The next generation of liposome delivery systems: recent experience with tumor-targeted, sterically-stabilized immunoliposomes and active-loading gradients.

Three topics are discussed. Enhanced anti-tumor efficacy of targeted doxorubicin-containing sterically-stabilized liposomes using an anti-beta1 integrin Fab' ligand. Use of tumor targeting with an internalizing ligand to improve the efficacy of a non-leaky cisplatin-containing sterically-stabilized liposome formulation. Formulation variables (remote-loading with dextran ammonium sulfate, rigid lipid bilayer) used to optimize in vivo performance of a liposomal camptothecin analog.

Influence of tumour size on uptake of(111)ln-DTPA-labelled pegylated liposomes in a human tumour xenograft model.

The relationship between tumour size and uptake of(111)In-DTPA-labelled pegylated liposomes has been examined in a human head and neck cancer xenograft model in nude mice. The mean tumour uptake of(111)In-labelled pegylated liposomes at 24 hours was 7.2 +/- 6.6% ID/g. Liposome uptake for tumours < 0.1 g, 0.1-1.0 g and > 1.0 g was 15.1 +/- 10.8, 5.9 +/- 2.2 and 3.0 +/- 1.3% ID/g, respectively. An inverse correlation between tumour weight and liposome uptake was observed by both Spearman's rank correlation test (r(s)= - 0.573, P< 0.001) and Pearson's correlation coefficient (r(s)= - 0.555, P< 0.001). For 18 tumours with macroscopic central necrosis, the ratio of uptake in the tumour rim relative to the necrotic tumour core was 11.2 +/- 6.4. Measurement of tumour vascular volume for tumours of various sizes revealed an inverse correlation between tumour weight and tumour vascular volume (Spearman's rank correlation test, r(s)= - 0.598, P< 0.001), consistent with poor or heterogeneous vascularization of larger tumours. These data have important implications for the clinical application of pegylated liposome targeted strategies for solid cancers which are discussed in detail.

Biodistribution and pharmacokinetics of 111In-DTPA-labelled pegylated liposomes in a human tumour xenograft model: implications for novel targeting strategies.

The biodistribution and pharmacokinetics of 111In-DTPA-labelled pegylated liposomes in tumour-bearing nude mice was studied to examine possible applications of pegylated liposome-targeted anti-cancer therapies. Nude mice received an intravenous injection of 100 microl of 111In-DTPA-labelled pegylated liposomes, containing 0.37-0.74 MBq of activity. The t1/2alpha and t1/2beta of 111In-DTPA-labelled pegylated liposomes were 1.1 and 10.3 h, respectively. Tumour uptake was maximal at 24 h at 5.5 +/- 3.0% ID g(-1). Significant reticuloendothelial system uptake was demonstrated with 19.3 +/- 2.8 and 18.8 +/- 4.2% ID g(-1) at 24 h in the liver and spleen, respectively. Other sites of appreciable deposition were the kidney, skin, female reproductive tract and to a lesser extent the gastrointestinal tract. There was no indication of cumulative deposition of pegylated liposomes in the lung, central nervous system, musculoskeletal system, heart or adrenal glands. In contrast, the t1/2alpha and t1/2beta of unencapsulated 111In-DTPA were 5 min and 1.1 h, respectively, with no evidence of accumulation in tumour or normal tissues. Incubation of 111In-DTPA-labelled pegylated liposomes in human serum for up to 10 days confirmed that they are very stable, with only minor leakage of their contents. The potential applications of pegylated liposomes in the arena of targeted therapy of solid cancers are discussed.

Hyperthermia induces doxorubicin release from long-circulating liposomes and enhances their anti-tumor efficacy.

PURPOSE: To examine the possibility that hyperthermia would accelerate drug release from long-circulating liposomes, and enhance their antitumor activity. METHODS AND MATERIALS: Liposomes were prepared by thin film hydration technique. Hyperthermia was induced by ultrasound apparatus and a water bath heating system. The antitumor efficacy of treatment against RIF-1 tumor in C3H mice was evaluated by the tumor growth delay assay. RESULTS: In vitro drug release experiments demonstrated that increase in temperature from 37 degrees C to 41 degrees C resulted in about a sixfold increase in doxorubicin (DOX) release in a 1-h period. Increasing the temperature to 43 degrees C, resulted in only a modest additional drug release. Drug uptake studies showed that local hyperthermic treatment immediately following the drug administration dramatically enhanced Stealth liposome-encapsulated doxorubicin (S-DOX) uptake by tumors, but did not do so for free DOX. At 42 degrees C and at a dose of 10 mg/kg, the accumulation of S-DOX was about 10-fold and 2.5-fold higher than that with free drug and S-DOX at 37 degrees C, respectively. The antitumor efficacy study confirmed our hypothesis that the addition of hyperthermia to the treatment of RIF-1 tumors with doxorubicin encapsulated in long-circulating liposomes would enhance antitumor effects. Two hyperthermia treatments given at 24-h intervals appeared to be the most promising method of combining heat and long-circulating liposomes. The increased antitumor activity was not accompanied by increased toxicity, as determined by the body weight of the mice. CONCLUSION: Local hyperthermic treatment is able to accelerate DOX release from long-circulating liposomes, increase tumor uptake, and enhance their antitumor efficacy. The combination of local hyperthermia and long-circulating liposomes appears to show considerable promise in the treatment of localized diseases.

Factors affecting the release rate of terbutaline from liposome formulations after intratracheal instillation in the guinea pig.

Maximum duration of bronchodilator efficacy in inhaled liposome-based formulations depends on optimizing the in vivo release rate of the encapsulated bronchodilator. We investigated the effect of several formulation variables on the pulmonary residence time of 3H-terbutaline sulfate liposomes administered intratracheally in guinea pigs, using an improved method enabling the measurement of pulmonary drug absorption for extended periods of time in conscious animals. Half-lives of liposome-encapsulated 3H-terbutaline disappearance from the lungs and airways after instillation ranged from 1.4 to 18 hr and were markedly affected by liposome size, cholesterol content, and phospholipid composition. This study demonstrates that liposomes can significantly prolong the residence time of bronchodilators in the lungs and that precise control over the pulmonary residence time of encapsulated bronchodilators can be achieved by controlling formulation variables.

Liposome disposition in vivo. VI: Delivery to the lung.

The effect of negatively charged liposome components and vesicle size on the time course and dose dependency of liposome disposition in mice was studied with a view to optimizing liposome delivery to the lung. The disposition of large multilamellar liposomes was followed using 125I-labeled p-hydroxybenzamidine phosphatidyl ethanolamine. Of the three negatively charged liposome compositions studied (phosphatidyl choline-X-cholesterol-alpha-tocopherol, molar ratio: 4:1:5:0.1; X = phosphatidyl serine, dipalmitoyl phosphatidic acid, or phosphatidyl glycerol), phosphatidyl serine liposomes resulted in the greatest accumulation in lungs. Lung levels decreased up to 95 h postdose, at which time 6% of the liposome dose present at 2 h still remained. The disposition of phosphatidyl serine-containing liposomes was independent of dose for the range 0.04-21 mumol/animal. When liposomes containing phosphatidyl choline were prepared using a variety of extrusion and dialysis conditions, a strong link between liposome size and lung accumulation was revealed. A maximum lung accumulation of 30.9% of the administered dose was achieved with no detectable gross pathological lung lesions up to 24 h postdose.

Delivery of therapeutic doses of doxorubicin to the mouse lung using lung-accumulating liposomes proves unsuccessful.

Addition of solid doxorubicin or solutions to pre-formed liposomes proved to be the optimal method for incorporating the drug into liposomes whilst maintaining their size distribution and hence ability to accumulate in the lung. Liposomes prepared in this way lost doxorubicin only slowly on dialysis but dilution with an equal volume of saline at 37 degrees C resulted in the loss of 80% of the incorporated doxorubicin within 30 min. These liposomes were thus ineffective in altering doxorubicin disposition in vivo and produced no enhanced activity compared with free drug and a non-lung-accumulating carrier liposome in the EMT6 cell-Balb/c mouse model lung tumour.

The use of a new radioactive-iodine labeled lipid marker to follow in vivo disposition of liposomes: comparison with an encapsulated aqueous space marker.

The in vivo disposition of multilamellar liposomes extruded at 0.6 micrometers (PC/DPPA/CH/ alpha-T = 4:1:5:0.1 molar ratio) when injected i.v. into mice has been examined utilizing a novel iodinatable phospholipid derivative as a lipid phase marker (p-hydroxybenzamidine phosphatidylethanolamine: 125I-BPE) and compared to that using 14C-inulin as an aqueous phase marker. At times up to 5 h post-dose the disposition of both markers was essentially identical with the exception of blood and intestine, where 125I-BPE levels were consistently higher than 14C-inulin levels. At time intervals from 5-72 h post-dose 125I-BPE levels in all the organs examined were lower than those of 14C-inulin. These differences in the behaviour of the two labels may be explained in terms of exchange of the iodinated lipids, excretion of released inulin and long term metabolism of the lipid marker. We conclude tha 125I-BPE is a useful marker for following liposome disposition in short-term studies particularly in view of the easily quantifiable nature of gamma-radioactivity which obviates the need for sample preparation.

Liposome disposition in vivo IV: the interaction of sequential doses of liposomes having different diameters.

In order to evaluate the degree to which liposomes of the same composition but of different size interact with organ binding sites, a series of pre-dose studies have been carried out in mice. Two sizes of multilamellar liposomes were used: large (L) liposomes averaging 0.5 micrometer in diameter and small (S) liposomes averaging 0.06 micrometer in diameter. Lipid composition was phosphatidylcholine/phosphatidic acid/cholesterol/alpha-tocopherol in the molar ratio 4:1:5:0.1. Experiments consisted of a saturating pre-dose of either non-radioactive L- or S-liposomes followed at various times by a test dose of radioactively labelled L- or S-liposomes. When pre-dose and test dose were of the same diameter, an interaction was observed. A pre-dose of L-liposomes also interacted with a test dose of S-liposomes but a pre-dose of S-liposomes did not interact with a test dose of L-liposomes. All disposition effects were reversible, returning to control values within 24 h. These results indicate that L- and S-liposomes of identical composition may associate with different hepatic binding sites and that a class of non-specific binding sites available to S-liposomes but not to L-liposomes may exist.

Liposome disposition in vivo. III. Dose and vesicle-size effects.

The effect of lipid dose (4,3-512.8 mumol total lipid/kg body weight), administered intravenously as liposomes encapsulating radioactive inulin, upon the ability of mouse organs to bind and/or take-up the radioactive label has been studied in vivo. Three different liposome diameters were investigated: 0.46 micrometers (L), 0.16 micrometers (M) and 0.058 micrometers(S). All liposomes were negatively charged with lipid composition of phosphatidylcholine/phosphatidic acid/cholesterol/alpha-tocopherol in the molar ration 4 : 1 : 5 : 0.1 or 4 : 1 : 1 : 0.05. Overall radioactive label disposition after 2 h was consistent with localization predominantly in the reticuloendothelial system. A saturation of liver with increasing lipid dose was demonstrated for all three sizes, together with a corresponding increase in blood levels. Spleen radioactivity increased with increasing dose of L- and M-liposomes, but decreased for increasing doses of S-liposomes. Levels in residual carcass exhibited no trend. It was noted that by adjusting liposomal lipid dose and vesicle diameter the percentage of administered dose present in blood could be varied 733-fold, that in spleen 9-fold, liver 4-fold. Stability in vivo was ranked L greater than M greater than S-liposomes. Correction for differences of in vivo stability reduced the differences in organ accumulation between the three liposome sizes. The organ accumulation pattern suggested a dose- and diameter-dependent mechanism for liposome disposition. It was expected that when doses of fixed liposome composition were expressed as number of liposomes or their total surface area, organ saturation patterns would be similar. However, re-plotting the percent dose values for liver and spleen versus the number of liposomes administered revealed a saturation pattern for L-, M- and S-liposomes which was different in each case. Plotting the data versus the total surface area of the dose revealed a similar disposition pattern for L-, M- and S-liposomes in liver and L- and M-liposomes in spleen. The data indicate that in addition to composition, the lipid dose, total liposomal surface area and effective mean diameter are important pharmacokinetic variables. Further, the optimization of the therapeutic index of an encapsulated agent or target-tissue delivery via liposomes will require consideration of both the surface area and diameter of the liposome doses together with liposome composition.

Liposome disposition in vivo: effects of pre-dosing with lipsomes.

The effect of a high, intravenous dose of extruded multilamellar liposomes (1.1 g lipid/kg body weight) upon the subsequent ability of mouse tissues to take-up or bind a second intravenous dose of similar liposomes encapsulating 14C-inulin has been studied in vivo. The first and second doses were separated by either 1,5 or 24 hours. All tissue levels were measured one hour after the second dose. Controls received only the second dose. When the two doses were separated by one hours, 14C-levels in liver were depressed 6-fold and blood levels rose 29-fold relative to controls. However spleen uptake of lipsomes increased to three times control levels. When the two doses were separated by 24 hrs, the first dose had only a minimal effect on the disposition of the second dose. The results are consistent with a reversible blockade of hepatic, but not spleenic uptake and/or binding sites, by the first dose and indicate that adjusting a liposome dose (i.e. number of lipsomes) or use of a drug-free liposome pre-dose may be a useful technique for reducing hepatic uptake, increasing the circulation life time and/or modifying the tissue disposition properteries of therapeutic liposomes without changing liposome composition or size.

Some characteristics of sn-glycero-3-phosphocholine diesterases from rat brain.

1. sn-Glycero-3-phosphocholine diesterase activities, glycerophosphohydrolase (EC 3.1.4.2) and choline phosphohydrolase (EC 3.1.4.38) from rat brain have been partially purified and characterized using sn-glycere-3-[32P]phosphocholine as substrate and separating the reaction products by anion-exchange chromatography and ionophoresis. 2. Rat brain contained particulate (75%) and soluble (25%) activity from both diesterases. No difference in pH optimum or metal ion requirement for the particulate compared to the soluble enzymes was observed. 3. Glycerophosphohydrolase (EC 3.1.4.2) was purified 60-fold, choline phosphohydrolase (EC E.1.4.38) 120-fold from rat brain supernatant fraction by DEAE-cellulose ion-exchange chromatography and sucrose density gradient centrifugation. The density gradient results in conjunction with dodecyl sulphate-polyacrylamide gel disc electrophoresis yielded molecular weight estimates of 230 000 (monomer 62 000) for choline phosphohydrolase and 120 000 (monomer 70 000) for glycerophosphohydrolase (EC 3.1.4.2). 4. Glycerophosphohydrolase (EC 3.1.4.2) has a pH optimum of 8.9 and a Km for sn-glycero-3-phosphocholine of 0.6 mM. The enzyme is inhibited by EDTA and reactivated by Ca2+. Choline phosphohydrolase (EC 3.1.4.38) has pH optimum 10.5, a Km of 2 mM and is unaffected by EDTA. Both enzymes require Ca2+ for maximum activity.

The fluidity of normal and virus-transformed cell plasma membrane.

1. The phospholipid composition and cholesterol/phospholipid ratio of plasma membrane is the same in normal as in transformed BHK (baby-hamster kidney) cells; no significant difference in length or degree of unsaturation of the contributing acyl chains is apparent. 2. The turnover of acetate-labelled phosphatidylcholine species in the plasma membrane of normal and transformed BHK cells is the same. 3. Intramembranous particles of normal and transformed 3T3-cell plasma membrane are randomly distributed, whether at 4degreesC or at 37degreesC, in sparse or in dense cultures. There is no correlation between distribution of particles and the movement of concanavalin A receptor sites. 4. It is concluded that transformation of fibroblastic cells by oncogenic viruses does not lead to major changes in the lipid fluidity of the plasma membrane. 5. Details of the phospholipid composition of nuclei, mitochondria and endoplasmic reticulum in normal and transformed BHK cells have been deposited as Supplementary Publication SUP 50061 (5 pages) at the British Library Lending Division, Boston, Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1976) 153, 5.

A novel pathway for phosphatidylcholine catabolism in rat brain homogenates.

Rat brain homogenates incubated with exogenous [32-P] phosphatidylcholine liberated: LYSO[32-P] phosphatidylcholine, sn-glycero-3-[32-P] phosphorylcholine, [32-P] phosphorylcholine, sn-gleycero-3-[32-P] phosphate and 32-Pi. Further investigation showed that [32-P] phosphorylcholine was released exclusively from sn-glycero-3-[32-P] phosphorylcholien by a novel diesterase activity. We propose that the enzyme be termed L-3-glycerylphosphinicocholine cholinephosphohydrolase (EC 3.1.4.-). Parallel experiments on rat liver homogenates and a P815Y mouse mastocytoma cell-lysate, revealed no diesterase activity.