Pharmacokinetics and pharmacodynamics of standard nerve agent medical countermeasures in Göttingen Minipigs.

Animal research continues to serve a critical role in the testing and development of medical countermeasures. The Göttingen minipig, developed for laboratory research, may provide many benefits for addressing research questions within chemical defense. Targeted development of the Göttingen minipig model could reduce reliance upon non-human primates, and improve study design, statistical power, and throughput to advance medical countermeasures for regulatory approval and fielding. In this vein, we completed foundational pharmacokinetics and physiological safety studies of intramuscularly administered atropine sulfate, pralidoxime chloride (2-PAM), and diazepam across a broad range of doses (1-6 autoinjector equivalent) using adult male Göttingen minipigs (n=11; n=4-8/study) surgically implanted with vascular access ports and telemetric devices to monitor cardiovascular, respiratory, arterial pressure, and temperature signals. Pharmacokinetic data were orderly and the concentration maximum mirrored available human data at comparably scaled doses clearly for atropine, moderately for 2-PAM, and poorly for diazepam. Time to peak concentration approximated 2, 7, and 20 min for atropine, 2-PAM, and diazepam, respectively, and the elimination half-life of these drugs approximated 2 hr (atropine), 3 hr (2-PAM), and 8 hr (diazepam). Atropine sulfate dose-dependently increased the magnitude and duration of tachycardia and decreased the PR and ST intervals (consistent with findings obtained from other species). Mild hypothermia was observed at the highest diazepam dose. Göttingen minipigs appear to provide a ready and appropriate large animal alternative to non-human primates, and further development and evaluation of novel nerve agent medical countermeasures and treatment strategies in this model are justified.

Assessment of naloxone as a therapeutic for inhaled carfentanil in the ferret.

Carfentanil is a powerful synthetic opioid that is approximately 100 times more potent than fentanyl and 10,000 times more potent than morphine. Carfentanil was originally intended to be used as a sedative for big game animals in a veterinary setting, but it is becoming increasingly recognized as a public health concern. We set out to investigate the effectiveness of naloxone against a potentially lethal dose of inhaled carfentanil in male ferrets. Ferrets were implanted with telemetry devices to study cardiac parameters and exposed to aerosolized carfentanil in a whole-body plethysmography chamber to record respiratory parameters. We observed profound respiratory depression in exposed animals, which led to apneic periods constituting 24-31 % of the exposure period. Concomitant with these apneic periods, we also observed cardiac abnormalities in the form of premature junctional contractions (PJCs). At our acute exposure dose, lethal in 3 % of our animals, naïve ferrets were unresponsive and incapacitated for a total of 126.1 ± 24.6 min. When administered intramuscularly at human equivalent doses (HEDs) of either 5 mg or 10 mg, naloxone significantly reduced the time that ferrets were incapacitated following exposure, although we observed no significant difference in the reduction of time that the animals were incapacitated between the treatment groups. Naloxone was able to quickly resolve the respiratory depression, significantly reducing the frequency of apneic periods in carfentanil-exposed ferrets. Our results suggest that naloxone, when administered via intramuscular injection following incapacitation, is a viable treatment against the effects of a potentially lethal dose of inhaled carfentanil.

Symptomatic treatment of botulism with a clinically approved small molecule.

Botulinum neurotoxins (BoNTs) are potent neuroparalytic toxins that cause mortality through respiratory paralysis. The approved medical countermeasure for BoNT poisoning is infusion of antitoxin immunoglobulins. However, antitoxins have poor therapeutic efficacy in symptomatic patients; thus, there is an urgent need for treatments that reduce the need for artificial ventilation. We report that the US Food and Drug Administration-approved potassium channel blocker 3,4-diaminopyridine (3,4-DAP) reverses respiratory depression and neuromuscular weakness in murine models of acute and chronic botulism. In ex vivo studies, 3,4-DAP restored end-plate potentials and twitch contractions of diaphragms isolated from mice at terminal stages of BoNT serotype A (BoNT/A) botulism. In vivo, human-equivalent doses of 3,4-DAP reversed signs of severe respiratory depression and restored mobility in BoNT/A-intoxicated mice at terminal stages of respiratory collapse. Multiple-dosing administration of 3,4-DAP improved respiration and extended survival at up to 5 LD50 BoNT/A. Finally, 3,4-DAP reduced gastrocnemius muscle paralysis and reversed respiratory depression in sublethal models of serotype A-, B-, and E-induced botulism. These findings make a compelling argument for repurposing 3,4-DAP to symptomatically treat symptoms of muscle paralysis caused by botulism, independent of serotype. Furthermore, they suggest that 3,4-DAP is effective for a range of botulism symptoms at clinically relevant time points.

A Randomized Cross-Over Trial Comparing the Effect of Intramuscular Versus Intranasal Naloxone Reversal of Intravenous Fentanyl on Odor Detection in Working Dogs.

Fentanyl is a potent opioid used clinically as a pain medication and anesthetic but has recently seen a sharp rise as an illicit street drug. The potency of fentanyl means mucous membrane exposure to a small amount of the drug can expose first responders, including working canines, to accidental overdose. Naloxone, a fast-acting opioid antagonist administered intranasally (IN) or intramuscularly (IM) is currently carried by emergency personnel in the case of accidental exposure in both humans and canines. Despite the fact that law enforcement relies heavily on the olfactory abilities of canine officers, the effects of fentanyl exposure and subsequent reversal by naloxone on the olfactory performance of canines are unknown. In a block-randomized, crossover trial, we tested the effects of IN and IM naloxone on the abilities of working dogs to recognize the odor of Universal Detection Calibrant (UDC) prior to, and two, 24, and 48 h after intravenous fentanyl sedation and naloxone reversal. No detectable influence of fentanyl sedation and naloxone reversal on the dogs' olfactory abilities was detected. We also found no difference in olfactory abilities when dogs received IN or IM naloxone. Together, results suggest no evidence that exposure to intravenous fentanyl followed by naloxone reversal impairs canine olfactory ability under these conditions.

Plasma Concentration of Meloxicam in Pediatric Rats.

In this study, we compared the plasma concentrations of meloxicam in pediatric rat pups (ages: 7, 14, 21, and 28 d) with those of young adult rats. Adult rats received 1.34 mg/kg SC meloxicam to determine the target peak plasma concentration (Cmax) for comparison with the pediatric animals. Pediatric rats received 1.34 mg/kg SC meloxicam, and in all age groups, Cmax met or exceeded that in adults (11.5 ±2.7 µg/mL). Plasma concentrations were similar between male and female pups within age groups, and peak plasma concentration was achieved more rapidly in rat pups than adults. The analgesic efficacy of this dose was not evaluated in this study.

Towards Development of Small Molecule Lipid II Inhibitors as Novel Antibiotics.

Recently we described a novel di-benzene-pyrylium-indolene (BAS00127538) inhibitor of Lipid II. BAS00127538 (1-Methyl-2,4-diphenyl-6-((1E,3E)-3-(1,3,3-trimethylindolin-2-ylidene)prop-1-en-1-yl)pyryl-1-ium) tetrafluoroborate is the first small molecule Lipid II inhibitor and is structurally distinct from natural agents that bind Lipid II, such as vancomycin. Here, we describe the synthesis and biological evaluation of 50 new analogs of BAS00127538 designed to explore the structure-activity relationships of the scaffold. The results of this study indicate an activity map of the scaffold, identifying regions that are critical to cytotoxicity, Lipid II binding and range of anti-bacterial action. One compound, 6jc48-1, showed significantly enhanced drug-like properties compared to BAS00127538. 6jc48-1 has reduced cytotoxicity, while retaining specific Lipid II binding and activity against Enterococcus spp. in vitro and in vivo. Further, this compound showed a markedly improved pharmacokinetic profile with a half-life of over 13 hours upon intravenous and oral administration and was stable in plasma. These results suggest that scaffolds like that of 6jc48-1 can be developed into small molecule antibiotic drugs that target Lipid II.

Silicon is a frequent component of atmospheric nanoparticles.

Nanoparticles are the largest fraction of aerosol loading by number. Knowledge of the chemical components present in nanoparticulate matter is needed to understand nanoparticle health and climatic impacts. In this work, we present field measurements using the Nano Aerosol Mass Spectrometer (NAMS), which provides quantitative elemental composition of nanoparticles around 20 nm diameter. NAMS measurements indicate that the element silicon (Si) is a frequent component of nanoparticles. Nanoparticulate Si is most abundant in locations heavily impacted by anthropogenic activities. Wind direction correlations suggest the sources of Si are diffuse, and diurnal trends suggest nanoparticulate Si may result from photochemical processing of gas phase Si-containing compounds, such as cyclic siloxanes. Atmospheric modeling of oxidized cyclic siloxanes is consistent with a diffuse photochemical source of aerosol Si. More broadly, these observations indicate a previously overlooked anthropogenic source of nanoaerosol mass. Further investigation is needed to fully resolve its atmospheric role.

Selective detection and characterization of nanoparticles from motor vehicles.

Numerous studies have shown that exposure to motor vehicle emissions increases the probability of heart attacks, asthma attacks, and hospital visits among at-risk individuals. However, while many studies have focused on measurements of ambient nanoparticles near highways, they have not focused on specific road-level domains, such as intersections near population centers. At these locations, very intense spikes in particle number concentration have been observed. These spikes have been linked to motor vehicle activity and have the potential to increase exposure dramatically. Characterizing both the contribution and composition of these spikes is critical in developing exposure models and abatement strategies. To determine the contribution of the particle spikes to the ambient number concentration, we implemented wavelet-based algorithms to isolate the particle spikes from measurements taken during the summer and winter of 2009 in Wilmington, Delaware, adjacent to a roadway intersection that approximately 28,000 vehicles pass through daily. These measurements included both number concentration and size distributions recorded once every second by a condensation particle counter (CPC*; TSI, Inc., St. Paul, MN) and a fast mobility particle sizer (FMPS). The high-frequency portion of the signal, consisting of a series of abrupt spikes in number concentration that varied in length from a few seconds to tens of seconds, accounted for 3% to 35% of the daily ambient number concentration, with spike contributions sometimes greater than 50% of hourly number concentrations. When the data were weighted by particle volume, this portion of the signal contributed an average of 10% to 20% to the daily concentration of particulate matter (PM) < or = 0.1 microm in aerodynamic diameter (PM0.1). The preferred locations for observing particle concentration spikes were those surrounding the measurement site at which motor vehicles accelerated after a red traffic light turned green. As the distance or transit time from emission to sampling increased, the size distribution shifted to a larger particle size, which confirmed the source assignments. To determine the distribution of emissions from individual vehicles, we correlated camera images with the spike contribution to particle number concentration at each time point. A small percentage of motor vehicles were found to emit a disproportionally large concentration of nanoparticles, and these high emitters included both spark-ignition (SI) and heavy-duty diesel (HDD) vehicles. In addition to characterizing the contribution of the spikes (local sources) to the ambient number concentration, we developed a method to determine the net contribution of motor vehicles (all sources) to the total mass concentration of ambient nanoparticles. To do this, we correlated the concentration of spikes with measurements of fast changes in the chemical composition of nanoparticles measured with the nano aerosol mass spectrometer (NAMS; built by the Johnston group). The NAMS irradiates individual, size-selected nanoparticles with a high-energy laser pulse to generate a mass spectrum consisting of multiply charged atomic ions. The elemental composition of each particle was determined from the ion signal intensities of each element. However, overlapping mass-to-charge ratios (m/z) at 4 m/z (O(+4) and C(+3)) and at 8 m/z (O(+2) and S(+4)) needed to be separated into their component ions to obtain a representative composition. To do this, we developed a method to deconvolute these ion signals using sucrose and ammonium sulfate [(NH4)2SO4] as calibration standards. With this approach, the differences between the expected and measured elemental mole fractions of carbon (C), oxygen (O), nitrogen (N), and sulfur (S) for a variety of test particles were generally much less than 10%. Ambient nanoparticles were found to consist mostly of C, O, N, and S. Many particles also contained silicon (Si). The elemental compositions were apportioned into molecular species that are commonly found in ambient aerosol: sulfate (SO4(2-)), nitrate (NO3-), ammonium (NH4+), carbonaceous matter, and when present, silicon dioxide (SiO2). Correlating NAMS chemical-composition measurements with spike contributions allowed for the development of a chemical profile representing motor vehicle emissions, which could be used to apportion their total contribution to the ambient nanoparticle mass. Particles originating from motor vehicles had compositions dominated by unoxidized carbonaceous matter, whereas non-motor vehicle particles consisted mostly of SO42-, NO3-, and oxidized carbonaceous matter. Motor vehicles were found to contribute up to 48% and 60% of the nanoparticle mass and number concentrations, respectively, in the winter measurement period, but only 16% and 49% of the nanoparticle mass and number concentrations, respectively, in the summer period. Chemical-composition profiles and contributions of SI versus HDD vehicles to the nanoparticle mass concentration were estimated by correlating still camera images, chemical composition, and spike contributions at each time point. The total mass contributions from SI and HDD vehicles were roughly equal, but the uncertainty in the split was large. The results of this study suggest that nanoparticle concentrations will be higher adjacent to an intersection than along the same roadway but further from an intersection. Possible ways to reduce the motor vehicle contribution to ambient nanoparticulate matter include minimizing stop-and-go activity at an intersection (i.e., vehicles accelerating after a red light turns green) and identifying the small fraction of motor vehicles that emit a disproportionally large number of nanoparticles.

Molecular transformations accompanying the aging of laboratory secondary organic aerosol.

The aging of fresh secondary organic aerosol (SOA), formed in a flow tube reactor by α-pinene ozonolysis, was studied by passing the fresh SOA into a second chamber for reaction with high levels of the hydroxyl radical. Two types of experiments were performed: (1) injection of a short plug of fresh SOA into the second chamber, where the particle mass and average O/C mole ratio were measured as a function of time after injection, and (2) injection of a continuous stream of fresh SOA into the second chamber, where particles were collected on a filter over a period of time for off line analysis by high performance mass spectrometry. These setups allowed the chemistry of SOA aging to be elucidated. The particle mass decreased and average O/C ratio increased with increasing aging time. Aged SOA showed an oligomer distribution shifted to lower molecular weight (fragmentation) and molecular formulas with higher O/C and lower H/C ratios (functionalization). Carbon oxidation states of individual molecules were higher for aged SOA, 0 to +2, than fresh SOA, -1 to 0. Tandem mass spectrometry of oligomers from fresh SOA showed small neutral losses associated with less oxidized functional groups such as aldehydes and ketones, while oligomers from aged SOA showed losses associated with more highly oxidized groups such as acids and peroxyacids. Product ion spectra of fresh SOA showed monomer building blocks with formulas corresponding to primary ozonolysis products such as pinic and pinonic acids, whereas aged SOA monomer building blocks corresponded to extremely oxidized products such as dimethyltricarballylic acid.

Quantitative and time-resolved nanoparticle composition measurements during new particle formation.

The chemical composition of 20 nm diameter particles was measured with the Nano Aerosol Mass Spectrometer (NAMS) in a rural/coastal environment during days when new particle formation (NPF) occurred and days when NPF did not occur. NAMS provides a quantitative measure of nanoparticle elemental composition with high time resolution. These measurements show that nanoparticle chemical composition is dynamic on both types of days and that changes in nanoparticle chemical composition do not necessarily correlate with changes in aerosol mass or number concentration. On NPF days, NAMS can distinguish between elements associated with particle formation and early mass growth from those associated with later mass growth. In the early stage of NPF, the particle phase sulphur mole fraction (S) increases simultaneously with the increase in gas phase sulphuric acid. This composition change occurs before the mode diameter has grown into the NAMS-measured size range and is quantitatively described by sulphuric acid condensation. The nitrogen mole fraction (N) also increases during this time period. The N/S mole ratio is approximately 2, indicating that particulate sulphate is fully neutralized. As the mode diameter passes into and through the NAMS-measured size range, N increases at a faster rate than S (N/S mole ratio increases above 2), indicating that a separate, nitrogen-based growth process exists, possibly involving aminium salts, inorganic nitrate and/or organonitrates. Carbonaceous matter is the most abundant component (-50% by mass) of the growing nanoparticles, but it is the inorganic species that are preferentially enhanced during NPF relative to other times of day. Concurrent measurements of cloud condensation nucleation activity during NPF events suggest that these newly formed particles are hygroscopic. Nanoparticle composition on non-NPF days also shifts toward a more inorganic composition during the daytime, but the chemical species are different from NPF days and the particles are less hygroscopic. Incorporation of S into growing nanoparticles is adequately explained by existing models, but currently no models exist to satisfactorily explain incorporation of nitrogen-containing species or carbonaceous matter.

Quantitative assessment of the sulfuric acid contribution to new particle growth.

The Nano Aerosol Mass Spectrometer (NAMS) was deployed to rural/coastal and urban sites to measure the composition of 20-25 nm diameter nanoparticles during new particle formation (NPF). NAMS provides a quantitative measure of the elemental composition of individual, size-selected nanoparticles. In both environments, particles analyzed during NPF were found to be enhanced in elements associated with inorganic species (nitrogen, sulfur) relative to that associated with organic species (carbon). A molecular apportionment algorithm was applied to the elemental data in order to place the elemental composition into a molecular context. These measurements show that sulfate constitutes a substantial fraction of total particle mass in both environments. The contribution of sulfuric acid to new particle growth was quantitatively determined and the gas-phase sulfuric acid concentration required to incorporate the measured sulfate fraction was calculated. The calculated values were compared to those calculated by a sulfuric acid proxy that considers solar radiation and SO(2) levels. The two values agree within experimental uncertainty. Sulfate accounts for 29-46% of the total mass growth of particles. Other species contributing to growth include ammonium, nitrate, and organics. For each location, the relative amounts of these species do not change significantly with growth rate. However, for the coastal location, sulfate contribution increases with increasing temperature whereas nitrate contribution decreases with increasing temperature.

Chemical composition of ambient nanoparticles on a particle-by-particle basis.

The nano aerosol mass spectrometer provides a quantitative measure of the elemental composition of individual, ambient nanoparticles in the 10-30 nm size range. In this work, carbon mole fraction plots are introduced as an efficient means of visualizing the full range of particle compositions in an ambient data set. These plots are constructed by plotting the composition of each particle in the data set, beginning with the particle having the highest carbon mole fraction and ending with the particle having the lowest carbon mole fraction. The method relies on the observation that the carbon content of an ambient particle is generally anticorrelated with oxygen, nitrogen, and sulfur. Carbon mole fraction plots allow internal vs external mixing of particle compositions to be assessed, and they provide a means of exploring the relationship between the oxidation of carbonaceous matter and the presence of inorganic species in a particle. It is shown that unoxidized carbonaceous matter exists primarily as externally mixed particles, whereas oxidized carbonaceous matter is found only in particles that also contain a significant amount of inorganic species. Particles containing oxidized carbonaceous matter are generally neutralized, whereas particles containing unoxidized carbonaceous matter or no carbon at all are acidic. Carbon mole fraction plots show how factor analysis methods such as the Adaptive Resonance Theory-2a algorithm (ART-2a) and positive matrix factorization partition a continuum of particle compositions into a few fixed composition profiles, and they provide a simple way to characterize how ambient particle compositions change with season and/or location.

Apportionment of motor vehicle emissions from fast changes in number concentration and chemical composition of ultrafine particles near a roadway intersection.

High frequency spikes in ultrafine number concentration near a roadway intersection arise from motor vehicles that accelerate after a red light turns green. The present work describes a method to determine the contribution of motor vehicles to the total ambient ultrafine particle mass by correlating these number concentration spikes with fast changes in ultrafine particle chemical composition measured with the nano aerosol mass spectrometer, NAMS. Measurements were performed at an urban air quality monitoring site in Wilmington, Delaware during the summer and winter of 2009. Motor vehicles were found to contribute 48% of the ultrafine particle mass in the winter measurement period, but only 16% of the ultrafine particle mass in the summer period. Chemical composition profiles and contributions to the ultrafine particle mass of spark vs diesel vehicles were estimated by correlating still camera images, chemical composition and spike contribution at each time interval.. The spark and diesel contributions were roughly equal, but the uncertainty in the split was large. The distribution of emissions from individual vehicles was determined by correlating camera images with the spike contribution to particle number concentration at each time interval. A small percentage of motor vehicles were found to emit a disproportionally large concentration of ultrafine particles, and these high emitters included both spark ignition and diesel vehicles.

Ultrafine particles near a roadway intersection: origin and apportionment of fast changes in concentration.

A wavelet-based algorithm was implemented to separate the high frequency portion of ambient nanoparticle measurements taken during the summer and winter of 2009 in Wilmington, Delaware. These measurements included both number concentration and size distributions recorded once every second by a condensation particle counter (CPC) and a fast mobility particle sizer (FMPS). The high frequency portion of the signal, consisting of a series of abrupt spikes in number concentration that varied in length from a few seconds to tens of seconds, accounted for 6-35% of the daily ambient number concentration with hourly contributions sometimes greater than 50%. When the data were weighted by particle volume, this portion of the signal contributed an average of 20% to the daily PM(0.1) concentration. Particle concentration spikes were preferentially observed from locations surrounding the measurement site where motor vehicles accelerate after a red traffic light turns green. As the distance or transit time from emission to sampling increased, the size distribution shifted to larger particle diameters.

Elemental composition of nanoparticles with the nano aerosol mass spectrometer.

The nano aerosol mass spectrometer (NAMS) irradiates individual, size selected nanoparticles with a high energy laser pulse to generate a mass spectrum consisting of multiply charged atomic ions. The elemental composition of the particle is determined from the ion signal intensities of each element, which requires deconvoluting isobaric ion signals at 4 m/z (O(4+) and C(3+)) and at 8 m/z (O(2+) and S(4+)). A method to deconvolute these ion signals using sucrose and ammonium sulfate as calibrants is presented. The approach is based on the assumption that the charge state distribution of a given element is independent of the chemical form of that element in the particle. Relative to previously reported methodology, the new approach permits accurate and precise determination of sulfur, which is crucial for interpretation of ambient nanoparticle data sets. With this approach, the differences between expected and measured elemental ratios of C, O, N, and S for a variety of test particles were generally much less than 10%, although a difference as high as 16% was observed.