99mTechnetium-stannous oxinate as marker of liposome formulations.

Liposomes associated with tin(II) dioxinate were prepared from egg yolk phosphatidylcholine and cholesterol as sterile and pyrogen-free multilamellar or unilamellar vesicles. Complexing of liposomal tin(II) dioxinate with 99mTc attained 98% of the added radioactivity. Thirty percent 99mTc were released during 24-h incubation in biological fluids. The absence of tin colloids seen by electron microscopy and the stability of liposomal phospholipid and tin(II) dioxinate during 72-h incubation at 37 degrees C in plasma and cerebrospinal fluid would allow safe and reliable scintigraphic liposome pharmacokinetic studies.

Liposome-incorporated dexamethasone palmitate inhibits in-vitro lymphocyte response to mitogen.

The use of liposomes for the pulmonary delivery of corticosteroid is an area that is under active investigation. We have recently developed a novel liposomal corticosteroid preparation based on the incorporation of dexamethasone palmitate (DMP) within the bilayer of small unilamellar vesicles (SUVs) made of egg yolk phosphatidylcholine (EPC) and cholesterol; molar ratio EPCC:cholesterol: DMP, 4:3:0.3. In the present study, the biological activity of DMP-SUVs was evaluated using the lymphocyte transformation test with peripheral blood mononuclear cells (PBMCs) and a gamma-interferon production assay. Results showed that DMP-SUVs (but not empty SUVs) inhibited [3H]thymidine uptake and gamma-interferon production by concanavalin A-stimulated PBMCs by 94 and 96%, respectively, at a concentration corresponding to 10(-6) M dexamethasone. The inhibition by DMP-SUVs was found to require a 24-h pre-incubation with unstimulated PBMCs, suggesting that interaction of SUVs with lymphocytes may be altered by mitogen stimulation. We conclude that our DMP liposomal preparation is biologically active and may be considered a promising alternative to conventional local glucocorticoid therapy.

Epidural administration of liposome-associated bupivacaine for the management of postsurgical pain: a first study.

STUDY OBJECTIVES: To explore the influence of liposomes on the pharmacodynamic action of bupivacaine and to determine whether postsurgical analgesic advantages can be obtained from epidural delivery of liposomal bupivacaine compared with the current formulation. DESIGN: Open, nonrandomized study. SETTING: Physiopathology laboratory, general operating theaters, and intensive care units of Reine Fabiola Hospital and Institut Médical de Traumatologie et Revalidation. PATIENTS: 26 ASA physical status II and III patients who had undergone major surgery (abdominal, vascular, urologic, thoracic, orthopedic). INTERVENTIONS: After completion of the operation, the patients were divided into 2 groups to receive 1 of 2 bupivacaine preparations epidurally for postsurgical pain: Group 1 (n = 12) received plain 0.5% bupivacaine with 1:200,000 epinephrine; Group 2 (n = 14) received liposomal 0.5% bupivacaine. MEASUREMENTS AND MAIN RESULTS: The following observations were made: onset and quality of analgesia, quality of motor block according to the Bromage scale, and sympathetic block. Onset time of sensory block averaged 15 minutes in both groups. Pain relief durations were 3.2 +/- 0.4 hours with plain bupivacaine and 6.25 +/- 1.13 hours with the liposomal preparation (p < 0.05). In the liposomal bupivacaine group, no motor block was recorded. Low sympathetic block occurred in all patients. Analgesia in a subset of patients following abdominal aortic surgery increased from 2.4 +/- 0.35 hours to 10.6 +/- 1.4 hours by encapsulation of bupivacaine (p < 0.01). There was no neurotoxicity or cardiotoxicity. CONCLUSIONS: The liposomal formulation of bupivacaine increased duration of analgesia without motor block or adverse side effects.

Motor blockade and absence of local nerve toxicity induced by liposomal bupivacaine injected into the brachial plexus of rabbits.

Bupivacaine has been encapsulated into multilameller liposomes in order to reduce its systemic toxicity and to lengthen its action. Low, constant and sustained plasma levels have been observed after epidural injection and brachial plexus administration to rabbits. The present experiment was performed in order to study the motor block effect and the possible neurotoxicity of bupivacaine encapsulated in multilamellar liposomes applied in vivo to intact peripheral nerve bundles. Five milliliters of sterile solutions containing 0.75% bupivacaine encapsulated in 42.5 and 85 mg of lipids (phosphatidylcholine and cholesterol in a molar ratio 4:3) were administered to 4 rabbits at the level of the brachial plexus. A motor blockade of the forelimb lasting for 10 hours was measured immediately after injection of the liposomal drug. Light microscopic analyses of the nerves after 2 and 7 days revealed weak inflammatory perineural infiltration. Electron microscopy showed no changes of the myelin sheaths and no alteration of unmyelinated fibers. It may be concluded that the pharmacologically active liposomal formulation of bupivacaine did not induce alterations of the nerve tissues.

Use of liposome-associated bupivacaine in a cancer pain syndrome.

Bupivacaine 0.25% encapsulated by multilamellar liposomes was administered epidurally to a patient suffering pain associated with lung cancer and the effect compared with a plain bupivacaine solution of the same concentration. Complete analgesia was produced for 4 h with the plain solution and 11 h with the liposomal formulation. No motor blockade or haemodynamic instability was observed with the liposome-associated bupivacaine.

Plasma concentrations of bupivacaine after brachial plexus administration of liposome-associated and plain solutions to rabbits.

Bupivacaine has been associated to multilamellar liposomes with the aim of altering circulating plasma concentrations after injection into the rabbit brachial plexus. Plasma concentrations of bupivacaine have been compared after administration of free drug (BP) or bupivacaine associated to multilamellar liposomes (BP-MLV) made of phosphatidylcholine and cholesterol (molar ratio 4:3). Under light general anaesthesia, one group of six rabbits received an axillary injection of 2.5 mg BP (1 ml, 0.25%), and a second received the same dose of BP-MLV. In both groups 3H bupivacaine was used as a marker. The brachial plexus was located using a nerve stimulator. Injection of the anaesthetic solutions invariably prevented the motor response of the paw. The arterial plasma concentrations of bupivacaine were determined after 5 to 240 min and after 24 hr by beta counting. In the MLV population, additional measurements were performed after 48 and 72 hr. The two plasma curves showed a plateau (0.2 microgram.ml-1) which was reached after five minutes in the BP group and after 90 min using BP-MLV. In the BP-MLV group, the plasma concentrations of bupivacaine were lower during the first ten minutes (P < 0.05), and higher after 24 hr (P < 0.05). Radioactivity decreased between 4 and 24 hr in the BP group and between one and two days in the BP-MLV population. It is concluded that elevated plasma drug concentrations were maintained for longer with BP-MLV than with BP. This could prolong the action of the local anaesthetic through a slow release.

Biodistribution of liposome-associated bupivacaine after extradural administration to rabbits.

After one extradural injection of 0.25% bupivacaine 0.3 ml and 3H-bupivacaine 0.005 mCi in multilamellar liposomes, no systemic radioactivity (plasma, liver, heart muscle) was obtained for 1 h, and the labelling was less than that of systemic distribution of plain bupivacaine for the following 3 h. In contrast, radioactivity in the lumbar spinal nerves peaked in the first hour and remained higher than that of plain bupivacaine for 4 h. No radioactivity was measured in cerebrospinal fluid. Small unilamellar vesicles incorporating 3H-cholesterol did not significantly label spinal nerves and central nervous structures indicating that the mode of action of liposomal bupivacaine did not involve uptake by nerve structures. Rapid uptake of radioactivity by spinal nerves suggested exchange of bupivacaine between liposomes and nerve sheaths.

Toxicity of bupivacaine encapsulated into liposomes and injected intravenously: comparison with plain solutions.

The acute central nervous system and cardiac toxicities of 0.25% bupivacaine, without adrenalin, encapsulated in multilamellar liposomes were compared with 0.25% plain solutions with and without adrenalin after intravenous infusion at a rate of 0.15 mg.kg-1 x min-1 with an increase of 0.036 mg.kg-1 x min-1 every 10 min. Three groups of six anesthetized, unventilated rabbits were studied. The doses of bupivacaine (in mg.kg-1) which produced seizure, ventricular tachycardia, and asystole were determined. The doses of bupivacaine inducing seizure and ventricular tachycardia were significantly higher for liposomal bupivacaine than for the two plain solutions. A statistical comparison of the cumulative lethal doses of bupivacaine 0.25% with adrenalin and of liposomal bupivacaine led to a P = 0.06. Adrenalin did not modify the systemic toxicity of the local anesthetic. This study showed a reduction of nervous and cardiac toxicity of bupivacaine encapsulated in multilamellar liposomes when infused intravascularly.

Biodistribution of liposomes after extradural administration in rodents.

We have mapped over 24 h the biodistribution of 99mTc-labelled multilamellar and small unilamellar liposomes in rabbits and rats by scintigraphic imaging after extradural injection. Multilamellar vesicles formed a depot at the site of injection; small unilamellar vesicles spread immediately along the extradural space and entered the systemic compartment 30 min after injection. Well-delineated liver and kidney labellings were seen after 24 h. The use of 3H-cholesterol-labelled small unilamellar vesicles suggested hepatic capture of intact liposomes with sizes averaging 0.05 microns drained unmodified into the systemic circulation through the extradural lymphatics. These results have led to the selection of multilamellar vesicles (0.1-15 microns size range) for clinical trials using liposome-associated local anaesthetics.

Neurotoxicological assessment after intracisternal injection of liposomal bupivacaine in rabbits.

UNLABELLED: Experiments were performed on rabbits randomly assigned to intracisternally receive 0.3 mL of plain bupivacaine 5 mg/mL, liposomal bupivacaine 5 mg/mL, bupivacaine-free liposomes, or isotonic phosphate-buffered saline. Mechanical ventilation was initiated or intravenous dopamine was infused when respiratory depression or hypotension occurred. Seven days after the injection, the whole spinal cord was removed and histopathologic characteristics were studied on transverse sections. All preparations were devoid of phosphatidylcholine hydrolysis or oxidation compounds. Solutions without bupivacaine produced transient irritative signs that required sedation in most rabbits. Despite the similar duration of respiratory depression in groups receiving liposomal or plain bupivacaine, liposomes produced significantly prolonged motor blockade (126 vs 70 min). Correction of hypotension after plain bupivacaine required a longer dopamine infusion and larger doses than after liposomal bupivacaine (28 vs 18 min and 74 vs 47 mg). Necrosis was observed in the cervical area of two rabbits (one in the liposomal bupivacaine group and another in the phosphate buffer group). No demyelinated areas were noted in spinal cord examinations. We conclude that liposomal bupivacaine leads to a less severe sympathetic block and to a prolonged motor block, whereas histologic changes are not significantly different among groups. IMPLICATIONS: Multilamellar liposomes containing bupivacaine administered intracisternally to rabbits produce spinal cord histopathologic changes not significantly different from those observed with plain bupivacaine. Sustained release of bupivacaine from liposomes is suggested by the prolonged motor blockade and the reduced severity of arterial hypotension. Use of these liposomes could prolong the local anesthetic effects of bupivacaine.

Scintigraphic visualization of intrathecal liposome biodistribution.

BACKGROUND: Liposomes containing local anaesthetics have been administered intrathecally and in the epidural space. Poor attention has been given to the pharmacokinetics of liposomes as drug carriers. Therefore, we observed the biodistribution of liposomes after intrathecal injection in rats by scintigraphic imaging during 24 h. METHODS: We administered 99mTc-labeled multilamellar (MLV) and small unilamellar vesicles (SUV) of defined size and volume dispersities into the cerebrospinal fluid at the lumbar level. Those vesicles were free of contamination by radiolabeled colloids as visualized by light and electron microscopy and of neurotoxic products from phosphatidylcholine hydrolysis and peroxidation, both during the preparation process and after 24 h incubation in cerebrospinal fluid at 37 degrees C in vitro. RESULTS: SUV immediately diffused from the lumbar site of injection to the head and were cleared between 1 and 24 h after injection. MLV were cleared more slowly from the spinal space and appeared in the head region 1 h after injection where they accumulated up to 24 h. These differences were explained in terms of vesicle sizes and volumes. SUV with 0.05 micron diameters were rapidly absorbed into the blood through the arachnoid granulations. In contrast, particles larger than the upper size limit of the arachnoid granulations permeability (+/- 8 microns) could accumulate in the head with a slow elimination rate. CONCLUSION: This difference in clearance from the intrathecal space outlines the importance of defining the size of the liposomes, the distribution of a tracer or a drug inside the liposomal preparation, the chemical stability and the absence of toxic degradation products of liposome formulations before clinical use.

Disposition of liposomal gentamicin following intrabronchial administration in rabbits.

Use of liposomes as carriers of gentamicin for intrabronchial pulmonary delivery was investigated in rabbits. Gentamicin, in isotonic glutamic acid buffer, pH 4.5, was encapsulated in multilamellar vesicles (MLVs) and administered intrabronchially. Higher drug concentrations were found at the pulmonary site of liposome instillation for 1 day as compared with free unencapsulated antibiotic. When time-course distributions of gentamicin given in the liposomal or free form were measured in bronchoalveolar lavages (BAL), similar accumulations were observed up to 4 h, but the drug remained longer (24 h) after administration of the liposomal formulation. Higher amounts of antibiotic were detected in BAL supernatant 1 h after instillation of plain gentamicin; this difference stopped being significant after 4 h. A microbiological assay outlined the bacteriostatic activity of gentamicin released from MLVs and recovered in BAL supernatant. Liposomal gentamicin accumulated in the BAL cell pellet 1 h after intrabronchial instillation; it decreased progressively but minute amounts were still detected after 1 day. On the contrary, no gentamicin was found in the pellet at any time after free drug administration. Comparison of aminoglycoside concentrations in plasma and kidneys indicated lower and constant levels when the liposomal form was instilled. Liposome encapsulation altered the disposition of gentamicin in a way suggesting improved pulmonary concentration and lower systemic toxicity.