Biomedical subjects

Robert O Williams

Publications and source records attributed to Robert O Williams.

At least 19 recordsLinked to original sources

PubMed2007

Hot-melt extrusion for enhanced delivery of drug particles.

With the recent advent of nanotechnology for pharmaceutical applications, drug particle engineering is the focus of increasing interest as a viable approach for overcoming solubility limitations of poorly water-soluble drugs. Although these particle engineering techniques have been proven successful for enhancing the dissolution properties of many poorly water-soluble drugs, there are limitations associated with them such as particle aggregation, morphological instability, and poor wettability. The aim of this study was to demonstrate a processing technique in which hot-melt extrusion (HME) is utilized to overcome these limitations. Micronized particles of amorphous itraconazole (ITZ) stabilized with PVP or HPMC were produced and subsequently melt extruded with poloxamer 407 and PEO 200 M to deaggregate and disperse the particles into the hydrophilic polymer matrix. Differential scanning calorimetry, X-ray diffraction, and scanning electron microscopy were used to demonstrate that the HME process did not alter the properties of the micronized particles. Dissolution testing conducted at sink conditions revealed that the dissolution rate of the micronized particles was improved by HME due to particle deaggregation and enhanced wetting. Supersaturation dissolution testing demonstrated that the ITZ-HPMC micronized particle extrudates provided superior supersaturation of ITZ compared to the ITZ-PVP micronized particle extrudates. Supersaturation dissolution testing incorporating a pH change (from pH 1.2 to 6.8 at 2 h) revealed that neither micronized particle extrudate formulation significantly reduced the rate of ITZ precipitation from supersaturated solution once pH was increased. Moreover, the two extrudate formulations performed very similarly when only considering dissolution testing from just before pH adjustment through the duration of testing at neutral pH. From oral dosing of rats, it was determined that the two extrudate formulations performed similarly in vivo as confirmed by their statistically equivalent AUC values. By correlating the results of supersaturation dissolution testing with pH change to the in vivo AUC, it appears that rapid precipitation of ITZ occurs upon entrance into the more neutral pH environment of the small intestine resulting in a brief opportunity for absorption. This suggests that perhaps the optimum formulation approach for ITZ is to control drug release so as to retard precipitation as pH is increased and extend the absorption window in the small intestine.

Animals↗

PubMed2006

Solid dispersions of itraconazole and enteric polymers made by ultra-rapid freezing.

The primary objective of the study is to investigate the influence of composition parameters including drug:polymer ratio and polymer type, and particle structure of enteric solid dispersions on the release of ITZ under sink and supersaturated dissolution conditions. Modulated differential scanning calorimetry (MDSC) was utilized to define the level of ITZ miscibility with each polymer. The compositions were completely miscible at 60% ITZ for both polymers and as high as 70% in HP-55. High potency composition glass transition temperatures (T(g)) correlated with predicted T(g)'s from the Gordon-Taylor equation, however, recrystallization exotherms revealed pure amorphous regions indicating that phase separation occurred during particle formation. Furthermore, in vitro studies including X-ray powder diffraction (XRD), scanning electron microscopy (SEM), surface area analysis (BET), and dissolution were performed to determine differences between low potency (completely miscible) and high potency (partially miscible) compositions. Dissolution studies on low potency ITZ compositions revealed that miscibility plays an active role in ITZ release under sink conditions, and square root diffusion through the enteric polymer is observed. Supersaturated dissolution profiles revealed high potency compositions had maximum saturation levels (C/Ceq(max)) between 10.6- and 8-times equilibrium solubility, but had higher cumulative extents of supersaturation, compared to low potency compositions which had C/Ceq(max) values of 15-19.6. However, these low potency compositions rapidly precipitated leading to significantly lower AUCs (p<0.05). The change in the miscibility of the solid dispersion had a pronounced effect of drug release (sink) while differences in potency influenced supersaturated dissolution profiles.

Acrylic Resins↗

PubMed2006

Drug nanoparticles by antisolvent precipitation: mixing energy versus surfactant stabilization.

Organic itraconazole (ITZ) solutions were mixed with aqueous solutions to precipitate sub-300 nm particles over a wide range of energy dissipation rates, even for drug loadings as high as 86% (ITZ weight/total weight). The small particle sizes were produced with the stabilizer poloxamer 407, which lowered the interfacial tension, increasing the nucleation rate while inhibiting growth by coagulation and condensation. The highest nucleation rates and slowest growth rates were found at temperatures below 20 degrees C and increased with surfactant concentration and Reynolds number (Re). This increase in the time scale for growth reduced the Damkohler number (Da) (mixing time/precipitation time) to low values even for modest mixing energies. As the stabilizer concentration increased, the average particle size decreased and reached a threshold where Da may be considered to be unity. Da was maintained at a low value by compensating for a change in one variable away from optimum conditions (for small particles) by manipulating another variable. This tradeoff in compensation variables was demonstrated for organic flow rate vs Re, Re vs stabilizer concentration, stabilizer feed location (organic phase vs aqueous phase) vs stabilizer concentration, and stabilizer feed location vs Re. A decrease in the nucleation rate with particle density in the aqueous suspension indicated that secondary nucleation was minimal. A fundamental understanding of particle size control in antisolvent precipitation is beneficial for designing mixing systems and surfactant stabilizers for forming nanoparticles of poorly water soluble drugs with the potential for high dissolution rates.

Itraconazole↗

PubMed2006

Evaluation of the USP dissolution test method A for enteric-coated articles by planar laser-induced fluorescence.

The USP drug release standard for delayed-release articles method A was evaluated using planar laser-induced fluorescence (PLIF). Prior authors have suggested that high pH "hot spots" could develop during the buffer medium addition of the method A enteric test. Additionally, previous studies have shown heterogeneous flow patterns and low-shear regions in the USP Apparatus II dissolution vessel, which may result in poor mixing of the buffer and acid media during the pH neutralization step of the method A enteric test. In this study, PLIF was used to evaluate the mixing patterns and evolution of pH neutralization during the buffer medium addition with rhodamine-B dye and the pH-sensitive dye fluorescein, respectively. Additionally, a comparison of the methods A and B enteric tests was performed with enteric-coated tablets containing rhodamine-B in the film so as to image the dissolution rate of the coating polymer with PLIF in order to determine if rapid buffer addition for the method A procedure accelerates the rate of film coat dissolution. Rapid addition of the 250 mL of buffer medium over 5 s to the 750 mL of acidic medium shows efficient mixing and pH neutralization due to the generation of large-scale stirring and enhanced turbulence resulting from the descending buffer medium. Slow addition near the paddle shaft over 5 min showed segregation in the recirculating region around the paddle shaft. In contrast, slow addition near the vessel wall introduces the medium into fluid outside of the recirculation region and enables transport over the entire vessel. Enteric-coated tablets tested according to method A with rapid medium addition and method B enteric tests performed identically, indicating no difference in polymer dissolution rate between the two tests. From the results of the PLIF imaging studies with rhodamine-B, fluorescein, and enteric-coated tablets, it was seen that "hot spots" affecting the dissolution performance of enteric dosage forms are not generated during the neutralization step of the method A enteric test namely when the media is added rapidly or outside of the recirculating region that surrounds the paddle shaft.

Delayed-Action Preparations↗

PubMed2006

Morphology of protein particles produced by spray freezing of concentrated solutions.

The mechanisms for the formation of high surface area lysozyme particles in spray freezing processes are described as a function of spray geometry and atomization, solute concentration and the calculated cooling rate. In the spray freeze-drying (SFD) process, droplets are atomized into a gas and then freeze upon contact with a liquid cryogen. In the spray freezing into liquid (SFL) process, a solution is sprayed directly into the liquid cryogen below the gas-liquid meniscus. A wide range of feed concentrations is examined for two cryogens, liquid nitrogen (LN2) and isopentane (i-C5). The particle morphologies are characterized by SEM micrographs and BET measurements of specific surface area. As a result of boiling of the cryogen (Leidenfrost effect), the cooling rate for SFL into LN2 is several orders of magnitude slower than for SFL into i-C5 and for SFD in the case of either LN2 or i-C5. For 50 mg/mL concentrated feed solutions, the slower cooling of SFL into LN2 leads to a surface area of 34 m(2)/g. For the other three cases with more rapid cooling rates, surface areas were greater than 100 m(2)/g. The ability to adjust the cooling rate to vary the final particle surface area is beneficial for designing particles for controlled release applications.

Algorithms↗

PubMed2006

Stable high surface area lactate dehydrogenase particles produced by spray freezing into liquid nitrogen.

Enzyme activities were determined for lactate dehydrogenase (LDH) powder produced by lyophilization, and two fast freezing processes, spray freeze-drying (SFD) and spray freezing into liquid (SFL) nitrogen. The 0.25 mg/mL LDH aqueous feed solutions included either 30 or 100 mg/mL trehalose. The SFL process produced powders with very high enzyme activities upon reconstitution, similar to lyophilization. However, the specific surface area of 13 m(2)/g for SFL was an order of magnitude larger than for lyophilization. In SFD activities were reduced in the spraying step by the long exposure to the gas-liquid interface for 0.1-1s, versus only 2 ms in SFL. The ability to produce stable high surface area submicron particles of fragile proteins such as LDH by SFL is of practical interest in protein storage and in various applications in controlled release including encapsulation into bioerodible polymers. The SFL process has been scaled down for solution volumes <1 mL to facilitate studies of therapeutic proteins.

Animals↗

PubMed2006

Novel ultra-rapid freezing particle engineering process for enhancement of dissolution rates of poorly water-soluble drugs.

An ultra-rapid freezing (URF) technology has been developed to produce high surface area powders composed of solid solutions of an active pharmaceutical ingredient (API) and a polymer stabilizer. A solution of API and polymer excipient(s) is spread on a cold solid surface to form a thin film that freezes in 50 ms to 1s. This study provides an understanding of how the solvent's physical properties and the thin film geometry influence the freezing rate and consequently the final physico-chemical properties of URF-processed powders. Theoretical calculations of heat transfer rates are shown to be in agreement with infrared images with 10ms resolution. Danazol (DAN)/polyvinylpyrrolidone (PVP) powders, produced from both acetonitrile (ACN) and tert-butanol (T-BUT) as the solvent, were amorphous with high surface areas (approximately 28-30 m2/g) and enhanced dissolution rates. However, differences in surface morphology were observed and attributed to the cooling rate (film thickness) as predicted by the model. Relative to spray-freezing processes that use liquid nitrogen, URF also offers fast heat transfer rates as a result of the intimate contact between the solution and cold solid surface, but without the complexity of cryogen evaporation (Leidenfrost effect). The ability to produce amorphous high surface area powders with submicron primary particles with a simple ultra-rapid freezing process is of practical interest in particle engineering to increase dissolution rates, and ultimately bioavailability.

Acetonitriles↗

PubMed2006

Targeted high lung concentrations of itraconazole using nebulized dispersions in a murine model.

PURPOSE: The purpose of this study was to investigate the delivery of itraconazole (ITZ) particles to a murine lung model by nebulization. METHODS: Three ITZ formulations were prepared and characterized in the dry state using contact angle, dissolution, X-ray powder diffraction, scanning electron microscopy, and Brunauer-Emmett-Teller surface area analysis. Aerodynamic particle size distributions and lung deposition studies in 14 outbred male ICR mice were performed using aqueous dispersions of all the formulations. A separate dosing uniformity study was also performed to qualify use of the chamber. RESULTS: All formulations had an aggregated particle size of approximately 30 microm in diameter. Two formulations showed that 80% of the drug dissolved in less than 5 min. The remaining ITZ formulation had a slower dissolution and the lowest total emitted dose from the nebulizer used. High concentrations of ITZ were shown to be present in the mouse lung during the lung deposition study, up to 16.8 +/- 0.13 microg/g (+/- SE) were achieved. Concentrations of up to 0.76 +/- 0.03 microg/g (+/- SE) could be maintained from the single nebulized dose for at least 24 h. CONCLUSION: An effective method of targeted delivery of ITZ to the deep lung is presented that may be useful for the treatment and prevention of acute fungal infections.

Administration, Inhalation↗

PubMed2006

Cryogenic liquids, nanoparticles, and microencapsulation.

The biopharmaceutical classification system (BCS) is used to group pharmaceutical actives depending upon the solubility and permeability characteristics of the drug. BCS class II compounds are poorly soluble but highly permeable, exhibiting bioavailability that is limited by dissolution. The dissolution rate of BCS class II drug substances may be accelerated by enhancing the wetting of the bulk powder and by reducing the primary particle size of the drug to increase the surface area. These goals may be achieved by nucleating drug particles from solution in the presence of stabilizing excipients. In the spray freezing into liquid (SFL) process, a drug containing solution is atomized and frozen rapidly to engineer porous amorphous drug/excipient particles with high surface areas and dissolution rates. Aqueous suspensions of nanostructured particles may be produced from organic solutions by evaporative precipitation into aqueous solution (EPAS). The suspensions may be dried by lyophilization. The particle size and morphology may be controlled by the type and level of stabilizers. In vivo studies have shown increased bioavailability of a wide variety of drugs particles formed by SFL or EPAS. For both processes, increased serum levels of danazol (DAN) were observed in mice relative to bulk DAN and the commercial product, Danocrine. Orally dosed itraconazole (ITZ) compositions, formed by SFL, produce higher serum levels of the drug compared to the commercial product, Sporanox oral solution. Additionally, nebulized SFL processed ITZ particles suspended in normal saline have been dosed via the pulmonary route and led to extended survival times for mice inoculated with Aspergillis flavus. SFL and EPAS processes produce amorphous drug particles with increased wetting and dissolution rates, which will subsequently supersaturate biological fluids in vivo, resulting in increased drug bioavailability and efficacy.

Administration, Inhalation↗

PubMed2006

Single dose and multiple dose studies of itraconazole nanoparticles.

The objective of this study was to determine and compare the lung and serum concentrations in mice following oral and pulmonary dosing of amorphous nanoparticulate itraconazole (ITZ) compositions as well as the Sporanox oral solution (itraconazole/Janssen). Second, the steady state partitioning of ITZ in lung tissue and circulatory compartments following repeated oral and pulmonary dosing was determined. The pulmonary formulation (ITZ-pulmonary) consisted of ITZ, polysorbate 80, and poloxamer 407 in a 1:0.75:0.75 ratio and the oral formulation (ITZ-oral) consisted of ITZ, PEG 8000, poloxamer 188, and sorbitan monooleate 80 in a 1:1:2:1 ratio. Mice were dosed every 12 h by nebulization with ITZ-pulmonary, or by oral gavage with ITZ-oral or Sporanox oral solution (n = 12 per study arm). ITZ-pulmonary achieved significantly greater (>10-fold) lung tissue concentrations compared to the Sporanox oral solution and ITZ-oral. There were no statistical differences between the two oral formulations. ITZ-pulmonary achieved significantly greater lung levels per unit serum concentration compared to the orally dosed ITZ compositions. High and sustained lung tissue concentrations were achieved via inhalation of an amorphous nanoparticulate ITZ-pulmonary composition while maintaining serum levels which are above the minimum lethal concentration (MLC) of Aspergillus fumigatus.

Administration, Oral↗

PubMed2006

Supersaturation produces high bioavailability of amorphous danazol particles formed by evaporative precipitation into aqueous solution and spray freezing into liquid technologies.

The bioavailability of high surface area danazol formulations was evaluated in a mouse model to determine what effect high supersaturation, as measured in vitro, has on the absorption of a poorly water soluble drug. Danazol, a biopharmaceutics classification system II (BCS II) compound, was used as the model drug. Evaporative precipitation into aqueous solution (EPAS) and spray freezing into liquid (SFL) technologies were used to prepare powders of danazol/PVP K-15 in a 1:1 ratio. The evaporative precipitation into aqueous solution (EPAS) and SFL compositions, physical mixture and commercial product were dosed by oral gavage to 28 male Swiss/ICR mice for each arm of the study. Samples were taken at time points ranging from 0.5 to 24 h. Pooled mouse serum was analyzed for danazol by high performance liquid chromatography (HPLC). Powders were analyzed for their ability to form supersaturated solutions through dissolution at concentrations of 1 mg/mL which was the dose delivered to the mouse models. Spray freezing into liquid (SFL) and EPAS compositions displayed higher C(max) at 392.5 ng/mL and 430.1 ng/mL, respectively, compared to the physical mixture (204.4 ng/mL) and commercially available danazol (199.3 ng/mL). The T(max) for all compositions studied was near the 1 h time point. The area under the curve (AUC) for the SFL composition was 2558 ng.h/mL compared to EPAS composition at 1534 ng.h/mL. The area under the curve (AUC) for the physical mixture and commercially available danazol were 672 ng.h/mL and 1519 ng.h/mL, respectively. The elimination rate constants for the EPAS composition, SFL composition, and physical mixture were similar at approximately 0.15 h(-1) where as the commercially available danazol capsules displayed an elimination rate constant of 0.103 h(-1). The extent of danazol absorption in the mouse model was higher for SFL composition compared to the less amorphous EPAS composition, physical mixture, and commercially available danazol powders. Both EPAS and SFL compositions were able to form supersaturated solutions. However, the SFL composition displayed a supersaturation of 33% above control and was able to maintain supersaturation for 90 min compared to the EPAS composition (27% supersaturation above control for 60 min). Through the use of a testing method for supersaturation, it was found that EPAS and SFL compositions achieve higher apparent solubilities when compared to the physical mixture and commercially available danazol capsules. Because of the greater extent of dissolution of the SFL composition, the bioavailability was enhanced in a mouse model.

Algorithms↗

PubMed2006

In vivo efficacy of aerosolized nanostructured itraconazole formulations for prevention of invasive pulmonary aspergillosis.

Aerosolized evaporative precipitation into aqueous solution and spray freezing into liquid nanostructured formulations of itraconazole as prophylaxis significantly improved survival relative to commercial itraconazole oral solution and the control in a murine model of invasive pulmonary aspergillosis. Aerosolized administration of nanostructured formulations also achieved high lung tissue concentrations while limiting systemic exposure.

Aerosols↗

PubMed2005

Encapsulation of protein nanoparticles into uniform-sized microspheres formed in a spinning oil film.

A new spinning oil film (SOF) solid-in-oil-in-oil emulsion process was developed to produce uniform-sized protein-loaded biodegradable microspheres. A thin SOF on a cylindrical rotor was used to shear droplets from a nozzle tip to control droplet size. The resulting microspheres with low polydispersity (6%) produced a low burst (6%-11%) release even at high loadings (13%-18% encapsulated solids, 8%-12% protein). The SOF process had a high yield and did not require the presence of water, which can cause protein denaturation, or surfactants, which may be unwanted in the final product. Amorphous protein and crystalline excipient solids were encapsulated into 3 different polymers, giving a homogenous drug distribution throughout the microspheres, and an essentially complete protein encapsulation efficiency (average = 99%). In contrast, large burst release was observed for polydisperse microspheres produced by a conventional emulsification technique, particularly for microspheres smaller than 25 mum in diameter, which gave 93% burst at 15% loading. The uniform encapsulation of high loadings of proteins into microspheres with low polydispersity in an anhydrous process is of practical interest in the development of controlled-release protein therapeutics.

Animals↗

PubMed2005

Stabilizer choice for rapid dissolving high potency itraconazole particles formed by evaporative precipitation into aqueous solution.

The objective of this study was to investigate the influence of stabilizer type on the physicochemical properties, including dissolution, of ultra-high potency powders containing itraconazole (ITZ) formed by evaporative precipitation into aqueous solution (EPAS). ITZ was dissolved in dichloromethane, which was then atomized through a heated coil at 80 degrees C into an aqueous solution over precise periods of time. Stabilizers were present in either the aqueous, organic or both phases. The dispersions were centrifuged and the supernatant was removed. Three hydrophilic stabilizers were investigated, including polysorbate 80, polyvinyl pyrrolidone and poloxamer 407. Rapid dissolving ultra-high potency of ITZ powders was successfully produced. Greater than 80% of ITZ was dissolved in 5 min compared to only 13% of ITZ bulk powders. The resulting stabilizer-coated drug particles had high drug-to-stabilizer ratios greater than 12, corresponding to potencies (wt drug/wt drug+wt surfactant) as high as 93%. An increase in dissolution rate was correlated with the amount of stabilizer adsorbed and the wettability. The combination of polysorbate 80 and poloxamer 407 present in the aqueous and organic phases, respectively, was superior in achieving high wetting and rapid dissolving ITZ powders. The ability to control the adsorption behavior of stabilizers by using synergistic combinations affords the opportunity to achieve high dissolution rates with higher potencies compared to previously reported values.

Adsorption↗

PubMed2005

Spray freezing into liquid versus spray-freeze drying: influence of atomization on protein aggregation and biological activity.

Protein aggregation and enzyme activity were compared for reconstituted lysozyme particles produced by two cryogenic technologies, spray freezing into liquid (SFL) and spray-freeze drying (SFD). The particles were characterized by enzyme activity measurements, scanning electron microscopy (SEM), light scattering, X-ray photoelectron spectroscopy (XPS) and BET specific surface area analysis. Highly porous microparticle aggregates of protein nanoparticles, observed by SEM, were produced by both processes. The smaller degree of protein aggregation and smaller losses in enzyme activity for the SFL process relative to the SFD process were due primarily to the spraying step. The higher stability of the SFL versus SFD powders was consistent with the smaller surface excess of lysozyme measured by XPS in SFL, resulting from the reduced time of exposure to the air-water interface during atomization. For pure lysozyme, the degree of aggregation and enzyme activity were comparable for lyophilization and SFL, despite the much larger particle surface area for SFL.

Enzyme Stability↗

PubMed2005

Uniform encapsulation of stable protein nanoparticles produced by spray freezing for the reduction of burst release.

Stable protein nanostructured particles, produced by spray freezing into liquid (SFL) nitrogen, were encapsulated uniformly into microspheres to reduce the burst release over the first 24 h. The denaturation and aggregation of these bovine serum albumin (BSA) high-surface area particles were minimal due to ultra-rapid freezing and the absence of a liquid-air interface. Upon sonication, these friable highly porous, solid protein particle aggregates broke up into submicron particles. These particles were encapsulated into DL-lactide/glycolide copolymer (PLGA) and poly(lactic acid) (PLA) microspheres by anhydrous solid-in-oil-in-oil (s/o/o) techniques. For 5% loading of protein, the burst release after 24 h was only 2.5-4.1%, that is, values fivefold to tenfold lower than those observed for larger more conventional BSA particles. At a loading of 10%, the burst was only 6 and 13% for PLGA and PLA, respectively, and at 15% loading it was only 12% for PLGA. As shown with confocal and scanning electron microscopy (SEM), the low burst is consistent with a uniform distribution of protein nanoparticles, which were about 100 times smaller than the microspheres. Changes in aggregation and secondary structure, which were monitored by size exclusion chromatography and FTIR, respectively, indicated only slight monomer loss (3.9%) and high structural integrity (38% alpha-helix) in the encapsulated protein.

Capsules↗

PubMed2005

Comparison of powder produced by evaporative precipitation into aqueous solution (EPAS) and spray freezing into liquid (SFL) technologies using novel Z-contrast STEM and complimentary techniques.

The objective of this study was to compare the properties of particles formed by nucleation and polymer stabilization (e.g. evaporative precipitation into aqueous solution (EPAS)) versus rapid freezing (e.g. spray freezing into liquid (SFL)). Powders formed by EPAS and SFL, composed of danazol and PVP K-15 in a 1:1 ratio, were characterized using X-ray powder diffraction, modulated differential scanning calorimetry (MDSC), contact angle determination, dissolution, scanning electron microscopy (SEM), environmental scanning electron microscopy (ESEM), BET specific surface area, and Z-contrast scanning transmission electron microscopy (STEM). Large differences in particle morphologies and properties were observed and explained in terms of the particle formation mechanisms. Both techniques produced amorphous powders with high T(g) and low contact angle values. However, STEM analysis showed highly porous bicontinuous nanostructured 30nm particles connected by narrow bridges for SFL versus aggregated 500 nm primary particles for EPAS. The combination of STEM and other characterization techniques indicates solid solutions were formed for the SFL powders consistent with rapid freezing. In contrast, the EPAS particle cores are enriched in hydrophobic API and the outer surface is enriched in the hydrophilic polymer, with less miscibility than in the SFL powders. Consequently, dissolution rates are faster for the SFL particles, although both techniques enhanced dissolution rates of the API.

Algorithms↗

PubMed2005

Design and evaluation of a restraint-free small animal inhalation dosing chamber.

The aim of research was to design a small, restraint free, low stress animal dosing chamber for inhalation studies, and to investigate distribution of a model drug within the chamber. A small animal dosing chamber was designed that consisted of a polymethylmethacrylate (PMMA) airtight box (40.6 x 11.4 x 21.6 cm) with a hinged top, having a nominal wall thickness of 1.25 cm. The chamber was designed to hold up to 14 mice, each having a floor area of approximately 63 cm2, in accordance with Institutional Animal Care and Use Committee (IACUC) guidelines. A "rodent proof" distribution fan was attached to the center of the hinged closure lid. The chamber was divided into 1 inch2 zones (120 in total) to enable a profile of drug distribution within the chamber to be obtained. Small holes were drilled into the side of the chamber and sealed using Parafilm to allow access to the sampling zones. Syringes (5 mL) with appropriate length polytetrafluoroethylene (PTFE) tubing were inserted into the holes to reach the sampling zones (eight on either side of the chamber giving a total of 16 zones). An aqueous caffeine solution (2% w/v) in glycerol (25% w/v) was prepared and nebulized into the chamber using an Aeroneb Pro nebulizer. Caffeine containing droplets were circulated into the chamber at a flow rate of 1.5 L/min(-1), and the air was recirculated in a closed system for a total of 20 minutes to ensure a high concentration of caffeine droplets throughout. Following nebulization, air samples (5 mL) were withdrawn from the 16 sampling zones of the sealed chamber. The process was repeated in quadruplet until a total of 64 sampling zones had been sampled. The entire experiment was also repeated with the absence of the "rodent-proof" distribution fan. Drug concentrations were calculated from a calibration curve of caffeine using UV absorbance at 272 nm. An average mass of caffeine (Standard Deviation; S.D.) of 5.0 (4.2) mg was detected throughout the chamber when the distribution fan was fitted, and caffeine 12.6 (9.7) mg was detected without the fan. This indicated that presence of the fan caused impingement of the drug on both the chamber walls and fan components; effectively removing nebulized drug from circulation within the chamber. The distribution of drug was plotted using a 3D graph; this revealed a lower concentration at the periphery and a higher concentration in the center of the chamber both with and without the distribution fan in place. In conclusion, a humane, nonrestraint rodent dosing chamber was designed for the efficient delivery of nebulized drugs for up to 14 mice simultaneously. The highest levels of the model drug caffeine were detectable throughout the small animal dosing chamber without the distribution fan. A circulation flow rate of 1.5 L/min(-1) was found to be adequate to distribute drug in the chamber. Surprisingly, the results demonstrate that avoiding the use of a distribution fan altogether maximizes the drug concentration within the chamber by reducing impingement of the nebulized drug. The small animal, restraint-free dosing chamber represents an advancement in reproducible dosing via the pulmonary route in the small animal model. The dosing chamber may be adapted to present the lung with an almost unlimited array of compounds, encompassing drugs, toxic compounds, and even pathogens, while still maintaining a relatively stress-free microenvironment for the test subject and furthermore, total safety for the operator.

Animals↗