Comparing therapeutic and prophylactic protection against the lethal effect of paraoxon.

Prophylactic and therapeutic efficacy against organophosphorus (OP) intoxication by pralidoxime (2-PAM) and atropine were studied and compared with sterically stabilized long-circulating liposomes encapsulating recombinant organophosphorus hydrolase (OPH), either alone or in various specific combinations, in paraoxon poisoning. Prophylactic and therapeutic properties of atropine and 2-PAM are diminished when they are used alone. However, their prophylactic effects are enhanced when they are used in combination. Present studies indicate that sterically stabilized liposomes (SL) encapsulating recombinant OPH (SL-OPH) alone can provide much better therapeutic and prophylactic protection than the classic 2-PAM + atropine combination. This protection was even more dramatic when SL-OPH was employed in combination with 2-PAM and/or atropine: the magnitude of prophylactic antidotal protection was an astounding 1022 LD(50) [920 mg/kg (LD(50) of paraoxon with antagonists)/ 0.95 mg/kg (LD(50) of control paraoxon)], and the therapeutic antidotal protection was 156 LD(50) [140 mg/kg (LD(50) of paraoxon with antagonists)/0.9 mg/kg (LD(50) of control paraoxon)]. The current study firmly establishes the value of using liposome encapsulating OPH.

Long circulating liposomes encapsulating organophosphorus acid anhydrolase in diisopropylfluorophosphate antagonism.

These studies are focused on antagonizing organophosphorous (OP) intoxications by a new conceptual approach using recombinant enzymes encapsulated within sterically stabilized liposomes to enhance diisopropylfluorophosphate (DFP) degradation. The OP hydrolyzing enzyme, organophosphorous acid anhydrolase (OPAA), encapsulated within the liposomes, was employed either alone or in combination with pralidoxime (2-PAM) and/or atropine. The recombinant OPAA enzyme, from the ALTEROMONAS: strain JD6, has high substrate specificity toward a wide range of OP compounds, e.g., DFP, soman, and sarin. The rate of DFP hydrolysis by liposomes containing OPAA (SL)* was measured by determining the changes in fluoride-ion concentration using a fluoride ion-selective electrode. This enzyme carrier system serves as a biodegradable protective environment for the OP-metabolizing enzyme (OPAA), resulting in an enhanced antidotal protection against the lethal effects of DFP. Free OPAA alone showed some antidotal protection; however, the protection with 2-PAM and/or atropine was greatly enhanced when combined with (SL)*.

In vitro studies on sterically stabilized liposomes (SL) as enzyme carriers in organophosphorus (OP) antagonism.

This study describes a new approach for organophosphorous (OP) antidotal treatment by encapsulating an OP hydrolyzing enzyme, OPA anhydrolase (OPAA), within sterically stabilized liposomes. The recombinant OPAA enzyme was derived from Alteromonas strain JD6. It has broad substrate specificity to a wide range of OP compounds: DFP and the nerve agents, soman and sarin. Liposomes encapsulating OPAA (SL)* were made by mechanical dispersion method. Hydrolysis of DFP by (SL)* was measured by following an increase of fluoride ion concentration using a fluoride ion selective electrode. OPAA entrapped in the carrier liposomes rapidly hydrolyze DFP, with the rate of DFP hydrolysis directly proportional to the amount of (SL)* added to the solution. Liposomal carriers containing no enzyme did not hydrolyze DFP. The reaction was linear and the rate of hydrolysis was first order in the substrate. This enzyme carrier system serves as a biodegradable protective environment for the recombinant OP-metabolizing enzyme, OPAA, resulting in prolongation of enzymatic concentration in the body. These studies suggest that the protection of OP intoxication can be strikingly enhanced by adding OPAA encapsulated within (SL)* to pralidoxime and atropine.

Antagonism of paraoxon intoxication by recombinant phosphotriesterase encapsulated within sterically stabilized liposomes.

This investigation effort is focused on increasing organophosphate (OP) degradation by phosphotriesterase to antagonize OP intoxication. For these studies, sterically stabilized liposomes encapsulating recombinant phosphotriesterase were employed. This enzyme was obtained from Flavobacterium sp. and was expressed in Escherichia coli. It has a broad substrate specificity, which includes parathion, paraoxon, soman, sarin, diisopropylfluorophosphate, and other organophosphorous compounds. Paraoxon is rapidly hydrolyzed by phosphotriesterase to the less toxic 4-nitrophenol and diethylphosphate. This enzyme was isolated and purified over 1600-fold and subsequently encapsulated within sterically stabilized liposomes (SL). The properties of this encapsulated phosphotriesterase were investigated. When these liposomes containing phosphotriesterase were incubated with paraoxon, it readily degraded the paraoxon. Hydrolysis of paraoxon did not occur when these sterically stabilized liposomes contained no phosphotriesterase. These sterically stabilized liposomes (SL) containing phosphotriesterases (SL)* were employed as a carrier model to antagonize the toxic effects of paraoxon by hydrolyzing it to the less toxic 4-nitrophenol and diethylphosphate. This enzyme-SL complex (SL)* was administered intravenously to mice either alone or in combination with pralidoxime (2-PAM) and/or atropine intraperitoneally. These results indicate that this carrier model system provides a striking enhanced protective effects against the lethal effects of paraoxon. Moreover when these carrier liposomes were administered with 2-PAM and/or atropine, a dramatic enhanced protection was observed.

Bond strength of light-cured glass ionomers to carious primary dentin.

The pre-cooperative or handicapped child with decay presents a special challenge to the practitioner and may require sedation or general anesthesia. Treatment with an interim restoration may delay treatment until the child is more mature and can accept dental treatment and is a more conservative approach than sedation, extractions or general anesthesia. Glass ionomer materials have been utilized for this application, but little is known about their retention to carious dentin. The purpose of this study was to determine whether the presence of artificial dentin decay will affect the shear bond strength of two light-cured glass ionomer materials. VariGlass and Vitrebond glass ionomer materials were attached to carious and non-carious primary dentin surfaces and bond strengths determined. There were no significant differences in shear bond strengths between the decayed and non-decayed surfaces [p < or = .001]. VariGlass had higher shear bond strengths than Vitrebond only after a pre-treatment with the PAA containing liquid. Pre-treatment with the liquid provided with each light-cured glass ionomer was beneficial in all instances except for Vitrebond on non-decayed surfaces.

Antagonism of the lethal effects of paraoxon by carrier erythrocytes containing phosphotriesterase.

Annealed murine erythrocytes were employed as a carrier model to antagonize the toxic effects of organophosphorus agents. These resealed cells containing a recombinant phosphotriesterase provided striking protection against the lethal effect of paraoxon, an active metabolite of an agricultural pesticide, parathion. Phosphotriesterase hydrolyzes paraoxon to the less-toxic 4-nitrophenol and diethylphosphate. This enzyme was encapsulated into carrier erythrocytes by hypotonic dialysis with subsequent resealing and annealing. These carrier cells were administered to mice either alone or in combination with pralidoxime (2-PAM) and/or atropine. The recipient animals were subsequently challenged with paraoxon and a marked protection was noted. Protection of free enzyme and encapsulated enzyme was compared and the encapsulated enzyme was found to persist longer and possess much greater efficacy. Less serum cholinesterase inhibition also was observed with this enhanced protection. These results indicate that the erythrocyte carrier alone is quite effective in the antagonism of organophosphorus intoxication. Moreover, when these carrier cells were administered in combination with 2-PAM and/or atropine, a marked synergism was observed.

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)

Spectrophotometric determination of paraoxonase within mouse carrier red blood cells.

A method has been developed to continuously measure paraoxonase activity spectrophotometrically in carrier red blood cells (RBCs) containing paraoxonase. This enzyme has a broad substrate specificity that includes parathion, paraoxon, soman, sarin, di-isopropyl fluorophosphate and many other organophosphorus compounds. Paraoxon is hydrolysed by paraoxonase to the less toxic 4-nitrophenol and diethyl phosphate. Determination of enzymic activity was based on the liberation of 4-nitrophenol in the presence of mouse RBCs. Paraoxonase was encapsulated within murine RBCs by hypotonic dialysis with subsequent resealing and annealing. The enzyme within resealed RBCs actively hydrolyses paraoxon in biological fluids to its less toxic metabolites. Paraoxonase incorporated within RBCs, like other enzymes, was found to be quite stable once encapsulated into RBCs and this formed the basis for this spectrophotometric method. Increasing absorbance at 400 nm indicated paraoxon hydrolysis and was the basis employed to determine enzymic activity. The Km of the enzyme within erythrocytes was 0.04 mM. This method offers a convenient, rapid and continuous way to monitor paraoxonase activity inside the carrier cell.

Encapsulation of rhodanese and organic thiosulfonates by mouse erythrocytes.

A series of organic thiosulfonates were synthesized and studied as sulfur donor substrates for rhodanese encapsulated within murine carrier erythrocytes. Previous studies have indicated that resealed erythrocytes containing rhodanese (CRBC) and sodium thiosulfate can rapidly metabolize cyanide to the less toxic thiocyanate. This thiosulfate-rhodanese system was very efficacious as a new conceptual approach to antagonize cyanide intoxication both in vitro and in vivo. However, its potential is restricted because of the limited availability of thiosulfate due to its poor permeability through RBC membrane. Present studies suggest that there are advantages in using alternative sulfur donors, i.e., organic thiosulfonates in this rhodanese-containing resealed erythrocyte system, since these compounds have higher lipid solubility than inorganic thiosulfates and can readily penetrate the red blood cell membrane. Therefore, this system could provide a virtually unlimited amount of sulfur donor to the encapsulated rhodanese even if the substrates are in solution outside the cells. Moreover, the rhodanese reaction rate of any of these organic thiosulfonates is much faster than the rate observed with the classic cyanide antidote, sodium thiosulfate. This CRBC system will continue to detoxify cyanide even when these encapsulated sulfur donors are depleted, as the lipid soluble organic thiosulfonate outside the cells will diffuse past the membrane into the cell to replenish the sulfur donor. The encapsulation efficiency for rhodanese is about 30%, and the velocity of the rhodanese reaction increases linearly with the volume of enzyme-laden erythrocytes. Similarly, reaction velocity increases linearly with substrate concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Antagonism of the lethal effects of cyanide by a synthetic water-soluble cobalt(III) porphyrin compound.

Efficacy of hydroxocobalamin (vitamin B12) as a cyanide antidote is limited by its high molecular weight (1355 g/mol) and by the competitive binding of the cobalamin dimethylbenzimidazole. The present study describes experiments with a lower molecular weight cobalt porphyrin that has a high affinity for cyanide, Co(III)-5,10,15,20-tetrakis(4-sulfonatophenyl) porphyrin (CoTPPS), which was prepared by the method of Herrmann et al. (1978). CoTPPS was synthesized and its efficacy as an antidote to the lethal effects of cyanide either alone or in various combinations with NaNO2 and/or Na2S2O3 was determined. The LD50 value for CoTPPS was found to be 334 mg/kg. These studies were conducted using the CoTPPS LD01, 200 mg/kg. The cyanide antagonists NaNO2 (0.1 g/kg, sc), Na2S2O3 (1.0 g/kg, ip), and CoTPPS (0.2 g/kg, ip) were administered at 45, 15, and 10 min respectively prior to graded doses of KCN (sc). The LD50 values for KCN in male Swiss-Webster mice were calculated by probit analysis at the 95% confidence level and the various treatments were compared by potency ratios. These results indicated that the administration of CoTPPS alone protects against the lethal effects of cyanide. Moreover, CoTPPS adds to the protection provided by Na2S2O3 and/or NaNO2. Efficacy of this antidote is probably related to the binding equilibrium between CoTPPS and cyanide.

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.

Encapsulation of phosphotriesterase within murine erythrocytes.

A new conceptual approach was employed to antagonize organophosphorus intoxication by using resealed carrier erythrocytes containing a recombinant phosphotriesterase. This enzyme has been reported to hydrolyze many organophosphorus compounds, including paraoxon, a potent cholinesterase inhibitor. Paraoxon is rapidly hydrolyzed by this enzyme to p-nitrophenol and diethylphosphate. Incorporation of phosphotriesterase within resealed murine erythrocytes was accomplished by hypotonic dialysis. The properties of this enzyme within these resealed erythrocytes were investigated. Addition of paraoxon to reaction mixtures containing these resealed erythrocytes loaded with phosphotriesterase resulted in the rapid hydrolysis of paraoxon. Hydrolysis of paraoxon did not occur when these carrier erythrocytes contained no phosphotriesterase. These in vitro studies suggest that carrier erythrocytes may be developed as an approach for the prophylactic and therapeutic antagonism of organophosphorus intoxication.

The encapsulation of squid diisopropylphosphorofluoridate-hydrolyzing enzyme within mouse erythrocytes.

This study describes the entrapment of squid-type diisopropylphosphorofluoridate-hydrolyzing enzyme (DFPase) within mouse red blood cells. These erythrocytes thereby gain the ability to rapidly hydrolyze alkylphosphate cholinesterase (ChE) inhibitors such as diisopropyl fluorophosphate (DFP). DFPase rapidly hydrolyzes DFP to diisopropyl phosphate. Resealed erythrocytes provide a stable carrier system that can preserve the activity of encapsulated enzymes against otherwise rapid in vivo degradation; thus, ChE inhibitors can be degraded to relatively nontoxic metabolites by these erythrocyte carriers. Squid DFPase was purified from the hepatopancreas of Atlantic squid and DFPase activity was determined by measuring changes in fluoride ion concentration using a fluoride ion selective electrode. Mouse erythrocytes in suspension with excess squid DFPase were dialyzed against hypotonic buffer to allow the encapsulation of the enzyme to occur. Cells were then resealed by returning the suspension to isosmotic with saline. Rate of DFP hydrolysis observed with these cells was much greater than the rate of nonenzymatic hydrolysis and was directly proportional to the amount of the erythrocyte suspension added to the assay solution. The rate of hydrolysis was first order in substrate. Erythrocyte controls showed no endogenous DFPase activity. These results suggest that enzyme entrapment may be developed as a method to prevent and antagonize organophosphate poisoning.

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.

The mechanism of cyanide intoxication and its antagonism.

The mechanism of cyanide intoxication has been attributed to the inhibition of cytochrome oxidase, thereby decreasing the tissue utilization of oxygen. One mechanism of cyanide antagonism is by sequestering cyanide with methaemoglobin to form cyanmethaemoglobin and another mechanism is detoxifying with a sulphur donor to thiocyanate. Questions have been raised with regard to these classical mechanisms. Oxygen with nitrite-thiosulphate antagonizes the lethal effects of cyanide. Theoretically, increased oxygen should serve no useful purpose, as it is the tissue utilization of oxygen which is inhibited. In the nitrite-thiosulphate antidotal combination, the proposal is made that the predominate antidotal action of nitrite is a vasogenic action, rather than methaemoglobin formation, because when methaemoglobin formation is inhibited by methylene blue the protective action of sodium nitrite persists. This suggests that methaemoglobin formation plays only a small part, if any, in the therapeutic antagonism of the lethal effects of cyanide. The roles and implications of sodium thiosulphate and non-rhodanese substrates in the detoxification mechanism are compared. Lastly, a new approach to cyanide antagonism has been initiated which involves the erythrocyte encapsulation of thiosulphate and sulphurtransferase as an antidote and prophylaxis against cyanide.

Stereospecific effect of naloxone hydrochloride on cyanide intoxication.

Cyanide intoxication in mice can be antagonized by the opiate antagonist, (-)naloxone HCl, alone or in combination with sodium thiosulfate and/or sodium nitrite. Potency ratios, derived from LD50 values, were compared in groups of mice pretreated with sodium nitrite (sc, 100 mg/kg), sodium thiosulfate (ip, 1 g/kg), and (-)naloxone HCl (sc, 10 mg/kg) either alone or in various combinations. These results indicate that naloxone HCl provides a significant protection against the lethal effects of potassium cyanide. The protective effect of sodium thiosulfate, but not sodium nitrite, was enhanced with (-)naloxone HCl. The combined administration of sodium nitrite and sodium thiosulfate was further enhanced with (-)naloxone HCl. This protective effect of naloxone HCl against the lethal effect of cyanide appears to be restricted to the (-)stereoisomer, as the (+)stereoisomer, the inactive opiate antagonist, is also inactive in protecting against the lethal effects of cyanide. The mechanism of antagonism is discussed.

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.