The Antioxidant/Nitric Oxide-Quenching Agent Cobinamide Prevents Aortic Disease in a Mouse Model of Marfan Syndrome.

Major pathologic changes in the proximal aorta underlie the life-threatening aortic aneurysms and dissections in Marfan Syndrome; current treatments delay aneurysm development without addressing the primary pathology. Because excess oxidative stress and nitric oxide/protein kinase G signaling likely contribute to the aortopathy, we hypothesized that cobinamide, a strong antioxidant that can attenuate nitric oxide signaling, could be uniquely suited to prevent aortic disease. In a well-characterized mouse model of Marfan Syndrome, cobinamide dramatically reduced elastin breaks, prevented excess collagen deposition and smooth muscle cell apoptosis, and blocked DNA, lipid, and protein oxidation and excess nitric oxide/protein kinase G signaling in the ascending aorta. Consistent with preventing pathologic changes, cobinamide diminished aortic root dilation without affecting blood pressure. Cobinamide exhibited excellent safety and pharmacokinetic profiles indicating it could be a practical treatment. We conclude that cobinamide deserves further study as a disease-modifying treatment of Marfan Syndrome.

Investigating the Replacement of Carboxylates with Carboxamides to Modulate the Safety and Efficacy of Platinum(II) Thioether Cyanide Scavengers.

Cyanide represents a persistent threat for accidental or malicious misuse due to easy conversion into a toxic gas and access to large quantities through several industries. The high safety index of hydroxocobalamin is a cornerstone quality as a cyanide scavenger. Unfortunately, intravenous infusion of hydroxocobalamin limits the utility in a mass casualty setting. We previously reported platinum(II) [Pt(II)] complexes with trans-directing sulfur ligands as an efficacious alternative to hydroxocobalamin when delivered by a bolus intramuscular injection in mice and rabbits. Thus, to enable Pt(II) as an alternative to hydroxocobalamin, a high safety factor is needed. The objective is to maintain efficacy and mitigate the risk for nephrotoxicity. Platinum amino acid complexes with the ability to form five- or six-membered rings and possessing either carboxylates or carboxamides are evaluated in vitro for cyanide scavenging. In vivo efficacy was evaulated in the zebrafish and mice cyanide exposure models. In addition, Pt(II) complex toxicity and pharmacokinetics were evaluated in a cyanide naive Sprague-Dawley model. Doses for toxicity are escalated to 5x from the efficacious dose in mice using a body surface area adjustment. The results show the carboxamide ligands display a time and pH dependence on cyanide scavenging in vitro and efficacy in vivo. Additionally, exchanging the carboxylate for carboxamide showed reduced indications of renal injury. A pharmacokinetic analysis of the larger bidentate complexes displayed rapid absorption by intramuscular administration and having similar plasma exposure. These findings point to the importance of pH and ligand structures for methionine carboxamide complexes with Pt(II).

The vitamin B12 analog cobinamide ameliorates azide toxicity in cells, Drosophila melanogaster, and mice.

CONTEXT: The azide anion (N3-) is highly toxic. It exists most commonly as sodium azide, which is used widely and is readily available, raising the potential for occupational incidents and use as a weapon of mass destruction. Azide-poisoned patients present with vomiting, seizures, hypotension, metabolic acidosis, and coma; death can occur. No specific azide antidote exists, with treatment being solely supportive. Azide inhibits mitochondrial cytochrome c oxidase and is likely oxidized to nitric oxide in vivo. Cytochrome c oxidase inhibition depletes intracellular adenosine triphosphate and increases oxidative stress, while increased nitric oxide causes hypotension and exacerbates oxidative damage. Here, we tested whether the cobalamin (vitamin B12) analog cobinamide, a strong and versatile antioxidant that also neutralizes nitric oxide, can reverse azide toxicity in mammalian cells, Drosophila melanogaster, and mice. RESULTS: We found cobinamide bound azide with a moderate affinity (Ka 2.87 × 105 M-1). Yet, cobinamide improved growth, increased intracellular adenosine triphosphate, and reduced apoptosis and malondialdehyde, a marker of oxidative stress, in azide-exposed cells. Cobinamide rescued Drosophila melanogaster and mice from lethal exposure to azide and was more effective than hydroxocobalamin. Azide likely generated nitric oxide in the mice, as evidenced by increased serum nitrite and nitrate, and reduced blood pressure and peripheral body temperature in the animals; the reduced temperature was likely due to reflex vasoconstriction in response to the hypotension. Cobinamide improved recovery of both blood pressure and body temperature. CONCLUSION: We conclude cobinamide likely acted by neutralizing both oxidative stress and nitric oxide, and that it should be given further consideration as an azide antidote.

Cobinamide is a strong and versatile antioxidant that overcomes oxidative stress in cells, flies, and diabetic mice.

Increased oxidative stress underlies a variety of diseases, including diabetes. Here, we show that the cobalamin/vitamin B12 analog cobinamide is a strong and multifaceted antioxidant, neutralizing superoxide, hydrogen peroxide, and peroxynitrite, with apparent rate constants of 1.9 × 108, 3.7 × 104, and 6.3 × 106 M-1 s-1, respectively, for cobinamide with the cobalt in the +2 oxidation state. Cobinamide with the cobalt in the +3 oxidation state yielded apparent rate constants of 1.1 × 108 and 8.0 × 102 M-1 s-1 for superoxide and hydrogen peroxide, respectively. In mammalian cells and Drosophila melanogaster, cobinamide outperformed cobalamin and two well-known antioxidants, imisopasem manganese and manganese(III)tetrakis(4-benzoic acid)porphyrin, in reducing oxidative stress as evidenced by: (i) decreased mitochondrial superoxide and return of the mitochondrial membrane potential in rotenone- and antimycin A-exposed H9c2 rat cardiomyocytes; (ii) reduced JNK phosphorylation in hydrogen-peroxide-treated H9c2 cells; (iii) increased growth in paraquat-exposed COS-7 fibroblasts; and (iv) improved survival in paraquat-treated flies. In diabetic mice, cobinamide administered in the animals' drinking water completely prevented an increase in lipid and protein oxidation, DNA damage, and fibrosis in the heart. Cobinamide is a promising new antioxidant that has potential use in diseases with heightened oxidative stress.

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.

Treatment of life-threatening H2S intoxication: Lessons from the trapping agent tetranitrocobinamide.

We sought to evaluate the efficacy of trapping free hydrogen sulfide (H2S) following severe H2S intoxication. Sodium hydrosulfide solution (NaHS, 20 mg/kg) was administered intraperitoneally in 69 freely moving rats. In a first group (protocol 1), 40 rats were randomly assigned to receive saline (n = 20) or the cobalt compound tetranitrocobinamide (TNCbi) (n = 20, 75 mg/kg iv), one minute into coma, when free H2S was still present in the blood. A second group of 27 rats received TNCbi or saline, following epinephrine, 5 min into coma, when the concentration of free H2S has drastically decreased in the blood. In protocol 1, TNCbi significantly increased immediate survival (65 vs 20 %, p < 0.01) while in protocol 2, administration of TNCbi led to the same outcome as untreated animals. We hypothesize that the decreased efficacy of TNCbi with time likely reflects the rapid spontaneous disappearance of the pool of free H2S in the blood following H2S exposure.

Analysis of bisaminotetrazole cobinamide, a next-generation antidote for cyanide, hydrogen sulfide and methanethiol poisoning, in swine plasma by liquid chromatography-tandem mass spectrometry.

Cyanide, hydrogen sulfide, and methanethiol are common toxic inhalation agents that inhibit mitochondrial cytochrome c oxidase and result in cellular hypoxia, cytotoxic anoxia, apnea, respiratory failure, cardiovascular collapse, seizure and potentially death. While all are occupational gas exposure hazards that have the potential to cause mass casualties from industrial accidents or acts of terrorism, only cyanide has approved antidotes, and each of these has major limitations, including difficult administration in mass-casualty settings. While bisaminotetrazole cobinamide (Cbi(AT)2) has recently gained attention because of its efficacy in treating these metabolic poisons, there is no method available for the analysis of Cbi(AT)2 in any biological matrix. Hence, in this study, a simple and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the analysis of Cbi(AT)2 in swine plasma. The method is extremely simple, consisting of protein precipitation, separation and drying of the supernatant, reconstitution in an aqueous solvent, and LC-MS/MS analysis. The method produced an LOD of 0.3 µM with a wide dynamic range (2 - 500 µM). Inter- and intraassay accuracies (100 ± 12 % and 100 ± 19 %, respectively) were acceptable and the precision (<12 % and < 9 % relative standard deviation, respectively) was good. The developed method was used to analyze Cbi(AT)2 from treated swine and the preliminary pharmacokinetic parameters showed impressive antidotal behavior, most notably a long estimated elimination half-life (t1/2 = 37.5 h). This simple and rapid method can be used to facilitate the development of Cbi(AT)2 as a therapeutic against toxic cyanide, hydrogen sulfide and methanethiol exposure.

Glyoxylate protects against cyanide toxicity through metabolic modulation.

Although cyanide's biological effects are pleiotropic, its most obvious effects are as a metabolic poison. Cyanide potently inhibits cytochrome c oxidase and potentially other metabolic enzymes, thereby unleashing a cascade of metabolic perturbations that are believed to cause lethality. From systematic screens of human metabolites using a zebrafish model of cyanide toxicity, we have identified the TCA-derived small molecule glyoxylate as a potential cyanide countermeasure. Following cyanide exposure, treatment with glyoxylate in both mammalian and non-mammalian animal models confers resistance to cyanide toxicity with greater efficacy and faster kinetics than known cyanide scavengers. Glyoxylate-mediated cyanide resistance is accompanied by rapid pyruvate consumption without an accompanying increase in lactate concentration. Lactate dehydrogenase is required for this effect which distinguishes the mechanism of glyoxylate rescue as distinct from countermeasures based solely on chemical cyanide scavenging. Our metabolic data together support the hypothesis that glyoxylate confers survival at least in part by reversing the cyanide-induced redox imbalances in the cytosol and mitochondria. The data presented herein represent the identification of a potential cyanide countermeasure operating through a novel mechanism of metabolic modulation.

Methyl mercaptan gas: mechanisms of toxicity and demonstration of the effectiveness of cobinamide as an antidote in mice and rabbits.

CONTEXT: Methyl mercaptan (CH3SH) is a colorless, toxic gas with potential for occupational exposure and used as a weapon of mass destruction. Inhalation at high concentrations can result in dyspnea, hypoventilation, seizures, and death. No specific methyl mercaptan antidote exists, highlighting a critical need for such an agent. Here, we investigated the mechanism of CH3SH toxicity, and rescue from CH3SH poisoning by the vitamin B12 analog cobinamide, in mammalian cells. We also developed lethal CH3SH inhalation models in mice and rabbits, and tested the efficacy of intramuscular injection of cobinamide as a CH3SH antidote. RESULTS: We found that cobinamide binds to CH3SH (Kd = 84 µM), and improved growth of cells exposed to CH3SH. CH3SH reduced cellular oxygen consumption and intracellular ATP content and activated the stress protein c-Jun N-terminal kinase (JNK); cobinamide reversed these changes. A single intramuscular injection of cobinamide (20 mg/kg) rescued 6 of 6 mice exposed to a lethal dose of CH3SH gas, while all six saline-treated mice died (p = 0.0013). In rabbits exposed to CH3SH gas, 11 of 12 animals (92%) treated with two intramuscular injections of cobinamide (50 mg/kg each) survived, while only 2 of 12 animals (17%) treated with saline survived (p = 0.001). CONCLUSION: We conclude that cobinamide could potentially serve as a CH3SH antidote.

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.

Remote Detection of HCN, HF, and Nerve Agent Vapors Based on Self-Referencing, Dye-Impregnated Porous Silicon Photonic Crystals.

A one-dimensional photonic crystal is prepared from porous silicon (pSi) and impregnated with a chemically specific colorimetric indicator dye to provide a self-referenced vapor sensor for the selective detection of hydrogen fluoride (HF), hydrogen cyanide (HCN), and the chemical nerve agent diisopropyl fluorophosphate (DFP). The photonic crystal is prepared with two stop bands: one that coincides with the optical absorbance of the relevant activated indicator dye and the other in a spectrally "clear" region, to provide a reference. The inner pore walls of the pSi sample are then modified with octadecylsilane to provide a hydrophobic interior, and the indicator dye of interest is then loaded into the mesoporous matrix. Remote analyte detection is achieved by measurement of the intensity ratio of the two stop bands in the white light reflectance spectrum, which provides a means to reliably detect colorimetric changes in the indicator dye. Indicator dyes were chosen for their specificity for the relevant agents: rhodamine-imidazole (RDI) for HF and DFP, and monocyanocobinamide (MCbi) for HCN. The ratiometric readout allows detection of HF and HCN at concentrations (14 and 5 ppm, respectively) that are below their respective IDLH (immediately dangerous to life and health) concentrations (30 ppm for HF; 50 ppm for HCN); detection of DFP at a concentration of 114 ppb is also demonstrated. The approach is insensitive to potential interferents such as ammonia, hydrogen chloride, octane, and the 43-component mixture of VOCs known as EPA TO-14A, and to variations in relative humidity (20-80% RH). Detection of HF and HCN spiked into the complex mixture EPA TO-14A is demonstrated. The approach provides a general means to construct robust remote detection systems for chemical agents.

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.

Metabolic interaction between amino acid deprivation and cisplatin synergistically reduces phosphoribosyl-pyrophosphate and augments cisplatin cytotoxicity.

Cisplatin is a mainstay of cancer chemotherapy. It forms DNA adducts, thereby activating poly(ADP-ribose) polymerases (PARPs) to initiate DNA repair. The PARP substrate NAD+ is synthesized from 5-phosphoribose-1-pyrophosphate (PRPP), and we found that treating cells for 6 h with cisplatin reduced intracellular PRPP availability. The decrease in PRPP was likely from (1) increased PRPP consumption, because cisplatin increased protein PARylation and PARP1 shRNA knock-down returned PRPP towards normal, and (2) decreased intracellular phosphate, which down-regulated PRPP synthetase activity. Depriving cells of a single essential amino acid decreased PRPP synthetase activity with a half-life of ~ 8 h, and combining cisplatin and amino acid deprivation synergistically reduced intracellular PRPP. PRPP is a rate-limiting substrate for purine nucleotide synthesis, and cisplatin inhibited de novo purine synthesis and DNA synthesis, with amino acid deprivation augmenting cisplatin's effects. Amino acid deprivation enhanced cisplatin's cytotoxicity, increasing cellular apoptosis and DNA strand breaks in vitro, and intermittent deprivation of lysine combined with a sub-therapeutic dose of cisplatin inhibited growth of ectopic hepatomas in mice. Augmentation of cisplatin's biochemical and cytotoxic effects by amino acid deprivation suggest that intermittent deprivation of an essential amino acid could allow dose reduction of cisplatin; this could reduce the drug's side effects, and allow its use in cisplatin-resistant tumors.

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.

Cobinamide is effective for treatment of hydrogen sulfide-induced neurological sequelae in a mouse model.

Hydrogen sulfide (H2 S) is a highly neurotoxic gas. Acute exposure can lead to neurological sequelae among survivors. A drug for treating neurological sequelae in survivors of acute H2 S intoxication is needed. Using a novel mouse model we evaluated the efficacy of cobinamide (Cob) for increasing survival of, and reducing neurological sequalae in, mice exposed to sublethal doses of H2 S. There were two objectives: (1) to determine the dose-response efficacy of Cob and (2) to determine the effective therapeutic time window of Cob. To explore objective 1, mice were injected intramuscularly with Cob at 0, 50, or 100 mg/kg at 2 min after H2 S exposure. For objective 2, mice were injected intramuscularly with 100 mg/kg Cob at 2, 15, and 30 min after H2 S exposure. For both objectives, mice were exposed to 765 ppm of H2 S gas. Cob significantly reduced H2 S-induced lethality in a dose-dependent manner (P < 0.05). Cob-treated mice exhibited significantly fewer seizures and knockdowns compared with the H2 S-exposed group. Cob also reversed H2 S-induced weight loss, behavioral deficits, neurochemical changes, cytochrome c oxidase enzyme inhibition, and neurodegeneration in a dose- and time-dependent manner (P < 0.01). Overall, these findings show that Cob increases survival and is neuroprotective in a mouse model of H2 S-induced neurological sequelae.

Oral Glycine and Sodium Thiosulfate for Lethal Cyanide Ingestion.

OBJECTIVE: Accidental or intentional cyanide ingestion is an-ever present danger. Rapidly acting, safe, inexpensive oral cyanide antidotes are needed that can neutralize large gastrointestinal cyanide reservoirs. Since humans cannot be exposed to cyanide experimentally, we studied oral cyanide poisoning in rabbits, testing oral sodium thiosulfate with and without gastric alkalization. SETTING: University research laboratory. SUBJECTS: New Zealand white rabbits. INTERVENTIONS: Seven animal groups studied; Groups 1-5 received high dose oral NaCN (50 mg, >LD100) and were treated immediately with oral (via nasogastric tube): 1) saline, 2) glycine, 3) sodium thiosulfate or 4) sodium thiosulfate and glycine, or 5) after 2 min with intramuscular injection of sodium nitrite and sodium thiosulfate plus oral sodium thiosulfate and glycine. Groups 6-7 received moderate dose oral NaCN (25 mg, LD70) and delayed intramuscular 6) saline or 7) sodium nitrite-sodium thiosulfate. MEASUREMENTS AND MAIN RESULTS: All animals in the high dose NaCN group receiving oral saline or glycine died very rapidly, with a trend towards delayed death in glycine-treated animals; saline versus glycine-treated animals died at 10.3+3.9 and 14.6+5.9 min, respectively (p=0.13). In contrast, all sodium thiosulfate-treated high dose cyanide animals survived (p<0.01), with more rapid recovery in animals receiving both thiosulfate and glycine, compared to thiosulfate alone (p<0.03). Delayed intramuscular treatment alone in the moderate cyanide dose animals increased survival over control animals from 30% to 71%. Delayed treatment in high dose cyanide animals was not as effective as immediate treatment, but did increase survival time and rescued 29% of animals (p<0.01 versus cyanide alone). CONCLUSIONS: Oral sodium thiosulfate with gastric alkalization rescued animals from lethal doses of ingested cyanide. The combination of oral glycine and sodium thiosulfate may have potential for treating high dose acute cyanide ingestion and merits further investigation. The combination of systemic and oral therapy may provide further options.

Cisplatin Analogs Confer Protection against Cyanide Poisoning.

Cisplatin holds an illustrious position in the history of chemistry most notably for its role in the virtual cure of testicular cancer. Here we describe a role for this small molecule in cyanide detoxification in vivo. Cyanide kills organisms as diverse as insects, fish, and humans within seconds to hours. Current antidotes exhibit limited efficacy and are not amenable to mass distribution requiring the development of new classes of antidotes. The binding affinity of the cyanide anion for the positively charged metal platinum is known to create an extremely stable complex in vitro. We therefore screened a panel of diverse cisplatin analogs and identified compounds that conferred protection from cyanide poisoning in zebrafish, mice, and rabbits. Cumulatively, this discovery pipeline begins to establish the characteristics of platinum ligands that influence their solubility, toxicity, and efficacy, and provides proof of concept that platinum-based complexes are effective antidotes for cyanide poisoning.

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.

The Vitamin B12 Analog Cobinamide Is an Effective Antidote for Oral Cyanide Poisoning.

INTRODUCTION: Cyanide is a major chemical threat, and cyanide ingestion carries a higher risk for a supra-lethal dose exposure compared to inhalation but provides an opportunity for effective treatment due to a longer treatment window and a gastrointestinal cyanide reservoir that could be neutralized prior to systemic absorption. We hypothesized that orally administered cobinamide may function as a high-binding affinity scavenger and that gastric alkalinization would reduce cyanide absorption and concurrently increase cobinamide binding, further enhancing antidote effectiveness. METHODS: Thirty New Zealand white rabbits were divided into five groups and were given a lethal dose of oral cyanide poisoning (50 mg). The survival time of animals was monitored with oral cyanide alone, oral cyanide with gastric alkalinization with oral sodium bicarbonate buffer (500 mg), and in combination with either aquohydroxocobinamide or dinitrocobinamide (250 mM). Red blood cell cyanide concentration, plasma cobinamide, and thiocyanate concentrations were measured from blood samples. RESULTS: In cyanide ingested animals, oral sodium bicarbonate alone significantly prolonged survival time to 20.3 ± 8.6 min compared to 10.5 ± 4.3 min in saline-treated controls, but did not lead to overall survival. Aquohydroxocobinamide and dinitrocobinamide increased survival time to 64 ± 41 (p < 0.05) and 75 ± 16.4 min (p < 0.001), respectively. Compared to aquohydroxocobinamide, dinitrocobinamide showed greater systemic absorption and reduced blood pressure. Dinitrocobinamide also markedly increased the red blood cell cyanide concentration. Under all conditions, the plasma thiocyanate concentration gradually increased with time. CONCLUSION: This study demonstrates a promising new approach to treat high-dose cyanide ingestion, with gastric alkalinization alone and in combination with oral cobinamide for treating a supra-lethal dose of orally administered cyanide in rabbits.

Hydrogen Sulfide--Mechanisms of Toxicity and Development of an Antidote.

Hydrogen sulfide is a highly toxic gas-second only to carbon monoxide as a cause of inhalational deaths. Its mechanism of toxicity is only partially known, and no specific therapy exists for sulfide poisoning. We show in several cell types, including human inducible pluripotent stem cell (hiPSC)-derived neurons, that sulfide inhibited complex IV of the mitochondrial respiratory chain and induced apoptosis. Sulfide increased hydroxyl radical production in isolated mouse heart mitochondria and F2-isoprostanes in brains and hearts of mice. The vitamin B12 analog cobinamide reversed the cellular toxicity of sulfide, and rescued Drosophila melanogaster and mice from lethal exposures of hydrogen sulfide gas. Cobinamide worked through two distinct mechanisms: direct reversal of complex IV inhibition and neutralization of sulfide-generated reactive oxygen species. We conclude that sulfide produces a high degree of oxidative stress in cells and tissues, and that cobinamide has promise as a first specific treatment for sulfide poisoning.