Adriano Chan

Cobinamide is superior to other treatments in a mouse model of cyanide poisoning

Context. Cyanide is a rapidly acting cellular poison, primarily targeting cytochrome c oxidase, and is a common occupational and residential toxin, mostly via smoke inhalation. Cyanide is also a potential weapon of mass destruction, with recent credible threats of attacks focusing the need for better treatments, as current cyanide antidotes are limited and impractical for rapid deployment in mass casualty settings. Objective. We have used mouse models of cyanide poisoning to compare the efficacy of cobinamide (Cbi), the precursor to cobalamin (vitamin B12), to currently approved cyanide antidotes. Cbi has extremely high affinity for cyanide and substantial solubility in water. Materials and Methods. We studied Cbi in both an inhaled and intraperitoneal model of cyanide poisoning in mice. Results. We found Cbi more effective than hydroxocobalamin, sodium thiosulfate, soldium nitrite, and the combination of sodium thiosulfate–sodium nitrite in treating cyanide poisoning. Compared to hydroxocobalamin, Cbi was 3 and 11 times more potent in the intraperitoneal and inhalation models, respectively. Cobinamide sulfite (Cbi-SO3) was rapidly absorbed after intramuscular injection, and mice recovered from a lethal dose of cyanide even when given at a time when they had been apneic for over 2 min. In range-finding studies, Cbi-SO3 at doses up to 2000 mg/kg exhibited no clinical toxicity. Discussion and Conclusion. These studies demonstrate that Cbi is a highly effective cyanide antidote in mouse models, and suggest it could be used in a mass casualty setting, because it can be given rapidly as an intramuscular injection when administered as Cbi-SO3. Based on these animal data Cbi-SO3 appears to be an antidote worthy of further testing as a therapy for mass casualties.

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.

Nitrosyl-cobinamide, a new and direct nitric oxide-releasing drug effective in vivo

A limited number of nitric oxide (NO)-generating drugs are available for clinical use for acute and chronic conditions. Most of these agents are organic nitrates, which do not directly release NO; tolerance to the drugs develops, in part, as a consequence of their conversion to NO. We synthesized nitrosyl-cobinamide (NO-Cbi) from cobinamide, a structural analog of cobalamin (vitamin B12). NO-Cbi is a direct NO-releasing agent that we found was stable in water, but under physiologic conditions, it released NO with a half-life of 30 mins to 1 h. We show in five different biological systems that NO-Cbi is an effective NO-releasing drug. First, in cultured rat vascular smooth muscle cells, NO-Cbi induced phosphorylation of vasodilator-stimulated phosphoprotein, a downstream target of cGMP and cGMP-dependent protein kinase. Second, in isolated Drosophila melanogaster Malpighian tubules, NO-Cbi–stimulated fluid secretion was similar to that stimulated by Deta-NONOate and a cGMP analog. Third, in isolated mouse hearts, NO-Cbi increased coronary flow much more potently than nitroglycerin. Fourth, in contracted mouse aortic rings, NO-Cbi induced relaxation, albeit to a lesser extent than sodium nitroprusside. Fifth, in intact mice, a single NO-Cbi injection rapidly reduced blood pressure, and blood pressure returned to normal after 45 mins; repeated NO-Cbi injections induced the expected fall in blood pressure. These studies indicate that NO-Cbi is a useful NO donor that can be used experimentally in the laboratory; moreover, it could be developed into a vasodilating drug for treating hypertension and potentially other diseases such as angina and congestive heart failure.

Cyanide Produced by Human Isolates of Pseudomonas aeruginosa Contributes to Lethality in Drosophila melanogaster

Some Pseudomonas aeruginosa strains are cyanogenic, and cyanide may contribute to the bacteriums virulence. Using human isolates of P. aeruginosa, we have shown that Drosophila melanogaster suspended above cyanogenic strains become motionless and develop bradycardia and that flies injected with cyanogenic bacterial strains die more rapidly than those injected with noncyanogenic strains. Flies exposed to cyanogenic strains had high cyanide and low adenosine triphosphate (ATP) concentrations in body extracts, and treatment with a cyanide antidote equalized survival of flies injected with cyanogenic and noncyanogenic strains. P. aeruginosa PAO1 strain with a mutation in the hydrogen cyanide synthase gene cluster was much less toxic to flies than the parental cyanogenic strain or 2 knock-in strains. Transgenic flies overexpressing rhodanese, which detoxifies cyanide by converting it to thiocyanate, were resistant to cyanide and the increased virulence of cyanogenic strains. We conclude that D. melanogaster is a good model for studying cyanide produced by P. aeruginosa.

The Cobalamin Precursor Cobinamide Detoxifies Nitroprusside-Generated Cyanide

Sodium nitroprusside is used to treat hypertensive emergencies and acute heart failure. It acts by releasing nitric oxide (NO), a highly potent vasodilator, but unfortunately, for each NO molecule released, five cyanide ions are released. Thus, nitroprusside therapy is limited by cyanide toxicity. Therefore, a cyanide scavenger could be beneficial when administering nitroprusside. Hydroxocobalamin, which has a relatively high binding affinity for cyanide, has been shown to reduce cyanide levels in nitroprusside-treated patients. Cobinamide, the penultimate precursor in hydroxocobalamin biosynthesis, has a much greater affinity for cyanide than cobalamin, and binds two cyanide ions. We now show that cobinamide is highly effective in neutralizing cyanide ions released by nitroprusside in cultured mammalian cells, Drosophila melanogaster, and mice. Cobinamide also binds NO, but at molar concentrations 2.5–5 times that of nitroprusside, it did not decrease NO concentrations or the physiological effectiveness of nitroprusside. We conclude that cobinamide could be a valuable adjunct to nitroprusside therapy.

The combination of cobinamide and sulfanegen is highly effective in mouse models of cyanide poisoning

Context. Cyanide is a component of smoke in residential and industrial fires, and accidental exposure to cyanide occurs in a variety of industries. Moreover, cyanide has the potential to be used by terrorists, particularly in a closed space such as an airport or train station. Current therapies for cyanide poisoning must be given by intravenous administration, limiting their use in treating mass casualties. Objective. We are developing two new cyanide antidotes – cobinamide, a vitamin B12 analog, and sulfanegen, a 3-mercaptopyruvate prodrug. Both drugs can be given by intramuscular administration, and therefore could be used to treat a large number of people quickly. We now asked if the two drugs would have an augmented effect when combined. Materials and methods. We used a non-lethal and two different lethal models of cyanide poisoning in mice. The non-lethal model assesses neurologic recovery by quantitatively evaluating the innate righting reflex time of a mouse. The two lethal models are a cyanide injection and a cyanide inhalation model. Results. We found that the two drugs are at least additive when used together in both the non-lethal and lethal models: at doses where all animals died with either drug alone, the combination yielded 80 and 40% survival in the injection and inhalation models, respectively. Similarly, drug doses that yielded 40% survival with either drug alone, yielded 80 and 100% survival in the injection and inhalation models, respectively. As part of the inhalation model, we developed a new paradigm in which animals are exposed to cyanide gas, injected intramuscularly with an antidote, and then re-exposed to cyanide gas. This simulates cyanide exposure of a large number of people in a closed space, because people would remain exposed to cyanide, even after receiving an antidote. Conclusion. The combination of cobinamide and sulfanegen shows great promise as a new approach to treating cyanide poisoning.

The vitamin B12 analog cobinamide is an effective hydrogen sulfide antidote in a lethal rabbit model

Abstract Background and purpose. Hydrogen sulfide (H2S) is a highly toxic gas for which no effective antidotes exist. It acts, at least in part, by binding to cytochrome c oxidase, causing cellular asphyxiation and anoxia. We investigated the effects of three different ligand forms of cobinamide, a vitamin B12 analog, to reverse sulfide (NaHS) toxicity. Methods. New Zealand white rabbits received a continuous intravenous (IV) infusion of NaHS (3 mg/min) until expiration or a maximum 270 mg dose. Animals received six different treatments, administered at the time when they developed signs of severe toxicity: Group 1—saline (placebo group, N = 9); Group 2—IV hydroxocobalamin (N = 7); Group 3—IV aquohydroxocobinamide (N = 6); Group 4—IV sulfitocobinamide (N = 6); Group 5—intramuscular (IM) sulfitocobinamide (N = 6); and Group 6—IM dinitrocobinamide (N = 8). Blood was sampled intermittently, and systemic blood pressure and deoxygenated and oxygenated hemoglobin were measured continuously in peripheral muscle and over the brain region; the latter were measured by diffuse optical spectroscopy (DOS) and continuous wave near infrared spectroscopy (CWNIRS). Results. Compared with the saline controls, all cobinamide derivatives significantly increased survival time and the amount of NaHS that was tolerated. Aquohydroxocobinamide was most effective (261.5 ± 2.4 mg NaHS tolerated vs. 93.8 ± 6.2 mg in controls, p < 0.0001). Dinitrocobinamide was more effective than sulfitocobinamide. Hydroxocobalamin was not significantly more effective than the saline control. Conclusions. Cobinamide is an effective agent for inhibiting lethal sulfide exposure in this rabbit model. Further studies are needed to determine the optimal dose and form of cobinamide and route of administration.

Cell cycle regulation of purine synthesis by phosphoribosyl pyrophosphate and inorganic phosphate.

Cells must increase synthesis of purine nucleotides/deoxynucleotides before or during S-phase. We found that rates of purine synthesis via the de novo and salvage pathways increased 5.0- and 3.3-fold respectively, as cells progressed from mid-G1-phase to early S-phase. The increased purine synthesis could be attributed to a 3.2-fold increase in intracellular PRPP (5-phosphoribosyl-α-1-pyrophosphate), a rate-limiting substrate for de novo and salvage purine synthesis. PRPP can be produced by the oxidative and non-oxidative pentose phosphate pathways, and we found a 3.1-fold increase in flow through the non-oxidative pathway, with no change in oxidative pathway activity. Non-oxidative pentose phosphate pathway enzymes showed no change in activity, but PRPP synthetase is regulated by phosphate, and we found that phosphate uptake and total intracellular phosphate concentration increased significantly between mid-G1-phase and early S-phase. Over the same time period, PRPP synthetase activity increased 2.5-fold when assayed in the absence of added phosphate, making enzyme activity dependent on cellular phosphate at the time of extraction. We conclude that purine synthesis increases as cells progress from G1- to S-phase, and that the increase is from heightened PRPP synthetase activity due to increased intracellular phosphate.

Nitrocobinamide, a New Cyanide Antidote That Can Be Administered by Intramuscular Injection

Currently available cyanide antidotes must be given by intravenous injection over 5-10 min, making them ill-suited for treating many people in the field, as could occur in a major fire, an industrial accident, or a terrorist attack. These scenarios call for a drug that can be given quickly, e.g., by intramuscular injection. We have shown that aquohydroxocobinamide is a potent cyanide antidote in animal models of cyanide poisoning, but it is unstable in solution and poorly absorbed after intramuscular injection. Here we show that adding sodium nitrite to cobinamide yields a stable derivative (referred to as nitrocobinamide) that rescues cyanide-poisoned mice and rabbits when given by intramuscular injection. We also show that the efficacy of nitrocobinamide is markedly enhanced by coadministering sodium thiosulfate (reducing the total injected volume), and we calculate that ∼1.4 mL each of nitrocobinamide and sodium thiosulfate should rescue a human from a lethal cyanide exposure.

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.