Nano-intercalated rhodanese in cyanide antagonism.

Present studies have focused on nano-intercalated rhodanese in combination with sulfur donors to prevent cyanide lethality in a prophylactic mice model for future development of an effective cyanide antidotal system. Our approach is based on the idea of converting cyanide to the less toxic thiocyanate before it reaches the target organs by utilizing sulfurtransferases (e.g., rhodanese) and sulfur donors in a close proximity by injecting them directly into the blood stream. The inorganic thiosulfate (TS) and the garlic component diallydisulfide (DADS) were compared as sulfur donors with the nano-intercalated rhodanese in vitro and in vivo. The in vivo and in vitro experiments showed that DADS is not a more efficient sulfur donor than TS. However, the utilization of external rhodanese significantly enhanced the in vivo efficacy of both sulfur donor-nitrite combinations, indicating the potential usefulness of enzyme nano-delivery systems in developing antidotal therapeutic agents.

Characterization of liposomal vesicles encapsulating rhodanese for cyanide antagonism.

The major mechanism of removing cyanide from the body is its enzymatic conversion by a sulfurtransferase, e.g. rhodanese, to the less toxic thiocyanate in the presence of a sulfur donor. Earlier results demonstrated that externally administered encapsulated rhodanese significantly enhances the in vivo efficacy of the given sulfur donor. Present studies are focused on liposomal carrier systems encapsulating rhodanese. Physicochemical properties, e.g. membrane rigidity, size distribution, surface potential, osmolarity, and viscosity, were determined for various liposomal lipid compositions and hydrating buffers to establish in vitro stability and in vivo fate. Lipid composition was also optimized to achieve maximum encapsulation efficiency.

Cyanide antagonism with carrier erythrocytes and organic thiosulfonates.

Previous studies reported that resealed erythrocytes containing rhodanese (CRBC) and NA2S2O3 rapidly metabolize cyanide to the less toxic thiocyanate both in vitro and in vivo. This provided a new conceptual approach to prevent and treat cyanide intoxication. Although the rhodanese-containing carrier cells with thiosulfate as the sulfur donor were efficacious, this approach has potential disadvantages, as thiosulfate has limited penetration of cell membrane and product inhibition of rhodanese can occur due to inorganic sulfite accumulation. In order to circumvent substrate limitation and product inhibition by sodium thiosulfate, organic thiosulfonates were explored. These thiosulfonates have higher lipid solubility than thiosulfate and therefore can replenish the depleted sulfur donor, as they can readily penetrate cell membranes. Also, product inhibition of rhodanese is less apt to occur. This change in sulfur donors should greatly enhance cyanide detoxication, replenish the sulfur donor, and minimize product inhibition of rhodanese. Present studies demonstrate the enhanced efficacy of exogenous organic thiosulfonates over sodium thiosulfate in the CRBC antidotal system to detoxify the lethal effects of cyanide either alone or in combinations with exogenously administered NaNO2. Murine carrier erythrocytes containing purified bovine liver rhodanese were administered intravenously into male Balb/C mice. Subsequently, butanethiosulfonate (BTS) or Na2S2O3 (ip), and NaNO2 (sc) were co-administered prior to KCN (sc). Potency ratios, derived from the LD50 values, were compared in groups of mice treated with CRBC-Na2S2O3 or CRBC-BTS either alone or in combination with NaNO2.(ABSTRACT TRUNCATED AT 250 WORDS)

Antagonism of cyanide intoxication with murine carrier erythrocytes containing bovine rhodanese and sodium thiosulfate.

Murine carrier erythrocytes containing bovine rhodanese and sodium thiosulfate are being explored as a new approach to antagonize the lethal effects of potassium cyanide in mice. Prior studies indicated that these carrier erythrocytes persist in the vascular system for the same length of time as normal erythrocytes and can enhance metabolism of cyanide to thiocyanate. The present studies demonstrate the ability of these carrier red blood cells containing rhodanese and thiosulfate to antagonize the lethal effects of cyanide either alone or in various combinations with sodium nitrite and/or sodium thiosulfate. Potency ratios are compared in groups of mice treated with sodium nitrite, sodium thiosulfate, and carrier erythrocytes containing rhodanese and sodium thiosulfate either alone or in various combinations prior to the administration of potassium cyanide. These results indicate that the administration of carrier erythrocytes containing rhodanese and thiosulfate alone can provide significant protection against the lethal effects of cyanide. These carrier erythrocytes potentiate the antidotal effect of sodium thiosulfate alone or the combination of sodium nitrite and sodium thiosulfate. The mechanisms of cyanide antagonism by these carrier erythrocytes and their broader conceptual significance to the antagonism of other chemical toxicants are discussed.

Antagonism of the lethal effects of cyanide with resealed erythrocytes containing rhodanese and thiosulfate.

A new concept has been presented for the antagonism of cyanide and possibly other chemical toxicants. Until now, only a half dozen truly specific "antidotes" were known. There are many other "antidotes" which merely prevent the absorption or enhance the elimination of a toxic compound rather than specifically destroying the substance to prevent its toxic effect. This new approach has considerable conceptual significance in toxicology, as it suggests the encapsulating other enzymes to degrade various other chemical toxicants. There are many chemical toxicants for which there are no specific antidotes, and the conceptual approach of employing erythrocyte-encapsulated enzyme provides an innovative, specific approach to antagonize the toxic and lethal effects of these chemicals.

In vivo studies on rhodanese encapsulation in mouse carrier erythrocytes.

Resealed erythrocytes containing sodium thiosulfate and rhodanese (CRBC) are being employed as a new approach in the antagonism of cyanide intoxication. In earlier in vitro studies, the behavior of red blood cells containing rhodanese and sodium thiosulfate was investigated with regard to their properties and their capability of metabolizing cyanide to thiocyanate. The present studies are concerned with the properties of these rhodanese-containing carrier erythrocytes in the intact animal. These carrier erythrocytes were administered intravenously and the survival of the encapsulated enzyme was compared with the administration (iv) of free exogenous enzyme. Also, the amount of leakage of the encapsulated rhodanese from the red blood cell was determined. The survival of the carrier red blood cell. prepared by hypotonic dialysis, was found to be characterized by a biphasic curve. There was an initial rapid loss of approximately 40 to 50% of the carrier cells with a t1/2 = 2.5 hr. Subsequently the remaining resealed annealed carrier erythrocytes persisted in the vascular system with a t1/2 = 8.5 days. When free exogenous rhodanese was administered directly into the vascular system, it was rapidly eliminated with a t1/2 = 53 min. Red blood cells containing sodium thiosulfate and rhodanese apparently are effective in vivo in the biotransformation of cyanide. In animals pretreated with encapsulated rhodanese and sodium thiosulfate, blood cyanide concentrations are appreciably decreased with a concomitant increase in thiocyanate ion, a metabolite of cyanide. When erythrocytes, which contained no rhodanese or sodium thiosulfate, were subjected to hypotonic dialysis, cyanide was not metabolized to any appreciable extent. Furthermore, carrier erythrocytes containing rhodanese and sodium thiosulfate were found to increase the protection against the lethal effects of cyanide by approximately twofold. The ability of these carrier erythrocytes alone to metabolize cyanide and to antagonize the lethal effects of cyanide reflects the potential of this new antidotal approach in the antagonism of chemical toxicants.

Rhodanese and sodium thiosulfate encapsulated in mouse carrier erythrocytes. II. In vivo survivability and alterations in physiologic and morphologic characteristics.

Biodegradable drug carrier mechanisms were employed in drug antagonism studies. Prior studies indicated that erythrocytes containing encapsulated rhodanese and sodium thiosulfate metabolized cyanide to thiocyanate in vitro. Studies were conducted to investigate the properties of these sulfurtransferase-loaded red blood cells in vivo by administering the carrier red blood cells intravenously. Approximately 40 to 50% of the cells were eliminated within the first few hours while the remaining loaded erythrocytes persisted in the circulation. The present studies were initiated to investigate the characteristics of the disposition of the loaded erythrocytes and to examine differences in the properties between carrier and noncarrier erythrocytes. Also, the disposition and viability of the erythrocytes in vivo were studied with relation to various biochemical, physiological, and morphological properties. These studies indicated that the carrier erythrocytes had a smaller cell volume and were more susceptible to hemolysis than normal erythrocytes. Morphologic studies by electron microscopy indicated that extensive morphologic changes occurred during the procedures after hypotonic dialysis, isotonicity adjustment, and resealing were completed. Differences were noted between those cells that were only resealed and those cells that were also subjected to annealing. The morphologic characteristics of most of the cells were restored to the "normal" morphologic appearance only after annealing. Annealed erythrocytes' in vivo survivability was correlated with the physical properties of these cells.

Encapsulation of thiosulfate: cyanide sulfurtransferase by mouse erythrocytes.

Murine carrier erythrocytes, prepared by hypotonic dialysis, were employed in the encapsulation of several compounds including [14C]sucrose, [3H]inulin, and bovine thiosulfate:cyanide sulfurtransferase (rhodanese), a mitochondrial enzyme which converts cyanide to thiocyanate. Approximately 30% of the added [14C]sucrose, [3H]inulin, and rhodanese was encapsulated by predialyzed erythrocytes, and a decrease in the mean corpuscular volume and mean corpuscular hemoglobin was observed. In the encapsulation of rhodanese a recovery of 95% of the erythrocytes was achieved and an 85% equilibrium was established. The addition of potassium cyanide (50 mM) to intact, rhodanese-loaded erythrocytes containing sodium thiosulfate resulted in its metabolism to thiocyanate. These results establish the potential use of erythrocytes as biodegradable drug carrier in drug antagonism.