Evaluation of the Long-Term Storage Stability of the Cyanide Antidote: Dimethyl Trisulfide and Degradation Product Identification.

This study reports the long-term storage stability of a formulation of the cyanide (CN) antidote dimethyl trisulfide (DMTS). The F3-formulated DMTS was stored in glass ampules at 4, 22, and 37 °C. Over a period of one year, nine ampules (n = 3 at each temperature) were analyzed by high-performance liquid chromatography (HPLC)-UV/vis at daily time intervals in the first week, weekly time intervals in the first month, and monthly thereafter for a period of one year to determine the DMTS content. No measurable loss of DMTS was found at 4 and 22 °C, and good stability was noted up to five months for samples stored at 37 °C. At 37 °C, a 10% (M/M) decrease of DMTS was discovered at the sixth month and only 30% (M/M) of DMTS remained by the end of the study; discoloration of the formulation and the growth of new peaks in the HPLC chromatogram were also observed. To identify the unknown peaks at 37 °C, controlled oxidation studies were performed on DMTS using two strong oxidizing agents: meta-chloroperoxybenzoic acid (mCPBA) and hydrogen peroxide (H2O2). Dimethyl tetrasulfide and dimethyl pentasulfide were observed as products using both of the oxidizing agents. Dimethyl disulfide was also observed as a product of degradation, which was further oxidized to S-methyl methanethiosulfonate only when mCPBA was used. HPLC-UV/vis and gas chromatography-mass spectrometry/solid phase microextraction analysis revealed good agreement between the degradation products of the stability study at 37 °C and those of disproportionation reactions. Furthermore, at 4 and 22 °C, chromatograms were remarkably stable over the one-year study period, indicating that the F3-formulated DMTS shows excellent long-term storage stability at T ≤ 22 °C.

Antidotal efficacies of the cyanide antidote candidate dimethyl trisulfide alone and in combination with cobinamide derivatives.

Formulation optimization and antidotal combination therapy are the two important tools to enhance the antidotal protection of the cyanide (CN) antidote dimethyl trisulfide (DMTS). The focus of this study is to demonstrate how the formulation with polysorbate 80 (Poly80), an excipient used in pharmaceutical technology, and the combinations with other CN antidotes having different mechanisms of action enhance the antidotal efficacy of the unformulated (neat) DMTS. The LD50 for CN was determined by the statistical Dixon up-and-down method on mice. Antidotal efficacy was expressed as antidotal potency ratio (APR). CN was injected subcutaneously one minute prior to the antidotes' injection intramuscularly. The APR values of 1.17 (dose: 25 mg/kg bodyweight) and 1.45 (dose: 50 mg/kg bodyweight) of the neat DMTS were significantly enhanced by the Poly80 formulation at both investigated doses to 2.03 and 2.33, respectively. The combination partners for the Poly80 formulated DMTS (DMTS-Poly80; 25 and 50 mg/kg bodyweight) were 4-nitrocobinamide (4NCbi) (20 mg/kg bodyweight) and aquohydroxocobinamide (AHCbi; 50, 100, and 250 mg/kg bodyweight). When DMTS-Poly80 (25 and 50 mg/kg bodyweight; APR = 2.03 and 2.33, respectively) was combined with 4NCbi (20 mg/kg bodyweight; APR = 1.35), significant increase in the APR values were noted at both DMTS doses (APR = 2.38 and 3.12, respectively). AHCbi enhanced the APR of DMTS-Poly80 (100 mg/kg bodyweight; APR = 3.29) significantly only at the dose of 250 mg/kg bodyweight (APR = 5.86). These studies provided evidence for the importance of the formulation with Poly80 and the combinations with cobinamide derivatives with different mechanisms of action for DMTS as a CN antidote candidate.

Development of magnetic carbon nanotubes for dispersive micro solid phase extraction of the cyanide metabolite, 2-aminothiazoline-4-carboxylic acid, in biological samples.

2-aminothiazoline-4-carboxylic acid (ATCA) is a minor metabolite of cyanide and is suggested to be a promising biomarker for cyanide exposure due to its specificity to cyanide metabolism and its excellent short- and long-term stability during storage. In this study, magnetic carbon nanotubes, including magnetic multi-walled carbon nanotubes (Mag-MWCNT) and magnetic single-walled carbon nanotubes (Mag-SWCNT) were synthesized as a novel sorbent for dispersive micro solid phase extraction (d-µSPE) to extract ATCA from biological matrices. ATCA spiked deionized water samples with the addition of the isotopic internal standard (ATCA - 13C, 15N) were subjected to Mag-CNT/d-µSPE to confirm extraction efficiency of this new technique. The extracted ATCA was derivatized and quantitated using gas chromatography/mass spectrometry (GC/MS) analysis. The extraction parameters were optimized and a detection limits of 15 and 25 ng/mL were obtained for synthetic urine and bovine blood respectively with a linear dynamic range of 30-1000 ng/mL. The optimized Mag-CNT/d-µSPE method facilitated efficient extraction of ATCA using 2 mg of Mag-MWCNT with a 10-minute extraction time. The current assay was also found to be effective for the extraction of ATCA with average recoveries of 97.7 ±â€¯4.0% (n = 9) and 96.5 ±â€¯12.1% (n = 9) from synthetic urine and bovine blood respectively. The approach of using Mag-CNT to facilitate d-µSPE offered a novel alternative to extract ATCA from complex biological matrices.

Correction to: Monitoring Dose Response of Cyanide Antidote Dimethyl Trisulfide in Rabbits Using Diffuse Optical Spectroscopy.

The online version of the original article can be found at.

Monitoring Dose Response of Cyanide Antidote Dimethyl Trisulfide in Rabbits Using Diffuse Optical Spectroscopy.

INTRODUCTION: Cyanide (CN) poisoning is a serious chemical threat from accidental or intentional exposures. Current CN exposure treatments, including direct binding agents, methemoglobin donors, and sulfur donors, have several limitations. Dimethyl trisulfide (DMTS) is capable of reacting with CN to form the less toxic thiocyanate with high efficiency, even without the sulfurtransferase rhodanese. We investigated a soluble DMTS formulation with the potential to provide a continuous supply of substrate for CN detoxification which could be delivered via intramuscular (IM) injection in a mass casualty situation. We also used non-invasive technology, diffuse optical spectroscopy (DOS), to monitor physiologic changes associated with CN exposure and reversal. METHODS: Thirty-six New Zealand white rabbits were infused with a lethal dose of sodium cyanide solution (20 mg/60 ml normal saline). Animals were divided into three groups and treated with saline, low dose (20 mg), or high dose (150 mg) of DMTS intramuscularly. DOS continuously assessed changes in tissue hemoglobin concentrations and cytochrome c oxidase redox state status throughout the experiment. RESULTS: IM injection of DMTS increased the survival in lethal CN poisoning. DOS demonstrated that high-dose DMTS (150 mg) reversed the effects of CN exposure on cytochrome c oxidase, while low dose (20 mg) did not fully reverse effects, even in surviving animals. CONCLUSIONS: This study demonstrated potential efficacy for the novel approach of supplying substrate for non-rhodanese mediated sulfur transferase pathways for CN detoxification via intramuscular injection in a moderate size animal model and showed that DOS was useful for optimizing the DMTS treatment.

Sealing Effects on the Storage Stability of the Cyanide Antidotal Candidate, Dimethyl Trisulfide.

BACKGROUND: Dimethyl trisulfide (DMTS) is a highly lipid-soluble cyanide (CN) antidote candidate molecule. In prior studies with various US FDA-approved co-solvents, surfactants, and their combinations, aqueous solutions containing 15% polysorbate 80 (Poly80) were found to effectively solubilize DMTS in formulations for intramuscular administration. However, DMTS formulated in 15% aqueous Poly80 solutions showed gradual losses over time when stored in vials with septum-based seals. OBJECTIVE: The present study tested whether storing DMTS formulations in hermetically sealed glass ampules could mitigate storage losses. METHODS: Samples consisted of 1-mL aliquots of a 50 mg/ml stock solution of DMTS in 15% aqueous Poly80. The control samples were stored using a vial-within-a-vial system-the inner and outer vials were sealed respectively, with a snap cap, and with a crimped septum. The hermetically sealed test samples were stored in fire-sealed glass ampules. The DMTS content was measured by HPLC-UV analysis at specific time points over a 100-day period. RESULTS: While the control samples exhibited systematic DMTS losses, no DMTS losses were observed from the test samples stored in hermetically sealed glass ampules over the 100-day testing period. CONCLUSION: DMTS formulated in 15% aqueous Poly80 solution has excellent stability when stored in fire-sealed glass ampules and thus has the potential to be effectively stored as an intramuscular CN countermeasure for mass casualty scenarios.

Methemoglobin Forming Effect of Dimethyl Trisulfide in Mice.

Dimethyl trisulfide (DMTS) is a natural organic trisulfide that has been patented as a promising antidotal candidate against cyanide (CN). The primary mode of action of DMTS is as a sulfur donor that enables the conversion of CN to thiocyanate. Recently, it was discovered that DMTS is capable of oxidizing hemoglobin (Hb) to methemoglobin (MetHb) in vitro. The goal of these experiments was to measure the extent of DMTS-induced MetHb formation in vivo. In these experiments, intramuscular (IM) injections of formulated DMTS were administered to mice. Following the IM injection, blood was drawn and analyzed for MetHb using a rapid spectrophotometric method. Methemoglobin levels peaked in a dose-dependent manner between 20 and 30 min., and then began dropping. The highest MetHb levels measured for the 50, 100, 200 and 250 mg/kg doses of DMTS were respectively 3.28, 6.12, 9.69, and 10.76% MetHb. These experiments provide the first experimental evidence that IM administered DMTS generates MetHb in vivo and provide additional evidence for the presence of a secondary therapeutic pathway for DMTS - CN scavenging by DMTS-generated MetHb.

From the Cover: In Vitro and In Vivo Blood-Brain Barrier Penetration Studies with the Novel Cyanide Antidote Candidate Dimethyl Trisulfide in Mice.

Recent in vitro and in vivo studies highlight the strong potential of dimethyl trisulfide (DMTS) as an antidote for cyanide (CN) intoxication. Due to its high oxygen demand, the brain is one of the main target organs of CN. The blood-brain barrier (BBB) regulates the uptake of molecules into the brain. In the literature, there is no data about the ability of DMTS to penetrate the BBB. Therefore, our aim was to test the in vitro BBB penetration of DMTS and its in vivo pharmacokinetics in blood and brain. The in vitro BBB penetration of DMTS was measured by using a parallel artificial membrane permeability assay (BBB-PAMPA), and a triple BBB co-culture model. The pharmacokinetics was investigated in a mouse model by following the DMTS concentration in blood and brain at regular time intervals following intramuscular administration. DMTS showed high penetrability in both in vitro systems (apparent permeability coefficients: BBB-PAMPA 11.8 × 10-6 cm/s; cell culture 158 × 10-6 cm/s) without causing cell toxicity and leaving the cellular barrier intact. DMTS immediately absorbed into the blood after the intramuscular injection (5 min), and rapidly penetrated the brain of mice (10 min). In addition to the observed passive diffusion in the in vitro studies, the contribution of facilitated and/or active transport to the measured high permeability of DMTS in the pharmacokinetic studies can be hypothesized. Earlier investigations demonstrating the antidotal efficacy of DMTS against CN together with the present results highlight the promise of DMTS as a brain-protective CN antidote.

Reaction of Dimethyl Trisulfide with Hemoglobin.

Dimethyl trisulfide (DMTS) is a promising antidotal candidate for cyanide intoxication. DMTS acts as a sulfur donor in the conversion of cyanide to the less-toxic thiocyanate. The alternate reaction pathways of DMTS in the blood are not well understood. We report changes in the hemoglobin absorption spectrum upon reaction with DMTS. These changes closely match those induced by the known methemoglobin former, sodium nitrite. The kinetics of methemoglobin formation with DMTS is slower than with sodium nitrite. These results support the hypothesis that a potentially significant side-reaction of the therapeutically administered DMTS is the oxidization of hemoglobin to methemoglobin.

Method development for detecting the novel cyanide antidote dimethyl trisulfide from blood and brain, and its interaction with blood.

The antidotal potency of dimethyl trisulfide (DMTS) against cyanide poisoning was discovered and investigated in our previous studies. Based on our results it has better efficacy than the Cyanokit and the Nithiodote therapies that are presently used against cyanide intoxication in the US. Because of their absence in the literature, the goal of this work was to develop analytical methods for determining DMTS from blood and brain that could be employed in future pharmacokinetic studies. An HPLC-UV method for detection of DMTS from blood, a GC-MS method for detection of DMTS from brain, and associated validation experiments are described here. These analytical methods were developed using in vitro spiking of brain and blood, and are suitable for determining the in vivo DMTS concentrations in blood and brain in future pharmacokinetic and distribution studies. An important phenomenon was observed in the process of developing these methods. Specifically, recoveries from fresh blood spiked with DMTS were found to be significantly lower than recoveries from aged blood spiked in the same manner with DMTS. This decreased DMTS recovery from fresh blood is important, both because of the role it may play in the antidotal action of DMTS in the presence of cyanide, and because it adds the requirement of sample stabilization to the method development process. Mitigation procedures for stabilizing DMTS samples in blood are reported.

Parenteral dosage form development and testing of dimethyl trisulfide, as an antidote candidate to combat cyanide intoxication.

This study focused on the solubility enhancement and the in vivo antidotal efficacy testing of a new potential cyanide (CN) countermeasure, dimethyl trisulfide (DMTS). Various FDA approved cyclodextrins (HPßCD, RMßCD, HPγCD), cosolvents (ethanol, polyethylene glycols, propylene glycol), surfactants (cremophor EL, cremophor RH 40, sodium cholate, sodium deoxycholate, polysorbate 80) and their combinations were applied. Based on the solubility enhancing potential of the tested systems, polysorbate 80 was chosen for further in vivo efficacy studies. A composition comprising 15% polysorbate 80 and 50 mg/ml DMTS with the applied DMTS dose of 100 mg/kg provided a therapeutic antidotal protection of 3.4 × LD50. For comparison, the present therapy of sodium thiosulfate (TS) with the dose of 100 mg/kg provided only 1.1 × LD50 protection, and at the dose of 200 mg/kg, the LD50 was enhanced by 1.3 times. No difference in the therapeutic protection by DMTS was detected when the concentration of polysorbate 80 was increased to 20% (3.2 × LD50 protection). These data demonstrate the potential importance of DMTS as a CN countermeasure, and the formulation comprising polysorbate 80 provides the base of an injectable intramuscular dosage form that can later serve as a CN antidotal kit suitable for mass scenario.

Determination of dimethyl trisulfide in rabbit blood using stir bar sorptive extraction gas chromatography-mass spectrometry.

Cyanide poisoning by accidental or intentional exposure poses a severe health risk. The current Food and Drug Administration approved antidotes for cyanide poisoning can be effective, but each suffers from specific major limitations concerning large effective dosage, delayed onset of action, or dependence on enzymes generally confined to specific organs. Dimethyl trisulfide (DMTS), a sulfur donor that detoxifies cyanide by converting it into thiocyanate (a relatively nontoxic cyanide metabolite), is a promising next generation cyanide antidote. Although a validated analytical method to analyze DMTS from any matrix is not currently available, one will be vital for the approval of DMTS as a therapeutic agent against cyanide poisoning. Hence, a stir bar sorptive extraction (SBSE) gas chromatography - mass spectrometry (GC-MS) method was developed and validated for the analysis of DMTS from rabbit whole blood. Following acid denaturation of blood, DMTS was extracted into a polydimethylsiloxane-coated stir bar. The DMTS was then thermally desorbed from the stir bar and analyzed by GC-MS. The limit of detection of DMTS using this method was 0.06µM with dynamic range from 0.5-100µM. For quality control standards, the precision, as measured by percent relative standard deviation, was below 10%, and the accuracy was within 15% of the nominal concentration. The method described here will allow further investigations of DMTS as a promising antidote for cyanide poisoning.

A Lipid Base Formulation for Intramuscular Administration of a Novel Sulfur Donor for Cyanide Antagonism.

This study represents a new formulation of the novel Cyanide (CN) antidote, Dimethyl trisulfide (DMTS), for intramuscular administration. This is a naturally occurring organosulfur molecule with the capability of reacting with CN more efficiently than the present sulfur donor type CN therapy of Thiosulfate (TS). Two types of micelles (PEG2000-DSPE and PEG2000-DSPE/TPGS) were prepared and tested for their ability to encapsulate the liquid, highly lipophilic and volatile drug, DMTS. The micellar encapsulation for DMTS does not only eliminate the possible muscle necrosis at the injection sites, but the rate of evaporation within the micelles is suppressed, that can provide a level of stability for the formulation. The method of micelle preparation was optimized and it was demonstrated that the PEG2000-DSPE preparation can dissolve up to 2.0 mg/ml of the antidote candidate. Keeping the injection volume minimized this could provide a maximum DMTS dose of 12.5 mg/kg. However, even this low dose of DMTS showed a remarkable in vivo therapeutic efficacy (2 X LD50 protection) in a mice model when injected intramuscularly. These in vitro and in vivo findings proved the efficacy of DMTS in combating CN intoxication, and the presented work gives valuable insight to micelle preparation and sets the bases for a more advanced future formulation of DMTS.

Dimethyl trisulfide: A novel cyanide countermeasure.

In the present studies, the in vitro and in vivo efficacies of a novel cyanide countermeasure, dimethyl trisulfide (DMTS), were evaluated. DMTS is a sulfur-based molecule found in garlic, onion, broccoli, and similar plants. DMTS was studied for effectiveness as a sulfur donor-type cyanide countermeasure. The sulfur donor reactivity of DMTS was determined by measuring the rate of the formation of the cyanide metabolite thiocyanate. In experiments carried out in vitro in the presence of the sulfurtransferase rhodanese (Rh) and at the experimental pH of 7.4, DMTS was observed to convert cyanide to thiocyanate with greater than 40 times higher efficacy than does thiosulfate, the sulfur donor component of the US Food and Drug Administration-approved cyanide countermeasure Nithiodote® In the absence of Rh, DMTS was observed to be almost 80 times more efficient than sodium thiosulfate in vitro The fact that DMTS converts cyanide to thiocyanate more efficiently than does thiosulfate both with and without Rh makes it a promising sulfur donor-type cyanide antidote (scavenger) with reduced enzyme dependence in vitro The therapeutic cyanide antidotal efficacies for DMTS versus sodium thiosulfate were measured following intramuscular administration in a mouse model and expressed as antidotal potency ratios (APR = LD50 of cyanide with antidote/LD50 of cyanide without antidote). A dose of 100 mg/kg sodium thiosulfate given intramuscularly showed only slight therapeutic protection (APR = 1.1), whereas the antidotal protection from DMTS given intramuscularly at the same dose was substantial (APR = 3.3). Based on these data, DMTS will be studied further as a promising next-generation countermeasure for cyanide intoxication.

Stability Characterization of a Polysorbate 80-Dimethyl Trisulfide Formulation, a Cyanide Antidote Candidate.

Novel cyanide countermeasures are needed for cases of a mass-exposure cyanide emergency. A lead candidate compound is dimethyl trisulfide (DMTS), which acts as a sulfur donor for rhodanese, thereby assisting the conversion of cyanide into thiocyanate. DMTS is a safe compound for consumption and, in a 15% polysorbate 80 (DMTS-PS80) formulation, has demonstrated good efficacy against cyanide poisoning in several animal models. We performed a stability study that investigated the effect of temperature, location of formulation preparation, and pH under buffered conditions. We found that while the stability of the DMTS component was fairly independent of which laboratory prepared the formulation, the concentration of DMTS in the formulation was reduced 36-58% over the course of 29 weeks when stored at room temperature. This loss typically increased with increasing temperatures, although we did not find statistical differences between the stability at different storage temperatures in all formulations. Further, we found that addition of a light buffer negatively impacted the stability, whereas the pH of that buffer did not impact stability. We investigated the factors behind the reduction of DMTS over time using various techniques, and we suggest that the instability of the formulation is governed at least partially by precipitation and evaporation, although a combination of factors is likely involved.

Intravascular Residence Time Determination for the Cyanide Antidote Dimethyl Trisulfide in Rat by Using Liquid-Liquid Extraction Coupled with High Performance Liquid Chromatography.

These studies represent the first report on the intravascular residence time determinations for the cyanide antidote dimethyl trisulfide (DMTS) in a rat model by using high performance liquid chromatography coupled with ultraviolet absorption spectroscopy (HPLC-UV). The newly developed sample preparation included liquid-liquid extraction by cyclohexanone. The calibration curves showed a linear response for DMTS concentrations between 0.010 and 0.30 mg/mL with R2 = 0.9994. The limit of detection for DMTS via this extraction method was 0.010 mg/mL, and the limit of quantitation was 0.034 mg/mL. Thus this calibration curve provided a tool for determining DMTS in the range between 0.04 and 0.30 mg/mL. Rats were given 20 mg/kg DMTS dose (in 15% Polysorbate 80) intravenously, and blood samples were taken 15, 60, 90, 120, and 240 min after DMTS injections. The data points were plotted as DMTS concentration in RBCs versus time, and the intravascular residence time was determined graphically. The results indicated a half-life of 36 min in a rat model, suggesting that the circulation time is long enough to provide a reasonable time interval for cyanide antagonism.

Simultaneous determination of 3-mercaptopyruvate and cobinamide in plasma by liquid chromatography-tandem mass spectrometry.

The current suite of Food and Drug Administration (FDA) approved antidotes (i.e., sodium nitrite, sodium thiosulfate, and hydroxocobalamin) are effective for treating cyanide poisoning, but individually, each antidote has major limitations (e.g., large effective dosage or delayed onset of action). To mitigate these limitations, next-generation cyanide antidotes are being investigated, including 3-mercaptopyruvate (3-MP) and cobinamide (Cbi). Analytical methods capable of detecting these therapeutics individually and simultaneously (for combination therapy) are essential for the development of 3-MP and Cbi as potential cyanide antidotes. Therefore, a liquid chromatography-tandem mass-spectrometry method for the simultaneous analysis of 3-MP and Cbi was developed. Sample preparation of 3-MP consisted of spiking plasma with an internal standard ((13)C3-3-MP), precipitation of plasma proteins, and derivatizing 3-MP with monobromobimane to produce 3-mercaptopyruvate-bimane. Preparation of Cbi involved denaturing plasma proteins with simultaneous addition of excess cyanide to convert each Cbi species to dicyanocobinamide (Cbi(CN)2). The limits of detection for 3-MP and Cbi were 0.5µM and 0.2µM, respectively. The linear ranges were 2-500µM for 3-MP and 0.5-50µM for Cbi. The accuracy and precision for 3-MP were 100±9% and <8.3% relative standard deviation (RSD), respectively. For Cbi(CN)2, the accuracy was 100±13% and the precision was <9.5% RSD. The method presented here was used to determine 3-MP and Cbi from treated animals and may ultimately facilitate FDA approval of these antidotes for treatment of cyanide poisoning.

Past, present and future of cyanide antagonism research: From the early remedies to the current therapies.

This paper reviews milestones in antidotal therapies for cyanide (CN) spanning early remedies, current antidotal systems and research towards next generation therapies. CN has been a part of plant defense mechanisms for millions of years. It became industrially important in the nineteenth century with the advent of CN assisted gold mining and the use of CN as a pest control agent. The biochemical basis of CN poisoning was actively studied and key mechanisms were understood as early as 1929. These fundamental studies led to a variety of antidotes, including indirect CN binders that generate methemoglobin, direct CN binders such as hydroxocobalamin, and sulfur donors that convert CN to the less toxic thiocyanate. Research on blood gases at the end of the twentieth century shed new light on the role of nitric oxide (NO) in the body. The discovery of NO's ability to compete with CN for enzymatic binding sites provided a previously missed explanation for the rapid efficacy of NO generating antidotes such as the nitrites. Presently used CN therapies include: methemoglobin/NO generators (e.g., sodium nitrite, amyl nitrite, and dimethyl aminophenol), sulfur donors (e.g., sodium thiosulfate and glutathione), and direct binding agents [(e.g., hydroxocobalamin and dicobalt salt of ethylenediaminetetraacetic acid (dicobalt edetate)]. A strong effort is being made to explore novel antidotal systems and to formulate them for rapid administration at the point of intoxication in mass casualty scenarios. New antidotes, formulations, and delivery systems are enhancing bioavailability and efficacy and hold promise for a new generation of improved CN countermeasures.

Cyanide toxicokinetics: the behavior of cyanide, thiocyanate and 2-amino-2-thiazoline-4-carboxylic acid in multiple animal models.

Cyanide causes toxic effects by inhibiting cytochrome c oxidase, resulting in cellular hypoxia and cytotoxic anoxia, and can eventually lead to death. Cyanide exposure can be verified by direct analysis of cyanide concentrations or analyzing its metabolites, including thiocyanate (SCN(-)) and 2-amino-2-thiazoline-4-carboxylic acid (ATCA) in blood. To determine the behavior of these markers following cyanide exposure, a toxicokinetics study was performed in three animal models: (i) rats (250-300 g), (ii) rabbits (3.5-4.2 kg) and (iii) swine (47-54 kg). Cyanide reached a maximum in blood and declined rapidly in each animal model as it was absorbed, distributed, metabolized and eliminated. Thiocyanate concentrations rose more slowly as cyanide was enzymatically converted to SCN(-). Concentrations of ATCA did not rise significantly above the baseline in the rat model, but rose quickly in rabbits (up to a 40-fold increase) and swine (up to a 3-fold increase) and then fell rapidly, generally following the relative behavior of cyanide. Rats were administered cyanide subcutaneously and the apparent half-life (t1/2) was determined to be 1,510 min. Rabbits were administered cyanide intravenously and the t1/2 was determined to be 177 min. Swine were administered cyanide intravenously and the t1/2 was determined to be 26.9 min. The SCN(-) t1/2 in rats was 3,010 min, but was not calculated in rabbits and swine because SCN(-) concentrations did not reach a maximum. The t1/2 of ATCA was 40.7 and 13.9 min in rabbits and swine, respectively, while it could not be determined in rats with confidence. The current study suggests that cyanide exposure may be verified shortly after exposure by determining significantly elevated cyanide and SCN(-) in each animal model and ATCA may be used when the ATCA detoxification pathway is significant.

Identification, solubility enhancement and in vivo testing of a cyanide antidote candidate.

Present studies focused on the in vitro testing, the solubility enhancement and the in vivo testing of methyl propyl trisulfide (MPTS), a newly identified sulfur donor to treat cyanide (CN) intoxication. To enhance the solubility of the lipophilic MPTS, various FDA approved co-solvents, surfactants and their combinations were applied. The order of MPTS solubility in the given co-solvents was found to be the following: ethanol >> PEG 200 ≈ PEG400 ≈ PEG300 > PG. The maximum solubility of MPTS was found at 90% ethanol of 177.11 ± 12.17 mg/ml. The order of MPTS solubility in different surfactants is Cremophor EL>Cremophor RH40>polysorbate 80>sodium deoxycholate>sodium cholate. The maximum solubility of 40.99 mg/ml was achieved with 20% Cremophor EL. A synergistic solubilizing effect encountered with the combination of 20% Cremophor EL+75% ethanol lead to a 2900-fold increase (compared to water solubility) in solubility. The in vivo efficacy using intramuscular administration was determined on a therapeutic mice model and expressed as a ratio of CN LD50 with and without the test antidote(s) (APR). Intramuscular administration was shown to be effective and the therapeutic antidotal protection by MPTS alone and MPTS+thiosulfate (TS) was significantly higher than the present therapy of TS.