50 microg protein/mouse) the immune response resulted in rapid elimination of a second dose of ovalbumin-coated PEG liposomes. Rapid liposome elimination was correlated to generation of high levels (> 9 microg/ml plasma) of circulating anti-ovalbumin IgG. In contrast, anti-ovalbumin antibodies were not detected when the liposomes used contained doxorubicin. Plasma elimination of these drug loaded protein coated liposomes decreased following repeated weekly i.v. doses, an effect that is consistent with liposomal doxorubicin mediated suppression of phagocytic cells in the liver.

3-(2-pyridyldithio)propionic acid hydrazide as a cross-linker in the formation of liposome-antibody conjugates.

Liposome antibody conjugates are potentially useful as a means of targeting drugs to specific tissues. A new protocol for the conjugation of IgG to maleimide-containing liposomes was developed using 3-(2-pyridyldithio)propionic acid hydrazide (PDPH) as a cross-linker. Periodate-oxidized antibody was treated with PDPH to yield a hydrazone derivative. Deprotection with DTT produced a thiolated antibody which was then conjugated to liposomes containing N-[4-(p-maleidophenyl)butyryl]-1,2-sn-distearoylphosphatidyleth anolamine. The liposome-antibody conjugates were found to have in vitro properties similar to those of conjugates formed by the traditional 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester (SPDP) protocol but were cleared less rapidly in circulation. The PDPH protocol presents a viable alternative to SPDP, particularly for antibodies sensitive to amine modification.

Liposomal doxorubicin.

Doxorubicin is a potent antineoplastic agent with activity against numerous human cancers. Encapsulation of doxorubicin inside a liposome alters bioavailability, biodistribution and thus its biological activity significantly. The physical properties of the liposome (size, lipid components and lipid dose) play a major role in determining drug retention and pharmacokinetics. The therapeutic benefits of liposomal doxorubicin will therefore depend on these physical characteristics. Here we review the toxicity and efficacy of liposomal doxorubicin determined for various liposome compositions (size, lipid composition and drug-to-lipid ratio). These physical properties can be independently varied using the transmembrane pH gradient-dependent drug encapsulation procedure. The results show that the toxicity of the formulation is related to drug retention in the circulation. The antitumor activity is more sensitive to the size of the liposomes. By optimizing these parameters, liposomal doxorubicin formulations can be optimized for improved therapeutic activity.

Accumulation of protein-coated liposomes in an extravascular site: influence of increasing carrier circulation lifetimes.

The primary objective of this work was to test whether increased blood levels and circulation lifetimes result in increased passive targeting of protein-coated liposomal drug carriers. The system used to evaluate this was based on i.v. injection of 100 nm of distearoyl phosphatidylcholine/cholesterol liposomes with covalently bound streptavidin. The circulation lifetime of these liposomes was increased by procedures that involved blockade of liposome uptake by phagocytic cells in the liver and/or the incorporation of a poly(ethylene glycol)-modified phospholipid [poly(ethylene glycol)2000-modified distearoyl phosphatidylethanolamine]. Blockade of liver phagocytic cells with a low predose (2 mg/kg of drug) of liposomal doxorubicin increased the circulation half-life of the streptavidin liposomes from less than 1 hr to greater than 3 hr. A further 2-fold increase in circulating half-life (to approximately 7.5 hr) was achieved by using liposomes with 2 mole % of poly(ethylene glycol)2000-modified phosphatidylethanolamine. In combination with RES blockade, the circulation lifetimes of poly(ethylene glycol)phosphatidylethanolamine containing streptavidin liposomes could be increased to greater than 12 hr. The ability of these liposomes to move from the plasma compartment to an extravascular compartment was measured by using the peritoneal cavity as a convenient, accessible, extravascular site. The tendency for liposomes to accumulate in this site was not, however, clearly dependent on circulating blood levels. Comparable levels of liposomes in the peritoneal cavity were achieved when using systems that exhibited significantly different circulation lifetimes.

The modulation of phosphatidylinositol biosynthesis in hamster hearts by methyl lidocaine.

Methyl lidocaine is an experimental anti-arrhythmic drug which has been shown to enhance the biosynthesis of phosphatidyl-inositol (PI) in the hamster heart. In this study, the effect of methyl lidocaine on enzymes involved in the biosynthesis of PI in the heart was examined. When the hamster heart was perfused with labelled methyl lidocaine, the majority of the compound was not metabolized after perfusion. The direct action of methyl lidocaine on an enzyme was studied by the presence of the drug in enzyme assays, whereas its indirect action was studied by assaying the enzyme activity in the heart after methyl lidocaine perfusion. CTP:phosphatidic acid cytidylyl-transferase, a rate-limiting enzyme in PI biosynthesis, was stimulated by methyl lidocaine in a direct manner. Kinetic studies revealed that methyl lidocaine caused a change in the affinity between the enzyme and phosphatidic acid and resulted in the enhancement of the reaction. Alternatively, acyl-CoA:lysophosphatidic acid acyltransferase, another key enzyme for PI biosynthesis, was not activated by the presence of methyl lidocaine. However, the enzyme activity was stimulated in hearts perfused with methyl lidocaine. The enhancement of the acyl-transferase by methyl lidocaine perfusion was found to be mediated via the adenylate cyclase cascade with the elevation of the cyclic AMP level. The stimulation of protein kinase A activity by cyclic AMP resulted in the phosphorylation and activation of the acyltransferase. Interestingly, the activity of protein kinase C was not stimulated by methyl lidocaine perfusion. We conclude that the enhancement of PI biosynthesis by methyl lidocaine in the hamster heart resulted from the direct activation of the cytidylyltransferase, as well as the phosphorylation and subsequent activation of the acyltransferase.

Effects of selenium supplement on the de novo biosynthesis of glycerolipids in the isolated rat heart.

The effect of selenium supplement on glycerolipid biosynthesis in the isolated rat heart was investigated. Selenium was administered to the rat by intraperitoneal injection of 4.33 mumol/kg per day for 3 consecutive days. Animals administered with an equal volume of saline were used as controls. Hearts from both animal groups were perfused in Krebs-Henseleit buffer containing labelled glycerol. Subsequent to perfusion, the radioactivity associated with each glycerolipid group was determined. Selenium supplement caused elevations in the labelling of phosphatidic acid and phosphatidylcholine but not in other phospholipids, diacylglycerol or triacylglycerol. The mechanisms for the enhancement of labelling into phosphatidic acid and phosphatidylcholine were examined. The activity of the enzymes responsible for the synthesis of phosphatidic acid in the rat heart was not changed by selenium supplement. However, a 51% increase in the acyl-CoA level was detected which might account for the elevated labelling of phosphatidic acid in the selenium supplemented animal. The 2-fold increase in the activity of CDPcholine:diacylglycerol cholinephosphotransferase might also account for the increase in the labelling of phosphatidylcholine in the heart of the selenium-supplemented rat. It is clear from this study that selenium plays a regulatory role in the control of cellular lipid metabolism.

Modulation of phosphatidylethanolamine biosynthesis by exogenous ethanolamine and analogues in the hamster heart.

In the hamster heart, exogenous ethanolamine is taken up by the heart and utilized for the biosynthesis of phosphatidylethanolamine. The role of the exogenous supply of ethanolamine on phosphatidylethanolamine biosynthesis was examined by perfusing hamster heart with various concentrations of labelled ethanolamine. Analysis of the radioactivity distributed in the ethanolamine-containing metabolites indicated that at low exogenous ethanolamine concentrations (< or = 0.1 microM), the conversion of phosphoethanolamine to CDP-ethanolamine was rate-limiting for phosphatidylethanolamine biosynthesis. However, perfusion with higher concentrations of ethanolamine (> or = 0.4 microM) resulted in the phosphorylation of ethanolamine becoming rate-limiting. Since the intracellular ethanolamine levels remained unchanged, the alterations in radioactivity distribution suggested that the newly imported ethanolamine was preferentially utilized for phosphatidylethanolamine biosynthesis. The effects of ethanolamine analogues on ethanolamine uptake and subsequent conversion to phosphatidylethanolamine at physiological concentrations of exogenous ethanolamine were examined. Monomethylethanolamine was found to inhibit ethanolamine uptake, the conversion of ethanolamine to phosphoethanolamine and incorporation of radioactivity into phosphatidylethanolamine. The accumulation of radioactivity in the ethanolamine fraction by monomethylethanolamine, despite of the inhibition of ethanolamine uptake, further confirms the rate-limiting role of ethanolamine kinase in the biosynthesis of phosphatidylethanolamine.

The effect of methyl-lidocaine on the biosynthesis of phospholipids de novo in the isolated hamster heart.

Methyl-lidocaine is an amphiphilic agent which has been used as an experimental anti-arrhythmic drug. When hamster hearts were perfused with labelled glycerol, the presence of methyl-lidocaine in the perfusate was found to enhance the labelling in phosphatidylserine, phosphatidylinositol, diacylglycerol and triacylglycerol. However, the labelling of phosphatidylcholine and phosphatidylethanolamine was not significantly changed by methyl-lidocaine treatment. Assays in vitro for the enzymes involved in the synthesis of neutral lipids and acidic phospholipids revealed that phosphatidate phosphatase and CTP: phosphatidate cytidylyltransferase activities were stimulated by methyl-lidocaine. The intracellular pool sizes of diacylglycerol and CDP-diacylglycerol were also elevated. We postulate that the enhanced syntheses of the neutral lipids and acidic phospholipids in the methyl-lidocaine-perfused heart were mediated via the direct activation of the key enzymes in the biosynthesis of these lipids de novo.

The quantitation of long-chain acyl-CoA in mammalian tissue.

Intracellular long-chain acyl-CoA esters are key metabolites in lipid metabolism. A rapid procedure was developed for the isolation of long-chain acyl-CoA from mammalian tissues. Acyl-CoA was extracted from the tissue with chloroform/methanol and separated from other lipid-containing metabolites by phase partition with solvents. The content and the molecular species of acyl-CoA were determined by gas-liquid chromatography. In rat liver and hamster heart, the total acyl-CoA content was estimated to be 83 +/- 11 and 61 +/- 9 nmol/g wet weight, respectively. The results obtained are comparable to those reported in previous studies. The relative ease of this procedure would permit the determination of acyl-CoA contents in a large number of samples.

Solubilization and modulation of acyl-CoA:1-acyl-glycerophosphocholine acyltransferase activity in rat liver microsomes.

The acylation of 1-acyl-glycerophosphocholine is an important mechanism for the maintenance of the asymmetrical distribution of acyl groups in phosphatidylcholine. The majority of acyl-CoA:1-acyl-glycerophosphocholine acyltransferase is located in the microsomal fraction. In this study, the rat liver microsomes were incubated with various detergents, and the solubilized enzyme was separated from the remainder by centrifugation. Sodium cholate, sodium deoxycholate and octylglucopyranoside caused the solubilization of 14-25% of the enzyme activity. The acyl specificity of the solubilized enzyme was similar to the insoluble enzyme, indicating that there was no selective solubilization of any acyl specific acyltransferase. The solubilized enzyme did not display any lipid requirement, and its activity was inhibited by phosphatidylcholine, phosphatidylethanolamine and 1,2-diacylglycerol. Kinetic studies with varying concentrations of acyl-CoAs revealed that the inhibition by 1,2-diacylglycerol was essentially uncompetitive. The modulation of acyltransferase activity by 1,2-diacylglycerol may be an important mechanism for controlling the acylation of lysophosphatidylcholine.

The effect of methyl lidocaine on lysophospholipid metabolism in hamster heart.

An important feature in the remodelling of fatty acyl chains in cellular phospholipids is the acylation of lysophospholipids. Since lysophospholipids are cytolytic at high concentrations, the acylation reaction may provide an alternate pathway for the removal of cellular lysophospholipids. However, the physiological role of the acylation process in the maintenance of lysophospholipid levels in mammalian tissues has not been clearly defined. In this study, methyl lidocaine was found to inhibit both lysophosphatidylcholine:acyl-CoA and lysophosphatidylethanolamine:acyl-CoA acyltransferase activities in the hamster heart, but the drug had no effect on the other lysophospholipid metabolic enzymes. When the heart was perfused with 0.5 mg methyl lidocaine/mL, acyltransferase activities were attenuated, but there was no change in the activities of phospholipase A or lysophospholipase. The levels of the major lysophospholipids in the heart were not altered by methyl lidocaine perfusion. When the hearts were perfused with labelled lysophospholipid in the presence of methyl lidocaine, there was a reduction in the formation of the phospholipid and an increase in the release of the free fatty acid. However, the labelling of lysophospholipid in the heart was not altered by methyl lidocaine. We postulate that the acylation reaction has no direct contribution to the maintenance of the lysophospholipid levels in the heart.