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.

Intramuscular aminotetrazole cobinamide as a treatment for inhaled hydrogen sulfide poisoning in a large swine model.

Hydrogen sulfide (H2 S), a high-threat chemical agent, occurs naturally in a variety of settings. Despite multiple incidents of exposures and deaths, no FDA-approved antidote exists. A rapid-acting, easy to administer antidote is needed. We conducted a randomized control trial in swine comparing intramuscular administration of aminotetrazole cobinamide (2.9 mL, 18 mg/kg) to no treatment following inhalation of H2 S gas. We found that aminotetrazole cobinamide administered 2 min after the onset of respiratory depression-defined as a tidal volume of less than 3 mL/kg for 2 consecutive minutes-yielded 100% survival, while all control animals died. Respiratory depression resolved in the treatment group within 3.6 ± 1.5 min (mean ± SD) of cobinamide administration, whereas control animals had intermittent gasping until death. Blood pressure and arterial oxygen saturation (SO2 ) returned to baseline values within 5 and 10 min, respectively, of cobinamide treatment, and plasma lactate concentration decreased to less than 50% of the highest value by the end of the experiment. In control animals, plasma lactate rose continuously until death. We conclude that intramuscular aminotetrazole cobinamide is effective in a large animal, inhalational model of acute, severe H2 S poisoning.

Cobamides.

Vitamin B12 is the only known essential human micronutrient made exclusively by prokaryotes. Kennedy and Taga introduce us to the world of cobamides-those cobalt-containing compounds, like B12, that appear to be the proprietary domain of our microbial partners.

Deuterium Kinetic Isotope Effects Resolve Low-Temperature Substrate Radical Reaction Pathways and Steps in B12-Dependent Ethanolamine Ammonia-Lyase.

The first-order reaction kinetics of the cryotrapped 1,1,2,2-2H4-aminoethanol substrate radical intermediate state in the adenosylcobalamin (B12)-dependent ethanolamine ammonia-lyase (EAL) from Salmonella enterica serovar Typhimurium are measured over the range of 203-225 K by using time-resolved, full-spectrum electron paramagnetic resonance spectroscopy. The studies target the fundamental understanding of the mechanism of EAL, the signature enzyme in ethanolamine utilization metabolism associated with microbiome homeostasis and disease conditions in the human gut. Incorporation of 2H into the hydrogen transfer that follows the substrate radical rearrangement step in the substrate radical decay reaction sequence leads to an observed 1H/2H isotope effect of approximately 2 that preserves, with high fidelity, the idiosyncratic piecewise pattern of rate constant versus inverse temperature dependence that was previously reported for the 1H-labeled substrate, including a monoexponential regime (T ≥ 220 K) and two distinct biexponential regimes (T = 203-219 K). In the global kinetic model, reaction at ≥220 K proceeds from the substrate radical macrostate, S•, and at 203-219 K along parallel pathways from the two sequential microstates, S1• and S2•, that are distinguished by different protein configurations. Decay from S•, or S1• and S2•, is rate-determined by radical rearrangement (1H) or by contributions from both radical rearrangement and hydrogen transfer (2H). Non-native direct decay to products from S1• is a consequence of the free energy barrier to the native S1• → S2• protein configurational transition. At physiological temperatures, this is averted by the fast protein configurational dynamics that guide the S1• → S2• transition.

Vitamin B12 modulates Parkinson's disease LRRK2 kinase activity through allosteric regulation and confers neuroprotection.

Missense mutations in Leucine-Rich Repeat Kinase 2 (LRRK2) cause the majority of familial and some sporadic forms of Parkinson's disease (PD). The hyperactivity of LRRK2 kinase induced by the pathogenic mutations underlies neurotoxicity, promoting the development of LRRK2 kinase inhibitors as therapeutics. Many potent and specific small-molecule LRRK2 inhibitors have been reported with promise. However, nearly all inhibitors are ATP competitive-some with unwanted side effects and unclear clinical outcome-alternative types of LRRK2 inhibitors are lacking. Herein we identify 5'-deoxyadenosylcobalamin (AdoCbl), a physiological form of the essential micronutrient vitamin B12 as a mixed-type allosteric inhibitor of LRRK2 kinase activity. Multiple assays show that AdoCbl directly binds LRRK2, leading to the alterations of protein conformation and ATP binding in LRRK2. STD-NMR analysis of a LRRK2 homologous kinase reveals the contact sites in AdoCbl that interface with the kinase domain. Furthermore, we provide evidence that AdoCbl modulates LRRK2 activity through disrupting LRRK2 dimerization. Treatment with AdoCbl inhibits LRRK2 kinase activity in cultured cells and brain tissue, and prevents neurotoxicity in cultured primary rodent neurons as well as in transgenic C. elegans and D. melanogaster expressing LRRK2 disease variants. Finally, AdoCbl alleviates deficits in dopamine release sustainability caused by LRRK2 disease variants in mouse models. Our study uncovers vitamin B12 as a novel class of LRRK2 kinase modulator with a distinct mechanism, which can be harnessed to develop new LRRK2-based PD therapeutics in the future.

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.

Efficacy of Intravenous Cobinamide Versus Hydroxocobalamin or Saline for Treatment of Severe Hydrogen Sulfide Toxicity in a Swine (Sus scrofa) Model.

BACKGROUND: Hydrogen sulfide (H2 S) is a potentially deadly gas that naturally occurs in petroleum and natural gas. The Occupational Health and Safety Administration cites H2 S as a leading cause of workplace gas inhalation deaths. Mass casualties of H2 S toxicity may be caused by exposure from industrial accidents or release from oil field sites. H2 S is also an attractive terrorism tool because of its high toxicity and ease with which it can be produced. Several potential antidotes have been proposed for hydrogen sulfide poisoning but none have been completely successful. OBJECTIVE: The objective was to compare treatment response assessed by the time to spontaneous ventilation among groups of swine with acute H2 S-induced apnea treated with intravenous (IV) cobinamide (4 mg/kg in 0.8 mL of 225 mmol/L solution), IV hydroxocobalamin (4 mg/kg in 5 mL of saline), or saline alone. METHODS: Twenty-four 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 would spontaneously ventilate with an FiO2 of 0.21. Sodium hydrosulfide (NaHS; concentration of 8 mg/mL) was begun at 1 mg/kg/min until apnea was confirmed for 20 seconds by capnography. This infusion rate was sustained for 1.5 minutes postapnea and then decreased to a maintenance rate for the remainder of the study to replicate sustained clinical exposure. Animals were randomly assigned to receive cobinamide (4 mg/kg), hydroxocobalamin (4 mg/kg), or saline and monitored for 60 minutes beginning 1 minute postapnea. G* power analysis using the Z-test determined that equal group sizes of eight animals were needed to achieve a power of 80% in detecting a 50% difference in return to spontaneous ventilations at α = 0.05. RESULTS: There were no significant differences in baseline variables. Moreover, there were no significant differences in the mg/kg dose of NaHS (5.6 mg/kg; p = 0.45) required to produce apnea. Whereas all of the cobinamide-treated animals survived (8/8), none of the control (0/8) or hydroxocobalamin (0/8)-treated animals survived. Mean (±SD) time to spontaneous ventilation in the cobinamide-treated animals was 3.2 (±1.1) minutes. CONCLUSIONS: Cobinamide successfully rescued the severely NaHS-poisoned swine from apnea in the absence of assisted ventilation.

The N Terminus of the PduB Protein Binds the Protein Shell of the Pdu Microcompartment to Its Enzymatic Core.

Bacterial microcompartments (MCPs) are extremely large proteinaceous organelles that consist of an enzymatic core encapsulated within a complex protein shell. A key question in MCP biology is the nature of the interactions that guide the assembly of thousands of protein subunits into a well-ordered metabolic compartment. In this report, we show that the N-terminal 37 amino acids of the PduB protein have a critical role in binding the shell of the 1,2-propanediol utilization (Pdu) microcompartment to its enzymatic core. Several mutations were constructed that deleted short regions of the N terminus of PduB. Growth tests indicated that three of these deletions were impaired MCP assembly. Attempts to purify MCPs from these mutants, followed by gel electrophoresis and enzyme assays, indicated that the protein complexes isolated consisted of MCP shells depleted of core enzymes. Electron microscopy substantiated these findings by identifying apparently empty MCP shells but not intact MCPs. Analyses of 13 site-directed mutants indicated that the key region of the N terminus of PduB required for MCP assembly is a putative helix spanning residues 6 to 18. Considering the findings presented here together with prior work, we propose a new model for MCP assembly.IMPORTANCE Bacterial microcompartments consist of metabolic enzymes encapsulated within a protein shell and are widely used to optimize metabolic process. Here, we show that the N-terminal 37 amino acids of the PduB shell protein are essential for assembly of the 1,2-propanediol utilization microcompartment. The results indicate that it plays a key role in binding the outer shell to the enzymatic core. We propose that this interaction might be used to define the relative orientation of the shell with respect to the core. This finding is of fundamental importance to our understanding of microcompartment assembly and may have application to engineering microcompartments as nanobioreactors for chemical production.

A Novel, Direct NO Donor Regulates Osteoblast and Osteoclast Functions and Increases Bone Mass in Ovariectomized Mice.

Most US Food and Drug Administration (FDA)-approved treatments for osteoporosis target osteoclastic bone resorption. Only PTH derivatives improve bone formation, but they have drawbacks, and novel bone-anabolic agents are needed. Nitrates, which generate NO, improved BMD in estrogen-deficient rats and may improve bone formation markers and BMD in postmenopausal women. However, nitrates are limited by induction of oxidative stress and development of tolerance, and may increase cardiovascular mortality after long-term use. Here we studied nitrosyl-cobinamide (NO-Cbi), a novel, direct NO-releasing agent, in a mouse model of estrogen deficiency-induced osteoporosis. In murine primary osteoblasts, NO-Cbi increased intracellular cGMP, Wnt/ß-catenin signaling, proliferation, and osteoblastic gene expression, and protected cells from apoptosis. Correspondingly, in intact and ovariectomized (OVX) female C57Bl/6 mice, NO-Cbi increased serum cGMP concentrations, bone formation, and osteoblastic gene expression, and in OVX mice, it prevented osteocyte apoptosis. NO-Cbi reduced osteoclasts in intact mice and prevented the known increase in osteoclasts in OVX mice, partially through a reduction in the RANKL/osteoprotegerin gene expression ratio, which regulates osteoclast differentiation, and partially through direct inhibition of osteoclast differentiation, observed in vitro in the presence of excess RANKL. The positive NO effects in osteoblasts were mediated by cGMP/protein kinase G (PKG), but some of the osteoclast-inhibitory effects appeared to be cGMP-independent. NO-Cbi increased trabecular bone mass in both intact and OVX mice, consistent with its in vitro effects on osteoblasts and osteoclasts. NO-Cbi is a novel direct NO-releasing agent that, in contrast to nitrates, does not generate oxygen radicals, and combines anabolic and antiresorptive effects in bone, making it an excellent candidate for treating osteoporosis. © 2016 American Society for Bone and Mineral Research.

Molecular and cellular effects of vitamin B12 forms on human trophoblast cells in presence of excessive folate.

Folic acid (FA) and iron are essential supplements during pregnancy. Similarly effects of vitamin B12 (B12) inadequacy and high folate and low B12 status, on pregnancy outcome are available. However there are no mandatory recommendations for B12. There are many forms of B12 viz. Cyanocobalamin (Cbl), Methylcobalamin (MeCbl), Adenosylcobalamin (AdCbl), and Hydroxycobalamin (HCbl) though there is limited consensus on which form has better efficacy. In the present study we have determined effect of various forms of B12 in the presence of two FA concentrations namely normal physiological (20ng/mL; NPFA) and supra-physiological (2000ng/mL; SPFA) concentration to mimic real time situation where FA is in excess due to supplementation. We assessed trophoblastic proliferation, viability, TNFα and EGFr mRNA expression, homocysteine, ß-hCG and MDA levels. Trophoblastic viability was significantly suppressed at SPFA concentration and was restored by B12 treatment with Cbl, AdCbl and combination of MeCbl+AdCbl. The mRNA expressions of TNFα were up-regulated, while EGFr were down-regulated at SPFA concentrations, as validated by RT-PCR. Treatment with MeCbl+AdCbl significantly decreased homocysteine and MDA levels at SPFA concentrations. High levels of FA alone had a detrimental effect on placental health and functions as reflected by decreased viability, EGFr expression and increased TNFα expression, homocysteine and MDA levels. Combination of B12 active forms i.e. MeCbl+AdCbl was found to be most effective in neutralising excess folate effect in-vitro.

Total Synthesis, Structure, and Biological Activity of Adenosylrhodibalamin, the Non-Natural Rhodium Homologue of Coenzyme B12.

B12 is unique among the vitamins as it is biosynthesized only by certain prokaryotes. The complexity of its synthesis relates to its distinctive cobalt corrin structure, which is essential for B12 biochemistry and renders coenzyme B12 (AdoCbl) so intriguingly suitable for enzymatic radical reactions. However, why is cobalt so fit for its role in B12 -dependent enzymes? To address this question, we considered the substitution of cobalt in AdoCbl with rhodium to generate the rhodium analogue 5'-deoxy-5'-adenosylrhodibalamin (AdoRbl). AdoRbl was prepared by de novo total synthesis involving both biological and chemical steps. AdoRbl was found to be inactive in vivo in microbial bioassays for methionine synthase and acted as an in vitro inhibitor of an AdoCbl-dependent diol dehydratase. Solution NMR studies of AdoRbl revealed a structure similar to that of AdoCbl. However, the crystal structure of AdoRbl revealed a conspicuously better fit of the corrin ligand for Rh(III) than for Co(III) , challenging the current views concerning the evolution of corrins.

Fermentative production of 1-propanol from d-glucose, l-rhamnose and glycerol using recombinant Escherichia coli.

Fermentative production of 1-propanol, which is one of the promising precursors of polypropylene production, from d-glucose, l-rhamnose and glycerol using metabolically engineered Escherichia coli was examined. To confer the ability to produce 1-propanol from 1,2-propanediol (1,2-PD) in recombinant E. coli, a part of the pdu regulon including the diol dehydratase and the propanol dehydrogenase genes together with the adenosylcobalamin (AdoCbl) regeneration enzyme genes of Klebsiella pneumoniae was cloned, and an expression vector for these genes (pRSF_pduCDEGHOQS) was constructed. Recombinant E. coli harboring pRSF_pduCDEGHOQS with 1,2-PD synthetic pathway (pKK_mde) genes, which was constructed in our previous report (Urano et al., Appl. Microbiol. Biotechnol., 99, 2001-2008, 2015), produced 16.1 mM of 1-propanol from d-glucose with a molar yield of 0.36 mol/mol after 72 h cultivation. 29.9 mM of 1-propanol was formed from l-rhamnose with a molar yield of 0.81 mol/mol using E. coli carrying only pRSF_pduCDEGHOQS. In addition, 1-propanol production from glycerol was achieved by addition of the ATP-dependent dihydroxyacetone kinase gene to E. coli harboring pKK_mde and pRSF_pduCDEGOQS. In all cases, 1-propanol production was achieved by adding only a small amount of AdoCbl.

Quantitative proteomics study of the neuroprotective effects of B12 on hydrogen peroxide-induced apoptosis in SH-SY5Y cells.

B12 belongs to the coumarin class of compounds that have been shown to have various physiological and pharmacological activities including anti-inflammatory, antibacterial, and antioxidant. In the present study, we characterised the neuroprotective effects of B12 against H2O2-induced neuronal cell damage in SH-SY5Y cells. Protein expression profiling in combination with pathway analysis was deployed to investigate the molecular events associated with the neuroprotective effects in human neuronal cells using a label-free quantitative proteomics approach. A total of 22 proteins were significantly differentially expressed in H2O2-damaged cells with or without B12 treatment. Bioinformatics analysis using the Cytoscape platform indicated that poly pyrimidine tract binding protein 1 (PTBP1) was highly associated with the protective effect, and western blotting verified that PTBP1 was up-regulated in H2O2 + B12 treatment group, compared with the H2O2 treated group. PTBP RNAi experiments knocked down PTBP expression, which cancelled out the protective effect of B12 on cell viability. Thus, we infer that B12 neuroprotective activity involves up-regulation of PTBP1 and its associated signalling networks following H2O2-induced apoptosis in SH-SY5Y cells. B12 or related compounds may prove to be useful therapeutic agents for the treatment of neurodegenerative diseases such as Alzheimer's and Parkinson's.

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.

Cobalamin coenzyme forms are not likely to be superior to cyano- and hydroxyl-cobalamin in prevention or treatment of cobalamin deficiency.

Methylcobalamin (MeCbl) and adenosylcobalamin (AdoCbl) are coenzymes for methionine synthase and methylmalonyl-CoA mutase, respectively. Hydroxylcobalamin (HOCbl) and cyanocobalamin (CNCbl) are frequently used for supplementation. MeCbl and AdoCbl have recently emerged as alternative forms in supplements. In the light of metabolic transformation of Cbl into its cofactor forms, this review discusses current evidence on efficacy and utility of different Cbl forms in preventing or treating Cbl deficiency. Cbl-transporting proteins bind and mediate the uptake of all aforementioned forms of Cbl. After internalization and lysosomal release, Cbl binds to the cytosolic chaperon MMACHC that is responsible for (i) flavin-dependent decyanation of [CN-Co(3+) Cbl to [Co(2+)]Cbl; (ii) glutathione-dependent dealkylation of MeCbl and AdoCbl to [Co(2+/1+)]Cbl; and (iii) glutathione-dependent decyanation of CNCbl or reduction of HOCbl under anaerobic conditions. MMACHC shows a broad specificity for Cbl forms and supplies the Cbl(2+) intermediate for synthesis of MeCbl and AdoCbl. Cobalamin chemistry, physiology, and biochemistry suggest that MeCbl and AdoCbl follow the same route of intracellular processing as CNCbl does. We conclude that supplementing MeCbl or AdoCbl is unlikely to be advantageous compared to CNCbl. On the other hand, there are obvious advantages of high parenteral doses (1-2 mg) of HOCbl in treating inborn errors of Cbl metabolism.

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.

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

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.