Deposition of Aerosolized Lucinactant in Nonhuman Primates.

Background: Lucinactant for inhalation is an investigational noninvasive, aerosolized surfactant replacement therapy for treatment of preterm neonates with respiratory distress syndrome. Lucinactant for inhalation consists of lyophilized lucinactant and the Aerosurf® Delivery System (ADS). The objective of this study was to characterize the total and regional pulmonary deposition of lucinactant delivered by the ADS in nonhuman primates (NHPs). Methods: Lucinactant was radiolabeled by the addition of technetium-99m (99mTc)-sulfur colloid. The radiolabeled aerosol was characterized and validated using a Mercer cascade impactor. An in vivo deposition study was performed in three cynomolgus macaques. Radiolabeled lucinactant was aerosolized using the ADS and delivered via nasal cannula under 5 cm H2O nasal continuous positive airway pressure (nCPAP) for 5-9 minutes. A two-dimensional planar image was acquired immediately after aerosol administration, followed by a three-dimensional single-photon emission computed tomography (SPECT) image and a second planar image. The images were analyzed to determine the pulmonary (lungs) and extrapulmonary (nose + mouth, trachea, stomach) distribution. The SPECT data were used to determine regional deposition. Results: The radiolabed lucinactant aerosol had a mass median aerodynamic diameter = 2.91 µm, geometric standard deviation (GSD) = 1.81, and an activity median aerodynamic diameter = 2.92 µm, GSD = 2.06. Aerosolized lucinactant was observed to deposit in the lungs (11.4%), nose + mouth (79.9%), trachea (7.3%), and stomach (1.4%). Analysis of the SPECT image demonstrated that the regional deposition within the lung was generally homogeneous. Aerosolized lucinactant was deposited in both the central (52.8% ± 1.2%) and peripheral (47.2% ± 1.2%) regions of the lungs. Conclusion: Aerosolized lucinactant, delivered using the ADS via constant flow nCPAP, is deposited in all regions of the lungs demonstrating that surfactant can be aerosolized and delivered noninvasively to NHPs.

Molecular imaging of brain localization of liposomes in mice using MALDI mass spectrometry.

Phospholipids have excellent biocompatibility and are therefore often used as main components of liposomal drug carriers. In traditional bioanalytics, the in-vivo distribution of liposomal drug carriers is assessed using radiolabeled liposomal constituents. This study presents matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) as an alternative, label-free method for ex-vivo molecular imaging of liposomal drug carriers in mouse tissue. To this end, indocyanine green as cargo and two liposomal markers, 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine conjugated with monodisperse polyethylene glycol (PEG36-DSPE) were incorporated into liposomal carriers and administered to mice. We used MALDI MSI of the two lipid markers in both positive and negative ion mode for visualization of liposome integrity and distribution in mouse organs. Additional MSI of hemoglobin in the same tissue slice and pixel-by-pixel computational analysis of co-occurrence of lipid markers and hemoglobin served as indicator of liposome localization either in parenchyma or in blood vessels. Our proof-of-concept study suggests that liposomal components and indocyanine green distributed into all investigated organs.

Nanodiscs as a therapeutic delivery agent: inhibition of respiratory syncytial virus infection in the lung.

There is increasing interest in the application of nanotechnology to solve the difficult problem of therapeutic administration of pharmaceuticals. Nanodiscs, composed of a stable discoidal lipid bilayer encircled by an amphipathic membrane scaffold protein that is an engineered variant of the human Apo A-I constituent of high-density lipoproteins, have been a successful platform for providing a controlled lipid composition in particles that are especially useful for investigating membrane protein structure and function. In this communication, we demonstrate that nanodiscs are effective in suppressing respiratory syncytial viral (RSV) infection both in vitro and in vivo when self-assembled with the minor pulmonary surfactant phospholipid palmitoyloleoylphosphatidylglycerol (POPG). Preparations of nanodiscs containing POPG (nPOPG) antagonized interleukin-8 production from Beas2B epithelial cells challenged by RSV infection, with an IC50 of 19.3 µg/mL. In quantitative in vitro plaque assays, nPOPG reduced RSV infection by 93%. In vivo, nPOPG suppressed inflammatory cell infiltration into the lung, as well as IFN-γ production in response to RSV challenge. nPOPG also completely suppressed the histopathological changes in lung tissue elicited by RSV and reduced the amount of virus recovered from lung tissue by 96%. The turnover rate of nPOPG was estimated to have a halftime of 60-120 minutes (m), based upon quantification of the recovery of the human Apo A-I constituent. From these data, we conclude that nPOPG is a potent antagonist of RSV infection and its inflammatory sequelae both in vitro and in vivo.

Lucinactant for the prevention of respiratory distress syndrome in premature infants.

Respiratory distress syndrome (RDS) is the leading cause of neonatal morbidity and mortality in premature infants. It is caused by surfactant deficiency and lung immaturity. Lucinactant is a synthetic surfactant containing sinapultide, a bioengineered peptide mimic of surfactant-associated protein B. A meta-analysis of clinical trials demonstrates that lucinactant is as effective as animal-derived surfactants in preventing RDS in premature neonates, and in vitro studies suggest it is more resistant to oxidative and protein-induced inactivation. Its synthetic origin confers lower infection and inflammation risks as well other potential benefits, which may make lucinactant an advantageous alternative to its animal-derived counterparts, which are presently the standard treatment for RDS.

Pharmacokinetics of temoporfin-loaded liposome formulations: correlation of liposome and temoporfin blood concentration.

Liposomal formulations of the highly hydrophobic photosensitizer temoporfin were developed in order to overcome solubility-related problems associated with the current therapy scheme. We have incorporated temoporfin into liposomes of varying membrane composition, cholesterol content, and vesicle size. Specifically, two phosphatidyl oligoglycerols were compared to PEG2000-DSPE with respect to the ability to prolong circulation half life of the liposomal carrier. We measured the resulting pharmacokinetic profile of the liposomal carrier and the incorporated temoporfin in a rat model employing a radioactive lipid label and (14)C-temoporfin. The data for the removal of liposomes and temoporfin were analyzed in terms of classical pharmacokinetic theory assuming a two-compartment model. This model, however, does not allow in a straightforward manner to distinguish between temoporfin eliminated together with the liposomal carrier and temoporfin that is first transferred to other blood components (e. g. plasma proteins) before being eliminated from the blood. We therefore additionally analyzed the data based on two separate one-compartment models for the liposomes and temoporfin. The model yields the ratio of the rate constant of temoporfin elimination together with the liposomal carrier and the rate constant of temoporfin elimination following the transfer to e. g. plasma proteins. Our analysis using this model demonstrates that a fraction of temoporfin is released from the liposomes prior to being eliminated from the blood. In case of unmodified liposomes this temoporfin release was observed to increase with decreasing bilayer fluidity, indicating an accelerated temoporfin transfer from gel-phase liposomes to e. g. plasma proteins. Interestingly, liposomes carrying either one of the three investigated surface-modifying agents did not adhere to the tendencies observed for unmodified liposomes. Although surface-modified liposomes exhibited improved pharmacokinetic properties with regard to the liposomal carrier, an enhanced temoporfin loss and elimination from the PEGylated-liposomes was observed. This effect was more pronounced for PEGylated liposomes than for the two oligo-glycerols. Our combined experimental-theoretical approach for in vivo plasma re-distribution of liposomal drugs may help to optimize colloidal drug carrier systems.

In vitro and in vivo entrapment of bupivacaine by lipid dispersions.

Intravenous lipid emulsion is recommended as treatment for local anesthetic intoxication based on the hypothesis that the lipophilic drug is entrapped by the lipid phase created in plasma. We compared a 15.6 mM 80/20 mol% phosphatidyl choline (PC)/phosphatidyl glycerol (PG)-based liposome dispersion with the commercially available Intralipid® emulsion in a pig model of local anesthetic intoxication. Bupivacaine-lipid interactions were studied by electrokinetic capillary chromatography. Multilamellar vesicles were used in the first in vivo experiment series. This series was interrupted when the liposome dispersion was discovered to cause cardiovascular collapse. The toxicity was decreased by an optimized sonication of the 50% diluted liposome dispersion (7.8 mM). Twenty anesthetized pigs were then infused with either sonicated PC/PG liposome dispersion or Intralipid®, following infusion of a toxic dose of bupivacaine which decreased the mean arterial pressure by 50% from baseline. Bupivacaine concentrations were quantified in blood samples using liquid chromatography/mass spectrometry. No significant difference in the context-sensitive plasma half-life of bupivacaine was detected (p=0.932). After 30 min of lipid infusion, the bupivacaine concentration was 8.2±1.5 mg/L in the PC/PG group and 7.8±1.8 mg/L in the Intralipid® group, with no difference between groups (p=0.591). No difference in hemodynamic recovery was detected between groups (p > 0.05).

Effects of a novel ultrasound contrast agent with long persistence on right ventricular pressure: Comparison with SonoVue.

This work investigated the effect of infusion of a self-made ultrasound contrast agent with long persistence (named ZHIFUXIAN) on rat right ventricular pressure and made a preliminary evaluation on the relative safety of the novel microbubbles. Normal saline, SonoVue and ZHIFUXIAN were injected through caudal vein at the total volume of 0.5ml for each injection. The right ventricular systolic pressure (RVSP) and end-diastolic pressure (RVEDP) were monitored and the changes of the pressure were compared with baseline readings. RVSP increased when saline, SonoVue or ZHIFUXIAN were injected, the greatest change being after SonoVue (about 2mmHg), but there was no statistical significance compared with baseline (P>0.05). There was no significant difference in RVSP between saline, SonoVue and ZHIFUXIAN at any time point. Also, there was no significant difference in RVEDP between groups at each time point and between different time points in each group. The results indicate that the self-made microbubbles effect on right ventricular hemodynamics is equivalent to that of normal saline at the same volume needed for effective enhanced imaging, demonstrating that it does not produce changes in right ventricular blood pressure under the study conditions. Pathological examination also showed it had no obvious influence on lung, liver and kidney.

Amphotericin B lipid preparations: what are the differences?

To reduce the in-vivo toxicity of the broad-spectrum antifungal drug amphotericin B, various lipid formulations of amphotericin B, ranging from lipid complexes to small unilamellar liposomes, have been developed and subsequently commercialized. These structurally diverse formulations differ in their serum pharmacokinetics as well as their tissue localisation, tissue retention and toxicity. These differences can affect the choice of formulation for a given infection, the time of initiation of treatment, and the dosing regimen. Although preclinical studies have shown similarities in the in-vitro and in-vivo antifungal activity of the formulations with comparable dosing, their acute and chronic toxicity profiles are not the same, and this has a significant impact on their therapeutic indices, especially in high-risk, immunosuppressed patients. With the recent introduction of new antifungal drugs to treat the increasing numbers of infected patients, the amphotericin B lipid formulations are now being studied to evaluate their potential in combination drug regimens. With proven efficacy demonstrated during the past decade, it is expected that amphotericin B lipid formulations will remain an important part of antifungal drug therapy.

Penetration of amphotericin B lipid formulations into pleural effusion.

The penetration of the amphotericin B (AMB) lipid formulations (liposomal AMB, AMB colloidal dispersion, and AMB lipid complex formulations) into pleural effusions in seven critically ill patients was assessed. AMB was detected in all pleural effusion samples at concentrations ranging from 0.02 to 0.43 microg/ml. The penetration ratio was 3 to 44%.

Efficacy of amphotericin B lipid complex in a rabbit model of coccidioidal meningitis.

OBJECTIVES: We compared the efficacy of treatments in a rabbit model of coccidioidal meningitis (CM). METHODS: Rabbits were infected intracisternally with Coccidioides immitis and treated with intravenous amphotericin B lipid complex (ABLC), deoxycholate amphotericin B (dAMB), oral fluconazole or diluent [sterile 5% dextrose in water (D5W)]. Survival and cfu in brain, spinal cord and CSF were determined and histology studied. Amphotericin B (AMB) concentrations in serum, CSF and tissue were determined by bioassay. RESULTS: Fluconazole-treated rabbits and controls lost weight and had decreased mobility. All treatments prolonged survival (P = 0.005) and reduced cfu in brain and spinal cord (P

Assessing the antifungal activity, pharmacokinetics, and tissue distribution of amphotericin B following the administration of Abelcet and AmBisome in combination with caspofungin to rats infected with Aspergillus fumigatus.

The purpose of this study was to assess the antifungal activity, pharmacokinetics, and tissue distribution of amphotericin B (AmpB) following the administration of Abelcet and AmBisome alone and in combination with Caspofungin to rats infected with Aspergillus fumigatus. Aspergillus fumigatus inoculum (2.1-2.5 x 10(7) colony forming units [CFU]) was injected via the jugular vein; 48 h later male albino Sprague-Dawley rats (350-400 g) were administered either a single intravenous (i.v.) dose of Abelcet (5 mg AmpB/kg; n = 6), AmBisome (5 mg AmpB/kg; n = 6), Caspofungin (3 mg/kg; n = 5), Abelcet (5 mg AmpB/kg) plus Caspofungin (3 mg/kg) (n = 6), AmBisome (5 mg AmpB/kg) plus Caspofungin (3 mg/kg) (n = 7), or physiologic saline (non-treated controls; n = 6) once daily for 4 days. Antifungal activity was assessed by organ CFU concentrations and plasma galactomannan levels. Plasma and tissue samples were taken from each animal for AmpB pharmacokinetic analysis and tissue distribution determinations. Abelcet treatment significantly decreased total fungal CFU concentrations recovered in all the organs added together by 73% compared to non-treated controls. Ambisome treatment significantly decreased total fungal CFU concentrations recovered in all the organs added together by 69% compared to non-treated controls. Caspofungin treatment significantly decreased total fungal CFU concentrations recovered in all the organs added together by 80% compared to non-treated controls. Abelcet plus Caspofungin treatment significantly decreased total fungal CFU concentrations recovered in all the organs added together by 81% compared to non-treated controls. Ambisome plus Caspofungin treatment significantly decreased total fungal CFU concentrations recovered in all the organs added together by 98% compared to non-treated controls. Abelcet treatment significantly decreased plasma galactomannan levels by 50 and 75% 96 h following the initiation of treatment in the absence and presence of Caspofungin co-therapy, respectively. AmBisome treatment significantly decreased plasma galactomannan levels by 73 and 78% 96 h following the initiation of treatment in the absence and presence of Caspofungin co-therapy, respectively. Co-administration of Caspofungin with Abelcet and AmBisome did not significantly alter the plasma concentration-time profile, pharmacokinetic parameters, and tissue distribution of AmpB. Taken together, our findings suggest that an alternative mechanism, possibly at the cellular level rather than altered AmpB disposition, may be an explanation for the differences in organ CFU concentrations following Abelcet plus Caspofungin versus AmBisome plus Caspofungin administration.

Aerosol deposition of lipid complex amphotericin-B (Abelcet) in lung transplant recipients.

Lung transplant recipients exhibit a high incidence of invasive aspergillosis. The inhalation of lipid complex amphotericin-B (Abelcet; ABLC) offers a possible prophylactic strategy. The goals of this study were to select the optimal nebulizer delivery system for ABLC and to measure deposited aerosol dose in 12 lung transplant recipients. In vitro testing was performed to select a nebulizer delivery system, and an empirical model was used to estimate lung deposition. Estimated pulmonary doses varied by as much as 2-fold between different nebulizers. Aerosol deposition testing was performed in six single and six double lung recipients, each of whom received one 7 mL (35 mg) nebulized dose of Technetium-labeled ABLC using the selected nebulizer. In single lung recipients, the average deposited doses were 3.9 +/- 1.6 mg (mean +/- S.D.) in the allograft versus 2.1 +/- 1.1 mg in the native lung. Double lung recipients deposited on average 2.8 +/- 0.8 mg (left lung) and 4.0 +/- 1.3 mg (right lung). The drug was well distributed throughout the lungs, but delivery to the native lung was in some cases suboptimal. These studies provide an important precursor to studies of the efficacy of inhaled ABLC as a prophylaxis of invasive aspergillosis after lung transplant.

Compartmentalized intrapulmonary pharmacokinetics of amphotericin B and its lipid formulations.

We investigated the compartmentalized intrapulmonary pharmacokinetics of amphotericin B and its lipid formulations in healthy rabbits. Cohorts of three to seven noninfected, catheterized rabbits received 1 mg of amphotericin B deoxycholate (DAMB) per kg of body weight or 5 mg of either amphotericin B colloidal dispersion (ABCD), amphotericin B lipid complex (ABLC), or liposomal amphotericin B (LAMB) per kg once daily for a total of 8 days. Following sparse serial plasma sampling, rabbits were sacrificed 24 h after the last dose, and epithelial lining fluid (ELF), pulmonary alveolar macrophages (PAM), and lung tissue were obtained. Pharmacokinetic parameters in plasma were derived by model-independent techniques, and concentrations in ELF and PAM were calculated based on the urea dilution method and macrophage cell volume, respectively. Mean amphotericin B concentrations +/- standard deviations (SD) in lung tissue and PAM were highest in ABLC-treated animals, exceeding concurrent plasma levels by 70- and 375-fold, respectively (in lung tissue, 16.24 +/- 1.62 versus 2.71 +/- 1.22, 6.29 +/- 1.17, and 6.32 +/- 0.57 microg/g for DAMB-, ABCD-, and LAMB-treated animals, respectively [P = 0.0029]; in PAM, 89.1 +/- 37.0 versus 8.92 +/- 2.89, 5.43 +/- 1.75, and 7.52 +/- 2.50 mug/ml for DAMB-, ABCD-, and LAMB-treated animals, respectively [P = 0.0246]). By comparison, drug concentrations in ELF were much lower than those achieved in lung tissue and PAM. Among the different cohorts, the highest ELF concentrations were found in LAMB-treated animals (2.28 +/- 1.43 versus 0.44 +/- 0.13, 0.68 +/- 0.27, and 0.90 +/- 0.28 microg/ml in DAMB-, ABCD-, and ABLC-treated animals, respectively [P = 0.0070]). In conclusion, amphotericin B and its lipid formulations displayed strikingly different patterns of disposition in lungs 24 h after dosing. Whereas the disposition of ABCD was overall not fundamentally different from that of DAMB, ABLC showed prominent accumulation in lung tissue and PAM, while LAMB achieved the highest concentrations in ELF.

The experience is CLEAR.

Conventional amphotericin B has been the 'gold standard' for antifungal efficacy, but nephrotoxicity problems limit its clinical utility. In the late 1990s, three lipid-based formulations of amphotericin B were introduced, all of which offered comparable efficacy and reduced renal complications. However, to date, robust safety and comparative efficacy data have been sparse. This paper briefly reviews the available clinical data on amphotericin B lipid complex (ABLC). Also, in detail, it reviews the findings of Collaborative Exchange of Antifungal Research (CLEAR), the most extensive dataset on systemic antifungal treatment with the lipid-based agent ABLC.

Population pharmacokinetics of amphotericin B lipid complex in neonates.

The pharmacokinetics of amphotericin B lipid complex (ABLC) were investigated in neonates with invasive candidiasis enrolled in a phase II multicenter trial. Sparse blood (153 samples; 1 to 9 per patient, 1 to 254 h after the dose) and random urine and cerebrospinal fluid (CSF) samples of 28 neonates (median weight [WT], 1.06 kg; range, 0.48 to 4.9 kg; median gestational age, 27 weeks; range, 24 to 41 weeks) were analyzed. Patients received intravenous ABLC at 2.5 (n = 15) or 5 (n = 13) mg/kg of body weight once a day over 1 or 2 h, respectively, for a median of 21 days (range, 4 to 47 days). Concentrations of amphotericin B were quantified as total drug by high-performance liquid chromatography. Blood data for time after dose (TAD) of <24 h fitted best to a one-compartment model with an additive-error model for residual variability, WT0.75 (where 0.75 is an exponent) as a covariate of clearance (CL), and WT as a covariate of volume of distribution (V). Prior amphotericin B, postnatal age, and gestational age did not further improve the model. The final model equations were CL (liters/h) = 0.399 x WT(0.75) (interindividual variability, 35%) and V (liters) = 10.5 x WT (interindividual variability, 43%). Noncompartmental analysis of pooled data with a TAD of >24 h revealed a terminal half-life of 395 h. Mean concentrations in the urine after 1, 2, and 3 weeks ranged from 0.082 to 0.430 microg/ml, and those in CSF ranged from undetectable to 0.074 microg/ml. The disposition of ABLC in neonates was similar to that observed in other age groups: weight was the only factor that influenced clearance. Based on these results and previously published safety and efficacy data, we recommend a daily dosage between 2.5 and 5.0 mg/kg for treatment of invasive Candida infections in neonates.

Amphotericin B lipid complex: in visceral leishmaniasis.

Amphotericin B lipid complex is a lipid formulation of amphotericin B, an antifungal drug with activity against Leishmania spp. Amphotericin B lipid complex appears to enhance uptake of amphotericin B by infected macrophages in patients with visceral leishmaniasis (VL). In randomised, open-label, dose-ranging studies, short-course treatment with once-daily amphotericin B lipid complex (5-15 mg/kg total cumulative dose over 5 days), administered by intravenous infusion, produced high rates of apparent (day 19) [93-100%] and definitive (6 months) [79-100%] cures in Indian patients with antimonial-resistant VL. Amphotericin B lipid complex appeared to be as effective as liposomal amphotericin B or the conventional deoxycholate formulation in a randomised, open-label study conducted in India in a mixed population of patients with previously untreated or antimonial-resistant VL. In patients with HIV infection and VL, amphotericin B lipid complex 3 mg/kg/day for 5 or 10 days appeared to be as effective as meglumine antimonate 20 mg/kg/day for 28 days in a small randomised pilot study in southern Europe. Amphotericin B lipid complex was generally well tolerated in patients with VL. Infusion-related reactions were the most common adverse events associated with amphotericin B lipid complex.

Corneal concentrations following systemic administration of amphotericin B and its lipid preparations in a rabbit model.

BACKGROUND: Amphotericin B (AmB) and various lipid formulations of AmB are available for the treatment of fungal infections of the eye. Currently, the recommended route of administration for the treatment of fungal keratitis is by topical application. Nevertheless, because of the risk of a difficult to treat exogenous fungal endophthalmitis, a combined topical and systemic treatment is frequently given when treating deep fungal keratitis. To date, little is known about the pure corneal availability of these drugs following systemic treatment. In this study, the corneal concentration following 7 daily doses of parenteral AmB lipid complex (ABLC) or liposomal AmB (L-AmB) was compared to that of AmB deoxycholate (D-AmB) in a rabbit model. METHODS: Following induction of uveitis in one rabbit eye by intravitreal injection of endotoxin, daily doses of D-AmB (1 mg/kg), ABLC (5 mg/kg) or L-AmB (5 mg/kg) were given intravenously on 7 consecutive days. Five or more rabbits per treatment were used. AmB concentrations were determined by high-performance liquid chromatography (HPLC) in corneas collected at autopsy 24 h after the 7th and final dose. Data were analyzed using the Mann-Whitney U test. RESULTS: After 7 days of treatment, mean corneal concentrations of AmB in the inflamed eyes were significantly higher (2.38 +/- 1.47 microg/g; p < 0.01) following treatment with L-AmB compared with ABLC (<0.1 microg/g) and D-AmB (0.46 +/- 0.2 microg/g). No AmB could be detected in the corneas of the non-inflamed eyes. CONCLUSION: In our rabbit model, AmB penetration into the cornea was significantly higher after systemic administration of L-AmB compared with conventional D-AmB or ABLC.

Adsorption of surfactant to bronchial epithelium: possible role of receptor 'unmasking' in asthma.

BACKGROUND: In a recent study in animals it has been shown how surface-active phospholipid (SAPL) in the form of a commercially available micronized (5 micromphi) dry powder (ALECT/PumactantT) was able to reduce afferent neural feedback to the brainstem in response to a methacholine challenge by the same order of magnitude as drugs commonly prescribed for asthma. The underlying theory assumed that adsorption of SAPL to bronchial epithelium masked irritant receptors eliciting the bronchoconstrictor reflex, thus providing a barrier to noxious stimuli entering the lungs. OBJECTIVE: To test the underlying assumption that SAPL was actually adsorbed (i.e., bound to bronchial epithelium), especially the major and most surface-active component of lung surfactant, namely dipalmitoyl phosphatidylcholine (DPPC). A secondary objective was to investigate any role of phosphatidylglycerol (PG) in promoting the adsorption of DPPC. METHODS: Radiolabeled DPPC dispersed ultrasonically in saline was used to incubate excised sections of porcine bronchial epithelium. The adsorbed DPPC was then quantified by rigorously rinsing the tissue of adhering fluid and then digesting it for beta-scintillation counting. Each test (n=8 runs) was repeated for ratios of DPPC:PG of 9:1, 7:3 (as per ALECT/PumactantT) and 1:1 for both dipalmitoyl PG (DPPG) and EggPG (as incorporated in ALECT/PumactantT). RESULTS: Despite rigorous rinsing postincubation, bronchial epithelium was found to adsorb DPPC at a level roughly equivalent to one close-packed monolayer; whereas both DPPG and EggPG promoted the adsorption of DPPC in a dose-dependent manner, reaching an approximate threefold increase for 7:3 DPPC:PG. CONCLUSION: DPPC adsorbs to bronchial epithelium in amounts necessary for the masking of receptors, and this adsorption (probably chemisorption) is quite strongly promoted by PG either in its indigenous state (DPPG) or in the form (EggPG) used in ALECT to suppress the sensitivity of bronchial irritant receptors in our previous study and in clinical trials just completed.

Accelerated clearance of a second injection of PEGylated liposomes in mice.

We recently reported that the firstly injected PEGylated liposomes dramatically affected the rate of blood clearance of secondly injected PEGylated liposomes in rats in a time interval of injection dependent manner [J. Control. Release (2003)]. Mice are frequently used in evaluations of the therapeutic efficacy of PEGylated liposomal formulations, but the pharmacokinetics of repeatedly injected PEGylated liposomes in mice is not fully understood. In this study, therefore, we examined in mice the effect of the repeated injection of PEGylated liposomes on their pharmacokinetics. An intravenous pretreatment with PEGylated liposomes produced a striking change in the biodistribution of the second dose which was given several days after the first injection. The first dose resulted in a reduction in the circulation half-life of the second dose. The degree of alteration was dependent on the time interval between the injections. The rapid clearance of the second dose was strongly related to hepatic clearance (CLh). This finding suggests that a considerable increase in hepatic accumulation accounts for this phenomenon. But, no liver injury or an increase in the number of Kupffer cells were detected in histopathological evaluations. Collectively, although the multiple injections of the PEGylated liposomes had no obvious physical effects, such as inflammation, their pharmacokinetic behavior was clearly altered in mice. The results obtained here have important implications not only with respect to the design and engineering of liposomes for human use, but for evaluating the therapeutic efficacy of liposomal formulations in experimental animal models as well.

Comparative toxicity and concentrations of intravitreal amphotericin B formulations in a rabbit model.

PURPOSE: To determine the toxicity of various doses of intravitreal amphotericin B deoxycholate, amphotericin B lipid complex (ABLC), and liposomal amphotericin B (L-AmB). METHODS: Fifty-two rabbits were divided into two treatment groups (groups A and B). Thirteen treatments were administered intravitreally to the 104 rabbit eyes. Treatments included a control plus 10, 20, 30, and 50 micro g amphotericin B deoxycholate, ABLC, and L-AmB. Eye examinations were performed before injection and on day 11 for group A and on day 18 for group B. At death, on days 13 and 21 in groups A and B, respectively, vitreous humor was aspirated and concentrations of amphotericin B were determined by high performance liquid chromatography (HPLC), followed by enucleation for histologic studies. RESULTS: Significantly more eyes treated with ABLC showed development of vitreal opacities than developed in eyes treated with amphotericin B deoxycholate or L-AmB (P < 0.05). Vitreal band formation was significantly higher in ABLC-treated eyes than in those treated with L-AmB, (P = 0.039). Vitreal inflammation was greater in eyes treated with L-AmB (75%), amphotericin B deoxycholate (78%), and ABLC (91%) than with the control (50%; P = 0.08). Retinal ganglion cell loss was greater in eyes treated with amphotericin B deoxycholate (81%), L-AmB (91%), and ABLC (97%) than with the control (38%; P = 0.003). Amphotericin B concentrations were measurable for all doses of the three formulations. CONCLUSIONS: Based on histologic data, increasing doses of all three agents appear to be associated with increasing toxicity, however based on ophthalmologic data, L-AmB appears to be less toxic than either amphotericin B deoxycholate or ABLC.