An Orally Bioavailable (Mice) Prodrug of Glutathione.

L-Cysteine-glutathione mixed disulfide (CySSG), a prodrug of glutathione (GSH), was found to be orally bioavailable in mice, and protected mice against a toxic dose of acetaminophen. If oral bioavailability can also be demonstrated in humans, a wide range of applicability for CySSG can be envisioned.

Sequestration and Elimination of Toxic Aldehydes.

It is well-known that aldehydes resulting from the in vivo oxidation of primary alcohols are toxic. Here, we experimentally demonstrate in rat models that the dipeptide cysteinylglycine (CG), formed in vivo from its oxidized product, cystinyl-bis-glycine (CbG), will sequester acetaldehyde and isoamyl aldehyde, two model aldehydes resulting from the oxidation of ethanol and isoamyl alcohol, respectively, and excrete them in urine as their respective conjugation products with CG. These data suggest that a whole series of toxic aldehydes can be sequestered and detoxified by CG and may prevent the flushing syndrome exhibited by individuals with a defective enzyme that converts acetaldehyde to acetate. The data also suggest the possibility of alleviating the hangover syndrome we believe to be caused by aldehydes, such as isoamyl aldehyde derived from short, branched-chain alcohols, present as congeners in certain alcoholic beverages. The sequestration of other toxic agents, such as cyanide, that can react with CG can also be envisioned.

In-vitro mercaptopyruvate sulfurtransferase species comparison in humans and common laboratory animals.

Cyanide is a metabolic poison that inhibits cytochrome c oxidase. Its broad applications in manufacturing and history as an agent of warfare/terror highlight the limitations in approved cyanide antidotes for mass casualties. Sulfanegen, a pre-clinical antidote for cyanide poisoning, exploits an endogenous detoxification pathway and should be amenable to mass-casualty scenarios. Because human studies are unethical, determination of appropriate animal species as models in translational studies for FDA approval under the "Animal Rule" are critical. Here, we compared the specific activities of mercaptopyruvate sulfurtransferase (MST, required for sulfanegen's activity), across common laboratory models of cyanide intoxication, and humans. Human MST activities in erythrocytes (measured as micromole pyruvate/min/106 rbc) were closest to those of Swiss-Webster mice and NZW rabbits. Similar species were selected for a more detailed tissue-specific comparison of MST activities. NZW Rabbits were closest to humans in the liver and kidney mitochondrial fractions, the Swiss-Webster mouse was closest to humans in the liver cytosolic fraction, while C57BL/6 mouse was closest in the kidney cytosolic fraction. These data comparing MST activities in animal models will help justify the use of those specific animals per the animal rule. Interestingly, statistically significant differences were found in MST activities of liver mitochondria between human smokers and non-smokers (p=0.0030).

Development of sulfanegen for mass cyanide casualties.

Cyanide is a metabolic poison that inhibits the utilization of oxygen to form ATP. The consequences of acute cyanide exposure are severe; exposure results in loss of consciousness, cardiac and respiratory failure, hypoxic brain injury, and dose-dependent death within minutes to hours. In a mass-casualty scenario, such as an industrial accident or terrorist attack, currently available cyanide antidotes would leave many victims untreated in the short time available for successful administration of a medical countermeasure. This restricted therapeutic window reflects the rate-limiting step of intravenous administration, which requires both time and trained medical personnel. Therefore, there is a need for rapidly acting antidotes that can be quickly administered to large numbers of people. To meet this need, our laboratory is developing sulfanegen, a potential antidote for cyanide poisoning with a novel mechanism based on 3-mercaptopyruvate sulfurtransferase (3-MST) for the detoxification of cyanide. Additionally, sulfanegen can be rapidly administered by intramuscular injection and has shown efficacy in many species of animal models. This article summarizes the journey from concept to clinical leads for this promising cyanide antidote.

Cyanide antidotes for mass casualties: water-soluble salts of the dithiane (sulfanegen) from 3-mercaptopyruvate for intramuscular administration.

Current cyanide antidotes are administered by IV infusion, which is suboptimal for mass casualties. Therefore, in a cyanide disaster, intramuscular (IM) injectable antidotes would be more appropriate. We report the discovery of the highly water-soluble sulfanegen triethanolamine as a promising lead for development as an IM injectable cyanide antidote.

Cyanide toxicity in juvenile pigs and its reversal by a new prodrug, sulfanegen sodium.

BACKGROUND: Cyanide (CN) toxicity is a serious clinical problem and can occur with sodium nitroprusside (SNP) administration, accidental smoke inhalation, industrial mishaps, and bio-terrorism. In this study, we induced severe CN toxicity independently with SNP or sodium cyanide (NaCN) in a juvenile pig model to demonstrate reversal of severe CN toxicity with a new antidote, sulfanegen sodium, a prodrug of 3-mercaptopyruvate. METHODS: SNP study: A pilot study in 11 anesthetized, mechanically ventilated juvenile pigs allowed us to determine the dose of SNP to induce CN toxicity. Blood CN, serum lactates, and blood gases were monitored. CN toxicity was defined as the occurrence of severe lactic acidosis accompanied by significant elevation in blood CN levels. Based on this pilot study, 8 anesthetized pigs received a high-dose i.v. infusion of SNP (100 mg/h) for 2 hours to induce CN toxicity. They were then randomized to receive either sulfanegen sodium or placebo. Four pigs received 3 doses of sulfanegen sodium (2.5 g i.v.) every hour after induction of severe CN toxicity, and 4 pigs received placebo. NaCN study: A pilot study was conducted in 4 spontaneously ventilating pigs sedated with propofol plus ketamine to demonstrate hemodynamic and metabolic stability for several hours. After this, 6 pigs were similarly sedated and given NaCN in bolus aliquots to produce CN toxicity ultimately resulting in death. Hemodynamics and metabolic variables were followed to define peak CN toxicity. In another group of 6 pigs, severe CN toxicity was induced by this method, and at peak toxicity, the animals were given sulfanegen sodium (2.5 g i.v.) followed by a repeat dose 60 minutes later in surviving animals. RESULTS: SNP study: The pilot study demonstrated the occurrence of a significant increase in blood CN levels (P < 0.05) accompanied by severe lactic acidemia (P < 0.05) in all pigs receiving a high dose of SNP. Administration of the sulfanegen antidote resulted in progressive significant reduction in blood lactate and CN levels with 100% survival (P < 0.05), whereas the placebo-treated pigs deteriorated and did not survive (P < 0.05). NaCN study: NaCN injection resulted in CN toxicity accompanied by severe lactic acidosis and mortality in all the pigs. Sulfanegen sodium reversed this toxicity and prevented mortality in all the pigs treated with this antidote. CONCLUSIONS: CN toxicity can be successfully induced in a juvenile pig model with SNP or NaCN. The prodrug, sulfanegen sodium, is effective in reversing CN toxicity induced by SNP or NaCN.

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 B(12) 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.

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

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

A novel paradigm for assessing efficacies of potential antidotes against neurotoxins in mice.

Historically, antidotal potencies of cyanide antagonists were measured as increases in the experimental LD(50) for cyanide elicited by the antidotes. This required the use of high doses of cyanide following pre-treatment with the putative antidote. Since IACUC guidelines at our institutions strongly discourage LD(50) determinations: we developed a new test paradigm that allowed for maximal survival of cyanide-treated animals with greatly reduced numbers of animals. Symptoms of cyanide toxicity include disruption of neuromuscular coordination, i.e., the righting reflex. Therefore, to establish a dose-response curve, the times required for recovery of this righting reflex with increasing doses of cyanide were measured. A cyanide dose that disrupted this righting reflex for approximately 1h with minimal deaths was then selected. Using this paradigm, the current cyanide antidotes, viz., nitrite plus thiosulfate and hydroxocobalamin, as well as some potential cyanide antidotes that we developed, were evaluated pre- and post-cyanide. This allowed, for the first time, the assessment of the post-cyanide effectiveness of the current antidotes against cyanide poisoning in a live animal. In addition, some prototype compounds were found to exhibit antidotal efficacy not only when injected i.p. following cyanide, but also when administered orally 30 min before cyanide. Pre-cyanide oral efficacy suggests that such compounds have the potential of being administered prophylactically before exposure to cyanide. This new test paradigm was found to be a powerful tool for assessing the efficacies of some novel antidotes against cyanide and should be equally applicable for evaluating putative antidotes for other neurotoxins.

Novel, orally effective cyanide antidotes.

A series of prodrugs of 3-mercaptopyruvate (3-MP), the substrate for the enzyme 3-mercaptopyruvate/cyanide sulfurtransferase (3-MPST) that converts cyanide to the nontoxic thiocyanate, which are highly effective cyanide antidotes, have been developed. These prodrugs of 3-MP are unique in being not only orally bioavailable, but may be administered up to an hour prior to cyanide as a prophylactic agent and are both rapid- or slow-acting when given parenterally.

Donors of HNO.

Recent comparisons of the pharmacological effects of nitric oxide (NO) and nitroxyl (HNO) donors have demonstrated that the responses to these redox-related nitrogen oxides are nearly universally dissimilar. These analyses have suggested the existence of mutually exclusive signaling pathways as a result of discrete chemical interactions of HNO and NO with a variety of critical biomolecules. Although the mechanisms of action are currently unresolved, the pharmacological responses to HNO are promising for clinical treatment of cardiovascular diseases such as heart failure, myocardial infarction and stroke. This review provides a detailed discussion of the most commonly utilized donors of HNO as well as a guideline for the characterization of novel donors.

p-Aminophenol-induced hepatotoxicity in hamsters: role of glutathione.

p-Aminophenol (PAP) is a widely used industrial chemical and a known nephrotoxin. Recently, it was found to also cause hepatotoxicity and glutathione (GSH) depletion in mice. The exact mechanism of liver toxicity is not known. The aims of this study were to determine whether PAP can cause acute hepatotoxicity in hamsters and to further investigate the role of GSH in PAP-induced toxicity. PAP was administered ip to hamsters in doses of 200-800 mg/kg. Liver damage at 24 h after PAP administration was assessed by elevations in plasma enzyme activities and histopathologic examination. GSH and cysteine (Cys) levels in liver at 4 h were determined by HPLC. PAP decreased hepatic GSH concentration to 8% and Cys to 30% of vehicle control values. It increased plasma glutamic pyruvic transaminase (GPT) activity by 47-fold and sorbitol dehydrogenase (SDH) activity by 113-fold. PAP also caused severe centrilobular hepatocellular necrosis. 2(RS)-n-Propylthiazolidine-4(R)-carboxylic acid (PTCA), a Cys precursor, attenuated the PAP-induced decreases in hepatic sulfhydryl levels; GSH and Cys were 39% and 78% of vehicle controls, respectively. PTCA also attenuated the PAP-induced elevations in plasma enzyme activities and hepatic necrosis. It was concluded that PAP hepatotoxicity is associated with depletion of hepatic GSH and can be prevented by PTCA.

Diverse antioxidants protect against acetaminophen hepatotoxicity.

The reactive oxygen species-sensitive transcription nuclear factor-kappaB (NF-kappaB) plays a pivotal role in the development of acetaminophen (APAP) hepatotoxicity. We investigated the efficacy of a diverse series of antioxidants in preventing APAP-induced hepatotoxicity. BALB/c mice were divided into four groups and provided with antioxidants incorporated into chow as follows: (1) control diet; or diet supplemented with (2) S-adenosylmethionine (SAMe); (3) green tea polyphenols (GrTP); or (4) (RS)-n-propylthiazolidine-4(R)-carboxylic acid (PTCA). After 5 days on these diets, the animals were further subdivided into (A) given an IP injection with APAP (750 mg/kg), or (B) kept as untreated controls. The animals were sacrificed at 0, 4 h, and 24 h following APAP administration. PAP/vehicle induced marked decreases in hepatic reduced glutathione (GSH) levels and endogenous SAMe concentrations (46%) when compared to controls. APAP also caused severe centrilobular necrosis and marked increase in serum enzyme ALT activity (38-fold). Oral administration of antioxidants significantly attenuated the APAP-induced liver damage and depletion of hepatic GSH. There were profound increases in serum TNF-alpha levels at 4 h following APAP administration in nonsupplemented compared to antioxidant-treated animals, but no significant differences noted after 24 h. Serum amyloid A increased in APAP-challenged mice irrespective of antioxidant treatment. Finally, hepatic SAMe concentrations were drastically decreased 24 h following APAP administration, and these decreases were attenuated by pretreatment with antioxidants. In conclusion, these orally administered antioxidants with dissimilar properties provided protection against liver damage, supporting the potential use of antioxidant therapy in patients with APAP toxicity. This is the first report that GrTP and oral administration of PTCA and SAMe can provide protection against APAP injury in this model.

Hepatoprotection by L-cysteine-glutathione mixed disulfide, a sulfhydryl-modified prodrug of glutathione.

L-Cysteine-glutathione disulfide, a ubiquitous substance present in mammalian cells, was shown to be highly effective in protecting mice against acetaminophen-induced hepatotoxicity. Since the corresponding D-cysteine-glutathione disulfide was totally ineffective in this regard, an enzymatic mechanism that provides glutathione directly to cells is postulated.

Double-prodrugs of L-cysteine: differential protection against acetaminophen-induced hepatotoxicity in mice.

A series of double-prodrugs of L-cysteine, designed to release L-cysteine in vivo and stimulate the biosynthesis of glutathione (GSH), were synthesized. To evaluate the hepatoprotective effectiveness of these double-prodrugs, male Swiss-Webster mice were administered acetaminophen (ACP) (2.45 mmol/kg (360 mg/kg), intraperitoneally (i.p.)). Prodrug (2.50 mmol/kg, i.p. or 1.25 mmol/kg, i.p., depending on the protocol) was administered 1 h before ACP as a priming dose. A supplementary dose of prodrug (2.5 mmol/kg, i.p. or 1.25 mmol/kg, i.p. depending on the protocol) was administered 0.5 h after ACP. The plasma alanine amino transferase (ALT) values, 24 h after ACP administration were transformed to logs and the 95% and 99% confidence intervals of the log values were plotted and compared for each group. Hepatoprotection was assessed by the degree of attenuation of plasma ALT levels. With these multiple dose schedules, the use of 2% carboxymethylcellulose as vehicle for the prodrugs was found to be detrimental; therefore, the prodrugs were dissolved in dilute aqueous base and the pH adjusted for administration. When a priming dose was given 1 h before ACP followed by a supplementary dose 0.5 h after ACP, only N,S-bis-acetyl-L-cysteine, where both the sulfhydryl and amino groups of L-cysteine were functionalized with the acetyl group, was found to be effective in protecting mice against the hepatotoxic effects of ACP. This suggests that these acetyl groups were rapidly hydrolyzed in vivo to liberate L-cysteine. In contrast, N-acetylation of 2(R,S)-methylthiazolidine-4(R)-carboxylic acid (MTCA) and its 2-n-propyl analog (PTCA), or N-acetylation of 2-oxothiazolidine-4-carboxylic acid (OTCA), reduced the hepatoprotective effects relative to the parent MTCA, PTCA, and OTCA, indicating that the release of L-cysteine in vivo from these N-acetylated thiazolidine prodrugs was metabolically unfavorable. The carbethoxy group, whether functionalized on the sulfhydryl or on the amino group of L-cysteine, or on the secondary amino group of MTCA, appears to be a poor "pro-moiety," since these carbethoxylated double-prodrugs of L-cysteine did not protect mice from ACP-induced hepatotoxicity.

Acetaminophen-induced suppression of hepatic AdoMet synthetase activity is attenuated by prodrugs of L-cysteine.

Administration of acetaminophen (ACP, 400 mg/kg, i.p.) to fasted, male Swiss-Webster mice caused a rapid 90% decrease in total hepatic glutathione (GSH) and a 58% decrease in mitochondrial GSH by 2 h post ACP. This was followed by a time-dependent decrease (72%) in hepatic AdoMet synthetase activity and rise in plasma ALT levels (>10000 U/l) at 24 h post ACP treatment. AdoMet synthetase activity was maintained at 82, 78 and 60% of controls, respectively, by the cysteine prodrugs PTCA, CySSME and NAC. Total hepatic and mitochondrial GSH levels were also protected from severe ACP-induced depletion by CySSME and MTCA. These results suggest that the maintenance of GSH homeostasis by cysteine prodrugs can protect mouse hepatic AdoMet synthetase, a sulfhydryl enzyme whose integrity is dependent on GSH, as well as the liver itself from the consequences of oxidative stress elicited by toxic metabolites of xenobiotics.