Nanoscale Probing of Liposome Encapsulating Drug Nanocrystal Using Atomic Force Microscopy-Infrared Spectroscopy.

Use of liposomes encapsulating drug nanocrystals for the treatment of diseases like cancer and pulmonary infections is gaining attention. The potential therapeutic benefit of these engineered formulations relies on maintaining the physical integrity of the liposomes and the stability of the encapsulated drug. With the significant advancement in the microscopic and analytical techniques, analysis of the size and size distribution of these nanosized vesicles is possible. However, due to the limited spatial resolution of conventional vibrational spectroscopy techniques, the chemical composition of individual nanosized liposome cannot be resolved. To address this limitation, we applied atomic force microscopy infrared spectroscopy (AFM-IR) to assess the chemical composition of individual liposomes encapsulating ciprofloxacin in dissolved and nanocrystalline form. Spatially resolved AFM-IR spectra acquired from individual liposomes confirmed the presence of peaks related to N-H bending vibration, C-N stretching and symmetric, and asymmetric vibration of the carboxyl group present in the ciprofloxacin. Our results further demonstrated the effectiveness of AFM-IR in differentiating the liposome containing ciprofloxacin in dissolved or nanocrystalline form. Spectra acquired from dissolved ciprofloxacin had peaks related to the ionised carboxyl group, i.e., at 1576 and 1392 cm-1, which were either absent or far weaker in intensity in the spectra of liposomal sample containing ciprofloxacin nanocrystals. These findings are highly significant for pharmaceutical scientists to ascertain the stability and physicochemical composition of individual liposomes and will facilitate the design and development of liposomes with greater therapeutic benefits.

Effective Treatment of Mycobacterium avium subsp. hominissuis and Mycobacterium abscessus Species Infections in Macrophages, Biofilm, and Mice by Using Liposomal Ciprofloxacin.

Nontuberculous mycobacteria (NTM) affect an increasing number of individuals worldwide. Infection with these organisms is more common in patients with chronic lung conditions, and treatment is challenging. Quinolones, such as ciprofloxacin, have been used to treat patients, but the results have not been encouraging. In this report, we evaluate novel formulations of liposome-encapsulated ciprofloxacin (liposomal ciprofloxacin) in vitro and in vivo Its efficacy against Mycobacterium avium and Mycobacterium abscessus was examined in macrophages, in biofilms, and in vivo using intranasal instillation mouse models. Liposomal ciprofloxacin was significantly more active than free ciprofloxacin against both pathogens in macrophages and biofilms. When evaluated in vivo, treatment with the liposomal ciprofloxacin formulations was associated with significant decreases in the bacterial loads in the lungs of animals infected with M. avium and M. abscessus In summary, topical delivery of liposomal ciprofloxacin in the lung at concentrations greater than those achieved in the serum can be effective in the treatment of NTM, and further evaluation is warranted.

Symposium Summary: "Breathe In, Breathe Out, Its Easy: What You Need to Know About Developing Inhaled Drugs".

Developing inhaled drugs requires knowledge of lung anatomy, cell biology, respiratory physiology, particle physics, and some plumbing. Although dose makes the poison, in the context of an inhaled drug, the "dose" is not easily defined. This lack of clarity around dose poses issues and challenges in the design of inhalation toxicology programs. To better understand dose, the influence of ventilation is discussed as are the perturbations in pulmonary function observed with inhalation exposure that can affect dose. Methods for determining inhaled drug deposition to arrive at an estimate of lung dose are examined. Equally important to understanding dose are the techniques used to deliver aerosols to animals. With a better understanding of dose and inhalation exposure, species-specific histopathologic lesions, both common background and toxicologically significant lesions, are reviewed. Finally, insight into how regulators synthesize and evaluate these complex findings to assess clinical safety risks is presented.

Liposome encapsulation of ciprofloxacin improves protection against highly virulent Francisella tularensis strain Schu S4.

Liposome-encapsulated ciprofloxacin for inhalation (CFI) was investigated as a putative postexposure therapeutic for two strains of Francisella tularensis. The efficacies of oral ciprofloxacin and intranasally instilled CFI could not be distinguished in a mouse model of infection with the F. tularensis live vaccine strain (LVS), where a single dose of either formulation offered full protection against a lethal challenge. However, mouse studies with the more virulent Schu S4 strain of F. tularensis demonstrated that a higher level of protection against a lethal aerosol infection is provided by CFI than by oral ciprofloxacin. In addition, using this infection model, it was possible to discriminate the efficacy of intranasally instilled CFI from that of aerosolized CFI, with aerosolized CFI providing full protection after just a single dose. The improved efficacy of CFI compared to oral ciprofloxacin is likely due to the high sustained concentrations of ciprofloxacin in the lung. In summary, CFI may be a promising therapy, perhaps enabling the prophylactic regimen to be shortened, for use in the event of a deliberate release of F. tularensis. The prophylactic efficacy of CFI against other biological warfare (BW) threat agents also warrants investigation.

Evaluation of liposomal ciprofloxacin formulations in a murine model of anthrax.

The in vivo efficacy of liposomal encapsulated ciprofloxacin in two formulations, lipoquin and apulmiq, were evaluated against the causative agent of anthrax, Bacillus anthracis. Liposomal encapsulated ciprofloxacin is attractive as a therapy since it allows for once daily dosing and achieves higher concentrations of the antibiotic at the site of initial mucosal entry but lower systemic drug concentrations. The in vivo efficacy of lipoquin and apulmiq delivered by intranasal instillation was studied at different doses and schedules in both a post exposure prophylaxis (PEP) therapy model and in a delayed treatment model of murine inhalational anthrax. In the mouse model of infection, the survival curves for all treatment cohorts differed significantly from the vehicle control. Ciprofloxacin, lipoquin and apulmiq provided a high level of protection (87-90%) after 7 days of therapy when administered within 24 hours of exposure. Reducing therapy to only three days still provided protection of 60-87%, if therapy was provided within 24 hours of exposure. If treatment was initiated 48 hours after exposure the survival rate was reduced to 46-65%. These studies suggest that lipoquin and apulmiq may be attractive therapies as PEP and as part of a treatment cocktail for B. anthracis.

Formation of ciprofloxacin nanocrystals within liposomes by spray drying for controlled release via inhalation.

The present study was conducted to harness spray drying technology as a novel method of producing Ciprofloxacin nanocrystals inside liposomes (CNL) for inhalation delivery. Liposomal ciprofloxacin dispersions were spray dried with sucrose as a lyoprotectant in different mass ratios (0.5:1, 1:1 and 2:1 sucrose to lipids), along with 2% w/w magnesium stearate and 5% w/w isoleucine as aerosolization enhancers. Spray drying conditions were: inlet air temperature 50 °C, outlet air temperature 33-35 °C, atomizer rate 742 L/h and aspirator 35 m3/h. After spray drying, the formation of ciprofloxacin nanocrystals inside the liposomes was confirmed by cryo- transmission electron microscopy. The physiochemical characteristics of the spray dried powder (particle size, morphology, crystallinity, moisture content, drug encapsulation efficiency (EE), in vitro aerosolization performance and drug release) were determined. The EE of the liposomes was found to vary between 44 and 87% w/w as the sucrose content was increased from 25 to 57% w/w. The powders contained partially crystalline particles with a volume median diameter of ~1 µm. The powders had low water content (~2% wt.) and were stable at high relative humidity. Aerosol delivery using the Osmohaler® inhaler at a flow rate of 100 L/min produced an aerosol fine particle fraction (% wt. <5 µm) of 58-64%. The formulation with the highest sucrose content (2:1 w/w sucrose to lipid) demonstrated extended ciprofloxacin release from liposomes (80% released within 7 h) in comparison to the original liquid formulation (80% released within 2 h). In conclusion, a stable and inhalable CNL powder with controlled drug release was successfully prepared by spray drying.

Ciprofloxacin nanocrystals liposomal powders for controlled drug release via inhalation.

This study was conducted to evaluate the feasibility of developing inhalable dry powders of liposomal encapsulated ciprofloxacin nanocrystals (LECN) for controlled drug release. Dry powders of LECN were produced by freeze-thaw followed by spray drying. The formulations contained sucrose as a lyoprotectant in different weight ratios (0.75:1, 1:1 and 2:1 sucrose to lipids), along with 2% magnesium stearate and 5% isoleucine as aerosolization enhancers. The powder physical properties (particle size, morphology, crystallinity, moisture content), in vitro aerosolization performance, drug encapsulation efficiency and in vitro drug release were investigated. The spray dried powders were comprised of spherical particles with a median diameter of ∼1 µm, partially crystalline, with a low water content (∼2% mass) and did not undergo recrystallization at high relative humidity. When dispersed by an Osmohaler® inhaler at 100 L/min, the powders showed a high aerosol performance with a fine particle fraction (% wt. <5 µm) of 66-70%. After reconstitution of the powders in saline, ciprofloxacin nanocrystals were confirmed by cryo-electron microscopy. The drug encapsulation efficiency of the reconstituted liposomes was 71-79% compared with the stock liquid formulation. Of the three formulations, the one containing a sucrose to lipids wt. ratio of 2:1 demonstrated a prolonged release of ciprofloxacin from the liposomes. In conclusion, ciprofloxacin nanocrystal liposomal powders were prepared that were suitable for inhalation aerosol delivery and controlled drug release.

Inhaled Liposomal Ciprofloxacin Protects against a Lethal Infection in a Murine Model of Pneumonic Plague.

Inhalation of Yersinia pestis can lead to pneumonic plague, which without treatment is inevitably fatal. Two novel formulations of liposome-encapsulated ciprofloxacin, 'ciprofloxacin for inhalation' (CFI, Lipoquin®) and 'dual release ciprofloxacin for inhalation' (DRCFI, Pulmaquin®) containing CFI and ciprofloxacin solution, are in development. These were evaluated as potential therapies for infection with Y. pestis. In a murine model of pneumonic plague, human-like doses of aerosolized CFI, aerosolized DRCFI or intraperitoneal (i.p.) ciprofloxacin were administered at 24 h (representing prophylaxis) or 42 h (representing treatment) post-challenge. All three therapies provided a high level of protection when administered 24 h post-challenge. A single dose of CFI, but not DRCFI, significantly improved survival compared to a single dose of ciprofloxacin. Furthermore, single doses of CFI and DRCFI reduced bacterial burden in lungs and spleens to below the detectable limit at 60 h post-challenge. When therapy was delayed until 42 h post-challenge, a single dose of CFI or DRCFI offered minimal protection. However, single doses of CFI or DRCFI were able to significantly reduce the bacterial burden in the spleen compared to empty liposomes. A three-day treatment regimen of ciprofloxacin, CFI, or DRCFI resulted in high levels of protection (90-100% survival). This study suggests that CFI and DRCFI may be useful therapies for Y. pestis infection, both as prophylaxis and for the treatment of plague.

Gamma scintigraphic evaluation of a miniaturized AERx pulmonary delivery system for aerosol delivery to anesthetized animals using a positive pressure ventilation system.

The purpose of this study was to characterize performance of a miniaturized AERx((R)) Pulmonary Delivery System designed for aerosol administration to large animal models. The miniaturized AERx System was developed through a systematic scaling down of the AERx System used for humans to allow for operation in certain animal models with lower inspiratory flow rates and inhaled volumes than those used for humans. We used gamma scintigraphy to characterize the in vivo particle deposition achieved with the miniaturized AERx System in two dogs. The dogs were 3-4 years old, and weighed 10.4 kg and 13.6 kg. Acepromazine was used as pre-anesthetic medication. Anesthesia was induced with 5% isoflurane. The trachea was intubated using an endotracheal tube (internal diameter 8.5 mm), and the dogs were ventilated using positive pressure during the exposure using the LRRI puff generator. An inhalation of aerosol was initiated by activation of the puff generator though the computer-controlled interface. Each dog inhaled approximately 0.8 L per puff, of which the aerosol volume comprised approximately 0.25 L, at a target flow rate of 15 L/min. The dogs were exposed to 10 AERx Strips in 10 puffs. The mass median aerodynamic diameter of the aerosolized formulation was approximately 1.25 microm with a fine particle fraction <3.5 microm of 0.976. The scintigraphic images showed uniform bilateral lung deposition following aerosol delivery with the AERx System. Total lung deposition for the two dogs was 10.7% and 18% of the loaded dose from the AERx Strip. The corresponding peripheral lung: inner lung (P/I) ratios were 0.83 and 0.75, suggestive of deposition in the deep lung. Only 0.1% to 0.2% of the loaded dose was exhaled. These results show the miniature AERx System can efficiently deliver aerosols to the deep lung of dogs. The miniaturized AERx System would be a valuable tool for conducting proof-of-concept studies as well as safety and tolerability analysis of inhaled drug candidates in large animal models.

The potential of liposome-encapsulated ciprofloxacin as a tularemia therapy.

Liposome-encapsulation has been suggested as method to improve the efficacy of ciprofloxacin against the intracellular pathogen, Francisella tularensis. Early work with a prototype formulation, evaluated for use against the F. tularensis live vaccine strain, showed that a single dose of liposomal ciprofloxacin given by the intranasal or inhalational route could provide protection in a mouse model of pneumonic tularemia. Liposomal ciprofloxacin offered better protection than ciprofloxacin given by the same routes. Liposomal ciprofloxacin has been further developed by Aradigm Corporation for Pseudomonas aeruginosa infections in patients with cystic fibrosis and non-cystic fibrosis bronchiectasis. This advanced development formulation is safe, effective and well tolerated in human clinical trials. Further evaluation of the advanced liposomal ciprofloxacin formulation against the highly virulent F. tularensis Schu S4 strain has shown that aerosolized CFI (Ciprofloxacin encapsulated in liposomes for inhalation) provides significantly better protection than oral ciprofloxacin. Thus, liposomal ciprofloxacin is a promising treatment for tularemia and further research with the aim of enabling licensure under the animal rule is warranted.

Evaluation of the AERx pulmonary delivery system for systemic delivery of a poorly soluble selective D-1 agonist, ABT-431.

PURPOSE: ABT-431 is a chemically stable, poorly soluble prodrug that rapidly converts in vivo to A-86929, a selective dopamine D-1 receptor agonist. This study was designed to evaluate the ability of the AERx pulmonary delivery system to deliver ABT-431 to the systemic circulation via the lung. METHODS: A 60% ethanol formulation of 50 mg/mL ABT-431 was used to prepare unit dosage forms containing 40 microL of formulation. The AERx system was used to generate a fine aerosol bolus from each unit dose that was collected either onto a filter assembly to chemically assay for the emitted dose or in an Andersen cascade impactor for particle size analysis. Plasma samples were obtained for pharmacokinetic analysis after pulmonary delivery and IV dosing of ABT-431 to nine healthy male volunteers. Doses from the AERx system were delivered as a bolus inhalation(s) (1, 2, 4, and 8 mg) and intravenous infusions were given over 1 hr (5 mg). Pharmacokinetic parameters of A-86929 were estimated using noncompartmental analysis. RESULTS: The emitted dose was 1.02 mg (%RSD = 11.0, n = 48). The mass median aerodynamic diameter of the aerosol was 2.9 +/- 0.1 microm with a geometric standard deviation of 1.3 +/- 0.1 (n = 15). Tmax (mean +/- SD) after inhalation ranged from 0.9 +/- 0.6 to 11.5 +/- 2.5. The mean absolute pulmonary bioavailibility (as A-86929) based on emitted dose ranged from 81.9% to 107.4%. CONCLUSIONS: This study demonstrated that the AERx pulmonary delivery system is capable of reproducibly generating fine nearly monodisperse aerosols of a small organic molecule. Aerosol inhalation utilizing the AERx pulmonary delivery system may be an efficient means for systemic delivery of small organic molecules such as ABT-431.