Identification of Platinum(II) Sulfide Complexes Suitable as Intramuscular Cyanide Countermeasures.

The development of rapidly acting cyanide countermeasures using intramuscular injection (IM) represents an unmet medical need to mitigate toxicant exposures in mass casualty settings. Previous work established that cisplatin and other platinum(II) or platinum(IV)-based agents effectively mitigate cyanide toxicity in zebrafish. Cyanide's in vivo reaction with platinum-containing materials was proposed to reduce the risk of acute toxicities. However, cyanide antidote activity depended on a formulation of platinum-chloride salts with dimethyl sulfoxide (DMSO) followed by dilution in phosphate-buffered saline (PBS). A working hypothesis to explain the DMSO requirement is that the formation of platinum-sulfoxide complexes activates the cyanide scavenging properties of platinum. Preparations of isolated NaPtCl5-DMSO and Na (NH3)2PtCl-DMSO complexes in the absence of excess DMSO provided agents with enhanced reactivity toward cyanide in vitro and fully recapitulated in vivo cyanide rescue in zebrafish and mouse models. The enhancement of the cyanide scavenging effects of the DMSO ligand could be attributed to the activation of platinum(IV) and (II) with a sulfur ligand. Unfortunately, the efficacy of DMSO complexes was not robust when administered IM. Alternative Pt(II) materials containing sulfide and amine ligands in bidentate complexes show enhanced reactivity toward cyanide addition. The cyanide addition products yielded tetracyanoplatinate(II), translating to a stoichiometry of 1:4 Pt to each cyanide scavenger. These new agents demonstrate a robust and enhanced potency over the DMSO-containing complexes using IM administration in mouse and rabbit models of cyanide toxicity. Using the zebrafish model with these Pt(II) complexes, no acute cardiotoxicity was detected, and dose levels required to reach lethality exceeded 100 times the effective dose. Data are presented to support a general chemical design approach that can expand a new lead candidate series for developing next-generation cyanide countermeasures.

Intramuscular cobinamide as an antidote to methyl mercaptan poisoning.

BACKGROUND: Methyl mercaptan occurs naturally in the environment and is found in a variety of occupational settings, including the oil, paper, plastics, and pesticides industries. It is a toxic gas and deaths from methyl mercaptan exposure have occurred. The Department of Homeland Security considers it a high threat chemical agent that could be used by terrorists. Unfortunately, no specific treatment exists for methyl mercaptan poisoning. METHODS: We conducted a randomized trial in 12 swine comparing no treatment to intramuscular injection of the vitamin B12 analog cobinamide (2.0 mL, 12.5 mg/kg) following acute inhalation of methyl mercaptan gas. Physiological and laboratory parameters were similar in the control and cobinamide-treated groups at baseline and at the time of treatment. RESULTS: All six cobinamide-treated animals survived, whereas only one of six control animals lived (17% survival) (p = 0.0043). The cobinamide-treated animals returned to a normal breathing pattern by 3.8 ± 1.1 min after treatment (mean ± SD), while all but one animal in the control group had intermittent gasping, never regaining a normal breathing pattern. Blood pressure and arterial oxygen saturation returned to baseline values within 15 minutes of cobinamide-treatment. Plasma lactate concentration increased progressively until death (10.93 ± 6.02 mmol [mean ± SD]) in control animals, and decreased toward baseline (3.79 ± 2.93 mmol [mean ± SD]) by the end of the experiment in cobinamide-treated animals. CONCLUSION: We conclude that intramuscular administration of cobinamide improves survival and clinical outcomes in a large animal model of acute, high dose methyl mercaptan poisoning.

Metabolism of Cyanide by Glutathione To Produce the Novel Cyanide Metabolite 2-Aminothiazoline-4-oxoaminoethanoic Acid.

The direct analysis of cyanide (HCN or CN- inclusively symbolized as CN) to confirm exposure has major limitations due to cyanide's volatility, reactivity, and short half-life in biological fluids. These limitations have led to the exploration of cyanide detoxification products for indirect verification of cyanide exposure. Although cyanide interacts strongly with sulfur-containing molecules, to date, biomarkers resulting from the interaction of cyanide with glutathione (GSH; i.e., a biologically abundant sulfur-donating biomolecule) have yet to be discovered. In this study, we studied the interaction of CN and GSH to produce 2-aminothiazoline-4-oxoaminoethanioc acid (ATOEA). An LC-MS/MS method was developed and validated to analyze ATOEA from plasma, producing a linear range of 0.5-50 µM, a limit of detection of 200 nM, and excellent precision and accuracy. ATOEA concentrations were significantly elevated in the plasma of animals following cyanide exposure. Moreover, the production of ATOEA from cyanide exposure was confirmed by detection of both ATOEA and ATOEA-13C15N in rabbit plasma ( N = 11 animals) following administration of NaCN:K13C15N (1:1), with a similar amount of ATOEA and ATOEA-13C15N formed ( R2 = 0.9924, p < 0.05). The concentration of ATOEA increased with cyanide dose and then decreased rapidly when an antidote was administrated. This study definitively showed that ATOEA is produced from interaction of CN and GSH and can serve as a biomarker of cyanide exposure.

Efficacy of Oral Administration of Sodium Thiosulfate and Glycine in a Large, Swine Model of Oral Cyanide Toxicity.

STUDY OBJECTIVE: Cyanide is a deadly poison, particularly with oral exposure, in which larger doses can occur before any symptoms develop. Multiple governmental agencies highlight oral cyanide as an agent that can be used in a terrorist attack because it can be easily weaponized and is readily available. Currently, there are no Food and Drug Administration-approved antidotes specifically for oral cyanide. An oral countermeasure that can neutralize and prevent absorption of cyanide from the gastrointestinal tract after oral exposure is needed. The objective of this study is to determine if the combination of glycine and sodium thiosulfate administered orally is effective in reducing mortality in a large, swine model of oral cyanide toxicity. METHODS: Nine swine (45 to 55 kg) were instrumented, sedated, and stabilized. Potassium cyanide (at 8 mg/kg) in saline solution was delivered as a onetime bolus through an orogastric tube. Three minutes after cyanide administration, animals that were randomized to the treatment group received sodium thiosulfate (508.2 mg/kg, 3.25-M solution) and glycine (30 mg/kg, 3.5-M solution) through an orogastric tube. Survival at 60 minutes was the primary outcome. We compared survival between groups by log-rank Mantel-Cox analysis and trended laboratory results and vital signs. RESULTS: At baseline and treatment, all animals were similar. Survival at 60 minutes was 100% in treated animals compared with 0% in the control group (P=.003). By the study end, defined as death or 60 minutes after cyanide administration, there was a significant difference in the lactate concentration between the treatment and control groups (control 9.43 mmol/L [SD 4.08]; treatment 1.66 mmol/L [SD 0.82]; difference between means 7.69 mmol/L [SD 2.07]; 95% confidence interval difference -14.05 to -1.32). Mean arterial pressure was significantly different between the treatment and control groups at study end (control 26 mm Hg [SD 6.7]; treatment 81 mm Hg [SD 14]; difference between means 55.2 mm Hg [SD 7.1]; 95% confidence interval difference 37.8 to 72.6). pH and oxygen saturation were also significantly different between the treatment and control groups at study end. CONCLUSION: The combination of oral sodium thiosulfate and glycine significantly improved survival and physiologic parameters in a large-animal model of oral cyanide toxicity.

Sodium Nitrite and Sodium Thiosulfate Are Effective Against Acute Cyanide Poisoning When Administered by Intramuscular Injection.

STUDY OBJECTIVE: The 2 antidotes for acute cyanide poisoning in the United States must be administered by intravenous injection. In the out-of-hospital setting, intravenous injection is not practical, particularly for mass casualties, and intramuscular injection would be preferred. The purpose of this study is to determine whether sodium nitrite and sodium thiosulfate are effective cyanide antidotes when administered by intramuscular injection. METHODS: We used a randomized, nonblinded, parallel-group study design in 3 mammalian models: cyanide gas inhalation in mice, with treatment postexposure; intravenous sodium cyanide infusion in rabbits, with severe hypotension as the trigger for treatment; and intravenous potassium cyanide infusion in pigs, with apnea as the trigger for treatment. The drugs were administered by intramuscular injection, and all 3 models were lethal in the absence of therapy. RESULTS: We found that sodium nitrite and sodium thiosulfate individually rescued 100% of the mice, and that the combination of the 2 drugs rescued 73% of the rabbits and 80% of the pigs. In all 3 species, survival in treated animals was significantly better than in control animals (log rank test, P<.05). In the pigs, the drugs attenuated an increase in the plasma lactate concentration within 5 minutes postantidote injection (difference: plasma lactate, saline solution-treated versus nitrite- or thiosulfate-treated 1.76 [95% confidence interval 1.25 to 2.27]). CONCLUSION: We conclude that sodium nitrite and sodium thiosulfate administered by intramuscular injection are effective against severe cyanide poisoning in 3 clinically relevant animal models of out-of-hospital emergency care.

Development of a fluorescence-based sensor for rapid diagnosis of cyanide exposure.

Although commonly known as a highly toxic chemical, cyanide is also an essential reagent for many industrial processes in areas such as mining, electroplating, and synthetic fiber production. The "heavy" use of cyanide in these industries, along with its necessary transportation, increases the possibility of human exposure. Because the onset of cyanide toxicity is fast, a rapid, sensitive, and accurate method for the diagnosis of cyanide exposure is necessary. Therefore, a field sensor for the diagnosis of cyanide exposure was developed based on the reaction of naphthalene dialdehyde, taurine, and cyanide, yielding a fluorescent ß-isoindole. An integrated cyanide capture "apparatus", consisting of sample and cyanide capture chambers, allowed rapid separation of cyanide from blood samples. Rabbit whole blood was added to the sample chamber, acidified, and the HCN gas evolved was actively transferred through a stainless steel channel to the capture chamber containing a basic solution of naphthalene dialdehyde (NDA) and taurine. The overall analysis time (including the addition of the sample) was <3 min, the linear range was 3.13-200 µM, and the limit of detection was 0.78 µM. None of the potential interferents investigated (NaHS, NH4OH, NaSCN, and human serum albumin) produced a signal that could be interpreted as a false positive or a false negative for cyanide exposure. Most importantly, the sensor was 100% accurate in diagnosing cyanide poisoning for acutely exposed rabbits.

Chemical and metabolomic screens identify novel biomarkers and antidotes for cyanide exposure.

Exposure to cyanide causes a spectrum of cardiac, neurological, and metabolic dysfunctions that can be fatal. Improved cyanide antidotes are needed, but the ideal biological pathways to target are not known. To understand better the metabolic effects of cyanide and to discover novel cyanide antidotes, we developed a zebrafish model of cyanide exposure and scaled it for high-throughput chemical screening. In a screen of 3120 small molecules, we discovered 4 novel antidotes that block cyanide toxicity. The most potent antidote was riboflavin. Metabolomic profiling of cyanide-treated zebrafish revealed changes in bile acid and purine metabolism, most notably by an increase in inosine levels. Riboflavin normalizes many of the cyanide-induced neurological and metabolic perturbations in zebrafish. The metabolic effects of cyanide observed in zebrafish were conserved in a rabbit model of cyanide toxicity. Further, humans treated with nitroprusside, a drug that releases nitric oxide and cyanide ions, display increased circulating bile acids and inosine. In summary, riboflavin may be a novel treatment for cyanide toxicity and prophylactic measure during nitroprusside treatment, inosine may serve as a biomarker of cyanide exposure, and metabolites in the bile acid and purine metabolism pathways may shed light on the pathways critical to reversing cyanide toxicity.

Development of sodium tetrathionate as a cyanide and methanethiol antidote.

CONTEXT: Hydrogen cyanide and methanethiol are two toxic gases that inhibit mitochondrial cytochrome c oxidase. Cyanide is generated in structural fires and methanethiol is released by decaying organic matter. Current treatments for cyanide exposure do not lend themselves to treatment in the field and no treatment exists for methanethiol poisoning. Sodium tetrathionate (tetrathionate), a product of thiosulfate oxidation, could potentially serve as a cyanide antidote, and, based on its chemical structure, we hypothesized it could react with methanethiol. RESULTS: We show that tetrathionate, unlike thiosulfate, reacts directly with cyanide in vitro under physiological conditions, and based on rabbit studies where we monitor cyanide poisoning in real-time, tetrathionate likely reacts directly with cyanide in vivo. We found that tetrathionate administered by intramuscular injection rescues >80% of juvenile, young adult, and old adult mice from exposure to inhaled hydrogen cyanide gas that is >80% lethal. Tetrathionate also rescued young adult rabbits from intravenously administered sodium cyanide. Tetrathionate was reasonably well-tolerated by mice and rats, yielding a therapeutic index of ∼5 in juvenile and young adult mice, and ∼3.3 in old adult mice; it was non-mutagenic in Chinese Hamster ovary cells and by the Ames bacterial test. We found by gas chromatography-mass spectrometry that both tetrathionate and thiosulfate react with methanethiol to generate dimethyldisulfide, but that tetrathionate was much more effective than thiosulfate at recovering intracellular ATP in COS-7 cells and rescuing mice from a lethal exposure to methanethiol gas. CONCLUSION: We conclude that tetrathionate has the potential to be an effective antidote against cyanide and methanethiol poisoning.

Rapid point of care analyzer for the measurement of cyanide in blood.

A simple, sensitive optical analyzer for the rapid determination of cyanide in blood in point of care applications is described. HCN is liberated by the addition of 20% H(3)PO(4) and is absorbed by a paper filter impregnated with borate-buffered (pH 9.0) hydroxoaquocobinamide (hereinafter called cobinamide). Cobinamide on the filter changes color from orange (λ(max) = 510 nm) to violet (λ(max) = 583 nm) upon reaction with cyanide. This color change is monitored in the transmission mode by a light emitting diode (LED) with a 583 nm emission maximum and a photodiode detector. The observed rate of color change increases 10 times when the cobinamide solution for filter impregnation is prepared in borate-buffer rather than in water. The use of a second LED emitting at 653 nm and alternate pulsing of the LEDs improves the limit of detection by 4 times to ~0.5 µM for a 1 mL blood sample. Blood cyanide levels of imminent concern (≥10 µM) can be accurately measured in ~2 min. The response is proportional to the mass of cyanide in the sample: smaller sample volumes can be successfully used with proportionate change in the concentration LODs. Bubbling air through the blood-acid mixture was found effective for mixing of the acid with the sample and the liberation of HCN. A small amount of ethanol added to the top of the blood was found to be the most effective means to prevent frothing during aeration. The relative standard deviation (RSD) for repetitive determination of blood samples containing 9 µM CN was 1.09% (n = 5). The technique was compared blind with a standard microdiffusion-spectrophotometric method used for the determination of cyanide in rabbit blood. The results showed good correlation (slope 1.05, r(2) 0.9257); independent calibration standards were used.

Efficacy of oral administration of sodium thiosulfate in a large, swine model of oral cyanide toxicity.

INTRODUCTION: Cyanide is a deadly poison, particularly with oral exposure where larger doses can occur before symptoms develop. Prior studies and multiple governmentagencies highlight oral cyanide as an agent with the potential for use in a terrorist attack. Currently, there are no FDA approved antidotes specific to oralcyanide. An oral countermeasure that can neutralize and prevent absorption of cyanide from the GI tract after oral exposure is needed. Our objective was toevaluate the efficacy of oral sodium thiosulfate on survival and clinical outcomes in a large, swine model of severe cyanide toxicity. METHODS: Swine (45-55kg) were instrumented, sedated, and stabilized. Potassium cyanide (8 mg/kg KCN) in saline was delivered as a one-time bolus via an orogastric tube. Three minutes after cyanide, animals randomized to the treatment group received sodium thiosulfate (510 mg/kg, 3.25 M solution) via orogastric tube. Our primary outcome was survival at 60 minutes after exposure. We compared survival between groups by log-rank, Mantel-Cox analysis and trended labs and vital signs. RESULTS: At baseline and time of treatment all animals had similar weights, vital signs, and laboratory values. Survival at 60 min was 100% in treated animals compared to 0% in the control group (p=0.0027). Animals in the control group became apneic and subsequently died by 35.0 min (20.2,48.5) after cyanide exposure. Mean arterial pressure was significantly higher in the treatment group compared to controls (p=0.008). Blood lactate (p=0.02) and oxygen saturation (p=0.02) were also significantly different between treatment and control groups at study end. CONCLUSION: Oral administration of sodium thiosulfate improved survival, blood pressure, respirations, and blood lactate concentrations in a large animal model of acute oral cyanide toxicity.

New facile method to measure cyanide in blood.

Cyanide, a well-known toxic substance that could be used as a weapon of mass destruction, is likely responsible for a substantial percentage of smoke inhalation deaths. The vitamin B(12) precursor cobinamide binds cyanide with high affinity, changing color and, correspondingly, its spectrophotometric spectrum in the ultraviolet/visible light range. Based on these spectral changes, we developed a new facile method to measure cyanide in blood using cobinamide. The limit of detection was 0.25 nmol, while the limit of quantitation was approximately 0.5 nmol. The method was reliable, requires minimal equipment, and correlated well with a previously established method. Moreover, we adapted it for rapid qualitative assessment of cyanide concentration, which could be used in the field to identify cyanide-poisoned subjects for immediate treatment.

Sulfanegen sodium treatment in a rabbit model of sub-lethal cyanide toxicity.

The aim of this study is to investigate the ability of intramuscular and intravenous sulfanegen sodium treatment to reverse cyanide effects in a rabbit model as a potential treatment for mass casualty resulting from cyanide exposure. Cyanide poisoning is a serious chemical threat from accidental or intentional exposures. Current cyanide exposure treatments, including direct binding agents, methemoglobin donors, and sulfur donors, have several limitations. Non-rhodanese mediated sulfur transferase pathways, including 3-mercaptopyruvate sulfurtransferase (3-MPST) catalyze the transfer of sulfur from 3-MP to cyanide, forming pyruvate and less toxic thiocyanate. We developed a water-soluble 3-MP prodrug, 3-mercaptopyruvatedithiane (sulfanegen sodium), with the potential to provide a continuous supply of substrate for CN detoxification. In addition to developing a mass casualty cyanide reversal agent, methods are needed to rapidly and reliably diagnose and monitor cyanide poisoning and reversal. We use non-invasive technology, diffuse optical spectroscopy (DOS) and continuous wave near infrared spectroscopy (CWNIRS) to monitor physiologic changes associated with cyanide exposure and reversal. A total of 35 animals were studied. Sulfanegen sodium was shown to reverse the effects of cyanide exposure on oxyhemoglobin and deoxyhemoglobin rapidly, significantly faster than control animals when administered by intravenous or intramuscular routes. RBC cyanide levels also returned to normal faster following both intramuscular and intravenous sulfanegen sodium treatment than controls. These studies demonstrate the clinical potential for the novel approach of supplying substrate for non-rhodanese mediated sulfur transferase pathways for cyanide detoxification. DOS and CWNIRS demonstrated their usefulness in optimizing the dose of sulfanegen sodium treatment.

Intramuscular cobinamide sulfite in a rabbit model of sublethal cyanide toxicity.

STUDY OBJECTIVE: Exposure to cyanide in fires and industrial exposures and intentional cyanide poisoning by terrorists leading to mass casualties is an ongoing threat. Current treatments for cyanide poisoning must be administered intravenously, and no rapid treatment methods are available for mass casualty cyanide exposures. Cobinamide is a cobalamin (vitamin B(12)) analog with an extraordinarily high affinity for cyanide that is more water-soluble than cobalamin. We investigate the use of intramuscular cobinamide sulfite to reverse cyanide toxicity-induced physiologic changes in a sublethal cyanide exposure animal model and determine the ability of an intramuscular cobinamide sulfite injection to rapidly reverse the physiologic effects of cyanide toxicity. METHODS: New Zealand white rabbits were given 10 mg sodium cyanide intravenously over 60 minutes. Quantitative diffuse optical spectroscopy and continuous-wave near-infrared spectroscopy monitoring of tissue oxyhemoglobin and deoxyhemoglobin concentrations were performed concurrently with blood cyanide level measurements and cobinamide levels. Immediately after completion of the cyanide infusion, the rabbits were injected intramuscularly with cobinamide sulfite (n=6) or inactive vehicle (controls, n=5). RESULTS: Intramuscular administration led to rapid mobilization of cobinamide and was extremely effective at reversing the physiologic effects of cyanide on oxyhemoglobin and within deoxyhemoglobin extraction. Recovery time to 63% of their baseline values in the central nervous system occurred within a mean of 1,032 minutes in the control group and 9 minutes in the cobinamide group, with a difference of 1,023 minutes (95% confidence interval 116 to 1,874 minutes). In muscle tissue, recovery times were 76 and 24 minutes, with a difference of 52 minutes (95% confidence interval 7 to 98 minutes). RBC cyanide levels returned toward normal significantly faster in cobinamide sulfite-treated animals than in control animals. CONCLUSION: Intramuscular cobinamide sulfite rapidly and effectively reverses the physiologic effects of cyanide poisoning, suggesting that a compact cyanide antidote kit can be developed for mass casualty cyanide exposures.

Intramuscular sodium tetrathionate as an antidote in a clinically relevant swine model of acute cyanide toxicity.

Background: Cyanide is a metabolic poison used in multiple industries and is a high threat chemical agent. Current antidotes require intravenous administration, limiting their usefulness in a mass casualty scenario. Sodium tetrathionate reacts directly with cyanide yielding thiosulfate and the non-toxic compound thiocyanate. Thiosulfate, in turn, neutralizes a second molecule of cyanide, thus, per mole, sodium tetrathionate neutralizes two moles of cyanide. Historical studies examined its efficacy as a cyanide antidote, but it has not been evaluated in a clinically relevant, large animal model, nor has it previously been administered by intramuscular injection.Objective: The objective of this study is to evaluate the efficacy of intramuscular sodium tetrathionate on survival and clinical outcomes in a large, swine model of severe cyanide toxicity.Methods: Anesthetized swine were instrumented for continuous monitoring of hemodynamics, then acclimated and breathing spontaneously prior to potassium cyanide infusion (0.17 mg/kg/min). At 6-min post-apnea (no breaths for 20 s), the cyanide infusion was terminated, and animals were treated with sodium tetrathionate (∼18 mg/kg) or normal saline control. Clinical parameters and laboratory values were evaluated at various time points until death or termination of the experiment (90 min post-treatment).Results: Laboratory values, vital signs, and time to apnea were similar in both groups at baseline and treatment. Survival in the sodium tetrathionate treated group was 100% and 17% in controls (p = 0.0043). All animals treated with sodium tetrathionate returned to breathing at a mean time of 10.85 min after antidote, and all but one control remained apneic through end of the experiment. Animals treated with tetrathionate showed improvement in blood lactate (p ≤ 0.002) starting at 30 min post-treatment. The average time to death in the control group is 63.3 ± 23.2 min. No systemic or localized adverse effects of intramuscular administration of sodium tetrathionate were observed.Conclusion: Sodium tetrathionate significantly improves survival and clinical outcomes in a large, swine model of acute cyanide poisoning.

Diagnosis of cyanide poisoning using an automated, field-portable sensor for rapid analysis of blood cyanide concentrations.

Cyanide (both HCN and CN- are represented by CN) has multiple industrial applications, is commonly found in some foods, and is a component of fire smoke. Upon exposure, CN blocks production of adenosine triphosphate, causing cellular hypoxia and cytotoxic anoxia, which can eventually result in death. Considering CN's quick onset of action and the long analysis times associated with current techniques, the objective of this study was to develop and validate a rapid and field-portable sensor to detect blood CN concentrations focusing on both concentration and diagnostic accuracy. The sensor takes advantage of the chemical properties of CN by converting it exclusively to HCN via acidification of whole blood. High-speed headspace transfer is used to deliver HCN to a capture solution where it is reacted with naphthalene dialdehyde and taurine to produce a fluorescent ß-isoindole product. Simple spectrofluorometric analysis of the product provides quantitative analysis of CN from whole blood in 60 s and requires only 25 µL of blood (obtainable via fingerstick). A limit of detection of 5 µM, a linear range of 10-200 µM (with ≥15 µM considered CN exposed), and excellent accuracy (100 ± 15%) and precision (≤15.2% relative standard deviation) were obtained. To evaluate the diagnostic accuracy of the sensor, rabbit blood samples (N = 190, including 24 blinded samples) were analyzed by both the sensor and a lab-based spectrophotometric method. An excellent positive correlation was obtained between the sensor and the lab-based method (R2 ˃ 0.995) confirming the concentration accuracy of the CN sensor. Moreover, the sensor produced no false positives or negatives when diagnosing CN poisoning.

Airway epithelium stimulates smooth muscle proliferation.

Communication between the airway epithelium and stroma is evident during embryogenesis, and both epithelial shedding and increased smooth muscle proliferation are features of airway remodeling. Hence, we hypothesized that after injury the airway epithelium could modulate airway smooth muscle proliferation. Fully differentiated primary normal human bronchial epithelial (NHBE) cells at an air-liquid interface were co-cultured with serum-deprived normal primary human airway smooth muscle cells (HASM) using commercially available Transwells. In some co-cultures, the NHBE were repeatedly (x4) scrape-injured. An in vivo model of tracheal injury consisted of gently denuding the tracheal epithelium (x3) of a rabbit over 5 days and then examining the trachea by histology 3 days after the last injury. Our results show that HASM cell number increases 2.5-fold in the presence of NHBE, and 4.3-fold in the presence of injured NHBE compared with HASM alone after 8 days of in vitro co-culture. In addition, IL-6, IL-8, monocyte chemotactic protein (MCP)-1 and, more markedly, matrix metalloproteinase (MMP)-9 concentration increased in co-culture correlating with enhanced HASM growth. Inhibiting MMP-9 release significantly attenuated the NHBE-dependent HASM proliferation in co-culture. In vivo, the injured rabbit trachea demonstrated proliferation in the smooth muscle (trachealis) region and significant MMP-9 staining, which was absent in the uninjured control. The airway epithelium modulates smooth muscle cell proliferation via a mechanism that involves secretion of soluble mediators including potential smooth muscle mitogens such as IL-6, IL-8, and MCP-1, but also through a novel MMP-9-dependent mechanism.

Determination of free cyano-cobinamide in swine and rabbit plasma by liquid chromatography tandem mass spectrometry.

In recent years, Cobinamide (Cbi) has shown promise as a therapeutic for cyanide poisoning. There are several forms of Cbi based on the identity of the ligands bound to the cobalt in Cbi and these different forms of Cbi have divergent behavior (e.g., the aquo and hydroxo forms of Cbi readily bind to proteins, limiting their distribution significantly, whereas [Cbi(CN)2] does not). While current analysis techniques only measure total Cbi, methods to elucidate the behavior of 'available' Cbi versus cyanide-complexed Cbi would be valuable for biomedical and pharmacokinetic studies. Therefore, a method was developed for the analysis of cyanide-complexed Cbi in plasma via liquid chromatography tandem mass spectrometry (LC-MS-MS). Plasma samples were prepared by denaturing proteins with 10% ammonium hydroxide in acetonitrile. The resulting mixture was centrifuged, and the supernatant was removed, dried, and reconstituted. Cyanide-complexed Cbi was then analyzed via LC-MS-MS. The limit of detection was 0.2 µM, and the linear dynamic range was between 1 and 200 µM. The accuracy was 100 ±â€¯17% and the precision, measured by relative standard deviation (%RSD), was ≤18.5%. Carryover, a severe problem when analyzing Cbi via liquid chromatography was eliminated using a polymeric-based stationary phase (PLRP-S) and a controlled washing protocol. The method allowed evaluation of the cyanide-bound and 'available' Cbi from treated animals and, when paired with a method for total Cbi analysis, allows for estimation of Cbi utilization when treating cyanide poisoning.

Hydrogen Sulfide Toxicity: Mechanism of Action, Clinical Presentation, and Countermeasure Development.

INTRODUCTION: Hydrogen sulfide (H2S) is found in various settings. Reports of chemical suicide, where individuals have combined readily available household chemicals to produce lethal concentrations of H2S, have demonstrated that H2S is easily produced. Governmental agencies have warned of potential threats of use of H2S for a chemical attack, but currently there are no FDA-approved antidotes for H2S. An ideal antidote would be one that is effective in small volume, readily available, safe, and chemically stable. In this paper we performed a review of the available literature on the mechanism of toxicity, clinical presentation, and development of countermeasures for H2S toxicity. DISCUSSION: In vivo, H2S undergoes an incomplete oxidation after an exposure. The remaining non-oxidized H2S is found in dissolved and combined forms. Dissolved forms such as H2S gas and sulfhydryl anion can diffuse between blood and tissue. The combined non-soluble forms are found as acid-labile sulfides and sulfhydrated proteins, which play a role in toxicity. Recent countermeasure development takes into account the toxicokinetics of H2S. Some countermeasures focus on binding free hydrogen sulfide (hydroxocobalamin, cobinamide); some have direct effects on the mitochondria (methylene blue), while others work by mitigating end organ damage by generating other substances such as nitric oxide (NaNO2). CONCLUSION: H2S exists in two main pools in vivo after exposure. While several countermeasures are being studied for H2S intoxication, a need exists for a small-volume, safe, highly effective antidote with a long shelf life to treat acute toxicity as well as prevent long-term effects of exposure.

Intramuscular dimethyl trisulfide: efficacy in a large swine model of acute severe cyanide toxicity.

BACKGROUND: Cyanide is a deadly compound used as a terrorist agent. Current FDA approved antidotes require intravenous administration, limiting their utility in a mass casualty scenario. Dimethyl trisulfide (DMTS), a sulfur-based molecule, binds cyanide converting it to the less toxic by-product thiocyanate. Studies evaluating efficacy in rodents have been performed, but a large, clinically relevant animal model has not been reported. OBJECTIVE: This study evaluates the efficacy of intramuscular DMTS on survival and clinical outcomes in a swine model of acute, severe cyanide toxicity. METHODS: Anesthetized swine were instrumented for continuous monitoring of hemodynamics. Prior to potassium cyanide infusion animals were acclimated and breathing spontaneously. At 5-minutes post-apnea animals were treated with DMTS or saline. Vital signs, hemodynamics, and laboratory values were evaluated at various time points. RESULTS: Baseline values and time to apnea were similar in both groups. Survival in the DMTS treated group was 83.3% and 0% in saline controls (p = .005). The DMTS group returned to breathing at a mean time of 19.3 ± 10 min after antidote, control animals did not return to breathing (CI difference 8.8, 29.8). At the end of the experiment or time of death, mean lactate was 9.41 mmol/L vs. 4.35 mmol/L (CI difference -10.94,0.82) in the saline and DMTS groups, respectively and pH was 7.20 vs. 7.37 (CI difference -0.04, 0.38). No adverse effects were observed at the injection site. CONCLUSION: Intramuscular administration of DMTS improves survival and clinical outcomes in our large animal swine model of acute cyanide toxicity.

Intramuscular cobinamide versus saline for treatment of severe hydrogen sulfide toxicity in swine.

INTRODUCTION: Hydrogen sulfide (H2S) is found in petroleum, natural gas, and decaying organic matter. Terrorist groups have attempted to use it in enclosed spaces as a chemical weapon. Mass casualty scenarios have occurred from industrial accidents and release from oil field sites. There is no FDA approved antidote for sulfide poisoning. We have previously reported that intravenous cobinamide is effective for sulfide poisoning. A rapid-acting antidote that is easy to administer intramuscularly (IM) would be ideal for use in a prehospital setting. In this study, we assessed survival in sulfide-poisoned swine treated with IM cobinamide. METHODS: Eleven swine (45-55 kg) were anesthetized, intubated, and instrumented with continuous femoral and pulmonary artery pressure monitoring. After stabilization, anesthesia was adjusted such that animals ventilated spontaneously with a FiO2 of 0.21. Sodium hydrosulfide (NaHS, 8 mg/mL) was infused intravenously at 0.9 mg/kg.min until apnea or severe hypotension. Animals were randomly assigned to receive cobinamide (4 mg/kg), or no treatment at the apnea/hypotension trigger. The NaHS infusion rate was sustained for 1.5 min post trigger, decreased to 0.2 mg/kg.min for 10 min, and then discontinued. RESULTS: The amount of NaHS required to produce apnea or hypotension was not statistically different in both groups (cobinamide: 9.0 mg/kg ±6.1; saline: 5.9 mg/kg ±5.5; mean difference: -3.1, 95% CI: -11.3, 5.0). All of the cobinamide treated animals survived (5/5), none of the control (0/6) animals survived (p < .01). Mean time to return to spontaneous ventilation in the cobinamide treated animals was 3.2 (±1.1) min. Time to return to baseline systolic blood pressure (±5%) in cobinamide-treated animals was 5 min. CONCLUSION: Intramuscular cobinamide was effective in improving survival in this large swine model of severe hydrogen sulfide toxicity.