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.

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.

Activity of a novel combined antiretroviral therapy of gemcitabine and decitabine in a mouse model for HIV-1.

The emergence of drug resistance threatens to limit the use of current anti-HIV-1 drugs and highlights the need to expand the number of treatment options available for HIV-1-infected individuals. Our previous studies demonstrated that two clinically approved drugs, decitabine and gemcitabine, potently inhibited HIV-1 replication in cell culture through a mechanism that is distinct from the mechanisms for the drugs currently used to treat HIV-1 infection. We further demonstrated that gemcitabine inhibited replication of a related retrovirus, murine leukemia virus (MuLV), in vivo using the MuLV-based LP-BM5/murine AIDS (MAIDS) mouse model at doses that were not toxic. Since decitabine and gemcitabine inhibited MuLV and HIV-1 replication with similar potency in cell culture, the current study examined the efficacy and toxicity of the drug combination using the MAIDS model. The data demonstrate that the drug combination inhibited disease progression, as detected by histopathology, viral loads, and spleen weights, at doses lower than those that would be required if the drugs were used individually. The combination of decitabine and gemcitabine exerted antiviral activity at doses that were not toxic. These findings indicate that the combination of decitabine and gemcitabine shows potent antiretroviral activity at nontoxic doses and should be further investigated for clinical relevance.

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.

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.

The effect of anesthesia by diethyl ether or isoflurane on activity of cytochrome P450 2E1 and P450 reductases in rat liver.

UNLABELLED: In this study we sought to determine whether exposure to the anesthetics diethyl ether and isoflurane influences the activity of hepatic cytochrome P450 2E1 and P450 reductases in the rat. Rats were fed a purified diet for 6 wk before anesthesia with 1 of 3 anesthetics: carbon dioxide, diethyl ether, or isoflurane. Cytochrome P450 2E1 and P450 reductases were measured in liver microsomes. No significant differences in enzyme activities were found among the groups. These results indicate that diethyl ether and isoflurane can be used to kill rats without inducing P450 enzymes. IMPLICATIONS: Rats were anesthetized with ether, isoflurane, or carbon dioxide and liver P450 enzymes were quantified by spectrophotometry. Based on the results of this study, rats can be anesthetized with isoflurane or diethyl ether for a short period without a change in the activity of P450 enzymes.

Effects of intracerebroventricular ethanol on ingestive behavior and induction of c-Fos immunoreactivity in selected brain regions.

The early changes in the central nervous system (CNS) following drinking of ethanol (ETOH) are poorly understood. It is known that chronic intracerebroventricular (ICV) administration of ethanol to rats induces preference for imbibed alcohol solutions. These results suggest that ICV ethanol could alter taste preference. In the present study, we tested whether ETOH[ICV] could induce a conditioned taste preference (CTP) or aversion (CTA) and alter c-Fos immunoreactivity (c-Fos-IR) in brain regions associated with feeding, aversion, and/or reward. Acute ETOH[ICV], as tested in the ETOH-naïve rat, did not induce CTA nor affect the amount of water imbibed by treated rats. The effects of ETOH[ICV] on intake and preference were determined using a novel palatable (i.e. sweet) noncaloric 0.1% saccharin solution. A single dose of ETOH[ICV] in the ETOH-nai;ve animal induced a CTP for saccharin. ETOH[ICV] significantly increased c-Fos-IR in a number of brain sites associated with feeding and reward including the bed nucleus of the stria terminalis, lateral dorsal area (BSTLD); nucleus accumbens, shell area (AcbSh); hypothalamic paraventricular nucleus (PVN); and lateral septum, ventral area (LSV). Thus, ETOH induced a CTP, not CTA, via central mechanisms; it increased c-Fos-IR in specific sites associated with feeding and reward.

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.