Enhanced thrombolysis, reduced coronary reocclusion and limitation of infarct size with liposomal prostaglandin E1 in a canine thrombolysis model.

OBJECTIVES: The purpose of this study was to test the hypothesis that liposomal prostaglandin E1 (TLC C-53) would result in more rapid thrombolysis, less reocclusion and smaller infarct size when administered with heparin and streptokinase in a canine thrombolysis model. BACKGROUND: In experimental animals, prostaglandin E1 has been shown to augment thrombolysis, improve coronary flow and reduce infarct size when infused directly into the left atrium. TLC C-53 is a stable preparation of prostaglandin E1 bound by phospholipid microspheres that produces fewer adverse hemodynamic effects during intravenous use. METHODS: To investigate the effects of TLC C-53 on coronary patency and infarct salvage, we studied 30 conditioned open chest dogs. After coil-induced left anterior descending coronary artery occlusion and 1 h of clot maturation, the dogs were randomly assigned to receive a 10-min intravenous infusion of either TLC C-53 (2 micrograms/kg body weight) or placebo. Both groups then received intravenous heparin and streptokinase. Hemodynamic variables and Doppler coronary flow were monitored, and myocardial blood flow was determined using radioactive microspheres. Infarct size was assessed with triphenyltetrazolium chloride staining. RESULTS: Thrombolysis time was accelerated from 79 +/- 38 to 47 +/- 9 min (mean +/- SD), and coronary patency was greater (100% vs. 50%) with TLC C-53 than with placebo (p < 0.05). Moreover, for arteries that recanalized, coronary Doppler flow and myocardial perfusion were more severely impaired with placebo. Infarct size as a percent of the area at risk was higher (p < 0.05) with placebo (51 +/- 15%) than with TLC C-53 (33 +/- 14%). Neutrophil infiltration into ischemic myocardium determined by myeloperoxidase assay was also significantly greater in the placebo group. CONCLUSIONS: TLC C-53 administered intravenously before thrombolytic therapy resulted in a significant acceleration of thrombolysis time, improvement in coronary patency and blood flow during reperfusion and a reduction in infarct size.

Liposome-bound prostaglandin E1 often prevents cyclic flow variations in stenosed and endothelium-injured canine coronary arteries.

BACKGROUND: Prostaglandin E1 is a potent vasodilator with anti-inflammatory and antiplatelet effects. We tested the hypothesis that prostaglandin E1 attenuates or prevents platelet aggregation-associated cyclic flow variations (CFVs) in severely stenosed and endothelium-injured coronary arteries. METHODS AND RESULTS: We induced CFVs in 21 dogs by placing an external constrictor around the left anterior descending coronary artery at the site where the endothelium had been mechanically injured. The blood flow velocity in the artery was monitored by a pulsed Doppler flow probe. Sixty minutes after CFVs were established, liposome-bound prostaglandin E1, a stable formulation, was administered intravenously to 12 dogs at incremental doses of 0.25, 0.5, 1, and 2 micrograms/kg body wt; it abolished CFVs in 8 of the 12 dogs (67%). After CFVs were eliminated, epinephrine was infused intravenously, and at a dose of 6.6 +/- 1.6 micrograms/min, it restored CFVs in 7 of 7 dogs. Control dogs (n = 9) were treated with free prostaglandin E1, which did not abolish CFVs in any dog. CONCLUSIONS: Liposome-bound but not free prostaglandin E1 effectively diminishes CFVs in severely stenosed and endothelium-injured canine coronary arteries.

Liposome-entrapped PGE1 posttreatment decreases IL-1 alpha-induced neutrophil accumulation and lung leak in rats.

We found that treatment with liposome-entrapped prostaglandin E1 (Lip-PGE1), but not with empty liposomes and/or free PGE1, decreased the leak of intravascularly administered 125I-labeled albumin into lungs of rats given interleukin-1 alpha (IL-1 alpha) intratracheally. Lip-PGE1 treatment also decreased lung myeloperoxidase activity, lung lavage neutrophil increases, and lung histological abnormalities found in rats given IL-1 alpha intratracheally. Interestingly, decreased lung leak and lung neutrophil accumulation occurred when Lip-PGE1 was given intravenously 2.5 h after, but not immediately before, intratracheal IL-1 alpha administration. When Lip-PGE1 treatment was given both before and 2.5 h after IL-1 alpha administration, lung leak was decreased to baseline levels. Lip-PGE1 treatment given 2.5 h after IL-1 alpha administration also decreased lung oxidized glutathione levels, which increased in rats given IL-1 alpha intratracheally. We conclude that postinsult treatment with Lip-PGE1 decreases lung leak, neutrophil recruitment, and oxidative responses in lungs of rats given IL-1 alpha intratracheally.

Initial clinical (phase I) trial of TLC D-99 (doxorubicin encapsulated in liposomes).

A liposome-encapsulated form of doxorubicin (TLC D-99), which was shown in preclinical toxicology to be less toxic to the gastrointestinal tract and myocardium than free doxorubicin, was administered by constant infusion (1.00-1.80 h) to 38 patients in single doses of 20, 30, 45, 60, 75, and 90 mg/m2 every 3 weeks and daily for 3 days at doses of 20, 25, and 30 mg/m2/day. The dose-limiting toxicity was leucopenia: the maximum tolerated doses were one at 90 mg/m2 and three at 25 mg/m2/day. Nausea, vomiting, and stomatitis were minimal or absent at each dose; alopecia was minor. Fever and chills were noted at most of the doses, and malaise was seen in some patients, especially at the higher doses. No hepatic, renal, or other organ toxicities were observed. Clinical cardiac toxicity was not observed in any patient; however, the cumulative doxorubicin dose was greater than 400 mg/m2 in only one patient. There was large variation among patients in estimated pharmacokinetic parameters and profiles. Higher plasma levels and dose intensities were achieved with TLC D-99 than were predicted for free doxorubicin. Liposome-encapsulated doxorubicin was well tolerated and produced less nausea, vomiting, and stomatitis than would be expected with free doxorubicin administered at equally myelosuppressive doses.

Anaphylaxis during induction of general anesthesia: subsequent evaluation and management.

Twenty-seven patients were referred for evaluation of anaphylaxis after induction of general anesthesia (GA) in which thiobarbiturates, muscle relaxants, or antibiotics were administered intravenously. Skin testing by the prick and intracutaneous methods was performed with dilutions of the thiobarbiturates and muscle relaxants; beta-lactam reagents were used in patients who had also received these drugs. No skin test reactivity was noted in 16 normal subjects. Skin tests were positive in 13 patients (thiobarbiturates in five, muscle relaxants in six, and antibiotics in two patients). Two patients were dermatographic and yielded indeterminate skin test results. Eleven of the 27 patients subsequently had GA; all patients received a premedication regimen of prednisone and diphenhydramine. Of three patients with negative skin tests, one experienced an arrhythmia, but no other signs attributable to anaphylaxis were noted. One patient with dermatographism had GA without a reaction. Positive skin tests implicated an agent that was avoided in seven patients; one of these patients experienced delayed urticaria/angioedema after the completion of GA. Thus, no patients developed anaphylaxis during subsequent GA for which agents producing positive skin tests were avoided, and a premedication regimen was used.

Use of liposomes as injectable-drug delivery systems.

The formation of liposomes and their application as delivery systems for injectable drugs are described. Liposomes are microscopic vesicles composed of one or more lipid membranes surrounding discrete aqueous compartments. These vesicles can encapsulate water-soluble drugs in their aqueous spaces and lipid-soluble drugs within the membrane itself. Liposomes release their contents by interacting with cells in one of four ways: adsorption, endocytosis, lipid exchange, or fusion. Liposome-entrapped drugs are distributed within the body much differently than free drugs; when administered intravenously to healthy animals and humans, most of the injected vesicles accumulate in the liver, spleen, lungs, bone marrow, and lymph nodes. Liposomes also accumulate preferentially at the sites of inflammation and infection and in some solid tumors; however, the reason for this accumulation is not clear. Four major factors influence liposomes' in vivo behavior and biodistribution: (1) liposomes tend to leak if cholesterol is not included in the vesicle membrane, (2) small liposomes are cleared more slowly than large liposomes, (3) the half-life of a liposome increases as the lipid dose increases, and (4) charged liposomal systems are cleared more rapidly than uncharged systems. The most advanced application of liposome-based therapy is in the treatment of systemic fungal infections, especially with amphotericin B. Liposomes are also under investigation for treatment of neoplastic disorders. Liposomes' uses in cancer therapy include encapsulation of known antineoplastic agents such as doxorubicin and methotrexate, delivery of immune modulators such as N-acetylmuramyl-L-alanine-D-isoglutamine, and encapsulation of new chemical entities that are synthesized with lipophilic segments tailored for insertion into lipid bilayers. Liposomal formulations of injectable antimicrobial agents and antineoplastic agents already are undergoing clinical testing, and most probably will receive approval for marketing in the early 1990s. Liposomal encapsulation of drugs represents a new drug delivery system that appears to offer important therapeutic advantages over existing methods of drug delivery.

The captured volume of multilamellar vesicles.

Characterization of classical 'hand-shaken' multilamellar lipid vesicles (MLVs) confirmed that these systems exclude solute during formation thus confounding previous captured volume measurements which typically have utilized solute as a merker of the occluded aqueous space. We used solvent rather than solute to determine the captured volume of these systems and obtained values at least twice those previously reported. We present here a captured volume and lamellarity profile of 'hand-shaken' MLVs and suggest that these parameters are dependent on the lipid concentration present during hydration.

Pharmacodynamics and pharmacokinetics of parenteral histamine (H2)-receptor antagonists.

Parenteral histamine (H2)-receptor antagonists are frequently used to prevent upper gastrointestinal bleeding caused by stress-induced gastric mucosal damage in critically ill patients. It is generally agreed that the goal of therapy in this syndrome is the consistent elevation of gastric pH levels above a certain value, often set at 4, in order to prevent the underlying mucosal damage from progressing to bleeding. The three H2-receptor antagonists currently available in a parenteral form and suitable for this mode of prophylaxis are cimetidine, ranitidine, and famotidine. The pharmacodynamic and pharmacokinetic properties of these agents, as they relate to their use in prevention of stress ulceration bleeding, are discussed here. These agents are more noted for their pharmacodynamic and pharmacokinetic similarities in acid suppression, elimination, and metabolism than for their differences. Ranitidine and famotidine are more potent than cimetidine, and famotidine has a slightly longer half-life than do cimetidine and ranitidine, but current dosing recommendations take these differences into account so that the agents have equivalent efficacy. Cimetidine and ranitidine have been widely used in this application. Less experience has been obtained, to date, with famotidine. Recent studies with primed, continuous infusions of cimetidine indicate that dosing schedule may be the key to obtaining better efficacy in prophylaxis of stress-related mucosal damage. Similar studies with ranitidine have not yielded results as promising as those with cimetidine, however, and few data are available on famotidine.

Breath pentane analysis as an index of lipid peroxidation: a functional test of vitamin E status.

Pentane, which evolves from the reaction involving omega-6 fatty acids, is a good index of lipid peroxidation. We describe a method for measuring breath pentane excretion in adult humans. After a 4-minute washout period, expired air was analyzed by gas chromatography. Breath was passed through a cooled loop of alumina to adsorb, concentrate, and release, on heating, pentane. Pentane was analyzed by a Porasil-D column with a derived calibration curve. The mean excretion of pentane in 10 normal adults was 6.34 +/- 0.96 pmol X kg-1 X min-1 (mean +/- SEM) and was significantly higher in five patients with plasma vitamin E deficiency (15.39 +/- 1.84 pmol X kg-1 X min-1). There was a significant negative correlation between pentane output and plasma vitamin E levels (r = -0.66, p less than 0.01). Moreover, breath pentane excretion was significantly decreased after a 10-d supplementation with vitamin E in five normal subjects. We conclude that breath pentane output is a sensitive, noninvasive, functional test for assessing vitamin E status.

Vitamin E and selenium status of patients receiving short-term total parenteral nutrition.

In a prospective protocol, plasma tocopherols, selenium (Se), Se-dependent glutathione peroxidase, platelet aggregation and erythrocyte hemolysis were measured in 23 control subjects, and 15 patients receiving total parenteral nutrition (TPN), before and after 2 wk of TPN unsupplemented with vitamin E and Se. The results indicate that short-term TPN did not alter status of these nutrients. However, TPN patients had significantly lower plasma levels of Se (p less than 0.01) and alpha-tocopherol (p less than 0.01) relative to control subjects. Low plasma levels, with no attendant decrease in function, suggest a marginal depletion. In view of this, and considering the low amount of vitamin E and Se supplied by the TPN solutions, supplementation with these nutrients is recommended.

Selenium and vitamin E stability in parenteral solutions.

Stability of alpha-tocopherol acetate and selenium in amino acid/dextrose solutions with SoluZyme or MVI-1000 vitamin injections was evaluated following exposure to fluorescent lighting and room temperature, and after flowing through an infusion apparatus. The stability of selenium in parenteral solutions for a 10-wk period was also determined. In each condition no significant loss of alpha-tocopherol acetate or selenium was observed. It was concluded that alpha-tocopherol acetate and selenium as selenious acid are stable in parenteral solutions and no significant loss occurs during delivery to patients.

Control of gastric pH with cimetidine: boluses versus primed infusions.

Previous studies suggest that antacids are more effective than intravenous cimetidine in maintaining the gastric pH above 4.0 in acutely ill patients. We hypothesized that this was because blood levels of cimetidine are not sustained at therapeutic levels with the bolus doses. The purpose of this study was to compare gastric pH and serum cimetidine levels when cimetidine was administered as bolus versus infusion. We studied 23 acutely ill patients who received intravenous cimetidine given as boluses and primed infusions. The gastric pH could be maintained above 4.0 with infusions of up to 50 mg/h (1200 mg/day) in 20 patients, compared with only 5 patients with bolus administrations of up to 300 mg every 6 h (1200 mg/day). The differences in ability to maintain the gastric pH above 4.0 were entirely due to the reduced ability of bolus infusion to maintain an adequate serum level. Neither technique could maintain the pH above 4.0 in 3 patients, all of whom had received cimetidine recently. A gastric pH greater than 4.0 correlated directly with a therapeutic serum cimetidine level. We conclude that infusions of cimetidine are better able to sustain therapeutic blood levels and, therefore, are superior to bolus cimetidine in maintaining gastric pH above 4.0. Some patients, however, may not respond to cimetidine even if therapeutic levels are achieved and may require supplemental antacids.

Novel multilayered lipid vesicles: comparison of physical characteristics of multilamellar liposomes and stable plurilamellar vesicles.

The preparation of a new kind of multilayered liposome, called a stable plurilamellar vesicle (SPLV), is described. Although SPLVs and classical multilamellar vesicles (MLVs) are made of the same materials and appear overtly similar in the electron microscope, the two types of vesicles differ as determined by stability, entrapment efficiency, electron spin resonance (ESR), NMR, X-ray diffraction, and biological effects. It is demonstrated that, contrary to what has been assumed, classical MLVs exclude solutes during their formation and, thus, are under a state of osmotic compression. By contrast, the SPLV process produces liposomes that are not compressed. The effects of osmotic compression are discussed. It is suggested that the state of osmotic stress is an important variable that distinguishes various types of liposomes and that has significant physical and biological consequences.

Total parenteral nutrition and complete bowel rest in the management of Crohn's disease.

The role of parenteral nutrition with complete bowel rest in the management of active Crohn's disease was evaluated retrospectively in 100 patients who were otherwise refractory to conventional medical management. Ninety patients received complete nutrient replacement and 10 received protein-sparing therapy. In 77 patients, a clinical remission was achieved. Analysis of subgroups revealed that the remission rate was equivalent in patients with subacute bowel obstruction (76%), inflammatory mass (82%), and otherwise uncomplicated severe active disease (89%). However, those patients with fistulae responded less well (63%). The location of the intestinal involvement with the disease did not influence the remission rate (73% in those with small bowel disease only and 78% in those with combined small and large bowel disease). All six patients with only large bowel involvement achieved a remission. In 81% of those patients with a remission, no corticosteroids were given, or the dose prior to TPN was maintained. The serum albumin improved significantly (p less than 0.001) from 3.2 +/- 0.1 to 3.6 +/- 0.1 g/dl with total parenteral nutrition, but there was no significant effect on the hematocrit (p greater than 0.5). The percentage of patients still in remission after 3 months and 1 yr of follow-up was 75 to 79 and 58 to 61%, respectively, in the three nonfistulous groups, and 46 and 36%, respectively, in those with fistulous disease. Thus total parenteral nutrition with complete bowel rest appears to be an effective therapeutic modality in the primary management of complicated Crohn's disease.

Novel liposome composition for a rapid colorimetric test for systemic lupus erythematosus.

This test for systemic lupus erythematosus utilizes a novel liposome composition entrapping the cation-responsive red dye Arsenazo III. In dilutions of normal sera the liposome membranes undergo a rearrangement when divalent cations are added, resulting in the release of the encapsulated dye and the rapid formation of a stable blue cation-dye complex. Microliter amounts of sera from patients with active lupus stabilize the liposome preparation such that the vesicles remain intact in the presence of the added divalent cations and thus maintain their red color for extended periods. The assay requires 1-min incubations in sera at room temperature and can be performed with standard microtiter plates, allowing the screening of large numbers of serum samples in a short time. Moreover, the unique absorption spectra of the complexed and uncomplexed dye allow for quantification of results.

Differential breakdown of phylogenetically diverse ribosomal RNA's inserted via liposomes into mammalian cells.

Liposomes were used to deliver ribosomal RNA's from the different organisms into cultivated mouse plasmacytoma cells. Ribosomal RNA from Escherichia coli was degraded intracellularly within 1 hour, whereas mouse and yeast ribosomal RNA's were degraded more slowly. This indicates that cells can discriminated between different ribosomal RNA's.