TOXICOLOGICAL SCIENCES 77, 258 262 (2004) DOI: 10.

1093/toxsci/kfg185 Advance Access publication July 11, 2003

Comparing Therapeutic and Prophylactic Protection against the Lethal Effect of Paraoxon
I. Petrikovics,* D. Papahadjopoulos, K. Hong, T.-C. Cheng,* S. I. Baskin,* ,1 J. Jiang, J. C. Jaszberenyi, B. A. Logue,* M. Szilasi, W. D. McGuinn, and J. L. Way
*U.S.A. Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, Maryland 21010; Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, California 94143; Technical University of Budapest, Hungary, H-1010, Debrecen, Hungary H-4031; Department of Medical Pharmacology and Toxicology, Texas A&M University, College of Medicine, College Station, Texas 77843; and University Medical School, Department of Pulmonology, Debrecen, Hungary H-4031 Received May 16, 2003; accepted June 24, 2003 Downloaded from http://toxsci.oxfordjournals.org/ at FMRP/USP/BIBLIOTECA CENTRAL on March 19, 2012

Prophylactic and therapeutic efcacy 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 specic 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 rmly establishes the value of using liposome encapsulating OPH. Key Words: organophosphorus hydrolase (OPH); OPA anhydrase; paraoxonase; paraoxon antagonism; sterically stabilized liposomes; stealth liposomes; long circulating liposomes; organophorus antagonism; prophylactic; therapeutic.

Cell carriers as biodegradable protective environments for organophosphorus (OP) enzymes to antagonize toxic effects of different toxins have attracted increasing attention recently. Usually, the body has large amounts of these OP metabolizing enzymes, but the distribution of toxin molecules to the enzyme localization is a limiting factor. To achieve maximal hydrolysis rate, recombinant fast enzymes should be available for the
1 To whom correspondence should be addressed at USAMRICD, Division of Pharmacology, 3100 Ricketts Point Rd., E3100, Aberdeen Proving Ground, MD 21010-5400. Fax: (410) 436-8377. E-mail: Steven.Baskin@apg.amedd. army.mil.

Toxicological Sciences vol. 77 no. 2 Society of Toxicology 2004; all rights reserved.

hydrolysis of the OP molecules in the body. Injection of puried free enzyme preparations directly into the bloodstream has serious limitations because of potential immunologic reactions and unfavorable physiological factors. Biodegradable enzyme carriers, which are permeable to toxin molecules, can efciently provide large amounts of the metabolizing enzymes in the circulation with minimal leakage and few immunologic reactions. Ihler et al. (1973) rst reported that enzymes and drugs could be successfully encapsulated within carrier red blood cells. DeLoach et al. (1980) optimized the red blood cell encapsulation by hypotonic dialysis procedures. In earlier studies, carrier resealed and annealed erythrocytes were successively employed as cell carriers in cyanide antagonism (Cannon et al., 1992; Leung et al., 1986, 1991; Petrikovics et al., 1995; Way et al., 1985) and in OP antagonism (Pei et al., 1994, 1995). In the sterically stabilized liposomes, also referred to as stealth liposomes, a protein is encapsulated in a biodegradable cell carrier to circumvent immune defenses, thus avoiding the phagocytic activity of the macrophages and the reticuloendothelial system (Allen, 1994; Oku and Namba, 1994; Papahadjopoulos et al., 1991; Woodle and Lasic, 1992). Previous studies reported the in vitro properties and in vivo prophylactic protective effects of sterically stabilized, long circulating liposomes encapsulating recombinant organophosphorus hydrolase (SL-OPH) to antagonize the lethal effects of paraoxon (Petrikovics et al., 1999). The present study compares the therapeutic and prophylactic effects of these OP antagonists. OP agents are believed to react with a serine hydroxyl group in the active site of acetylcholinesterase (AChE), thereby inactivating this hydrolytic enzyme. This causes an accumulation of acetylcholine esters, followed by a cholinergic overstimulation. A new antidotal mechanism involved in OP intoxication involves the degradation of organophosphorus compounds using OP-hydrolyzing enzymes. In this way, the OP molecules are degraded before they phosphorylate AChE. Other OP antagonists [pralidoxime (2-PAM), atropine] exert a protective effect, but they do not destroy the OP compounds. 2-PAM can exert its antidotal effect as an OP antagonist by reactivating the

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259

inhibited phosphorylated AChE. The 2-PAM can control the nicotinic symptoms caused by the OP agents, and the atropine counteracts the muscarinic effects of the accumulating neurotransmitter acetylcholine by competing with the OP molecules for the acetylcholine receptor site. The combination of 2-PAM and atropine with the OP-hydrolyzing enzyme in an appropriate biodegradable carrier protective environment provides a far better protection against the lethal effects of OP agents than the components alone. The therapeutic protection against OP intoxication, particularly in combination with SL-OPH has not been reported and is of considerable importance.
MATERIALS AND METHODS
Chemicals. Paraoxon (ca. 90%) was purchased from Sigma (St Louis, MO), and was further puried by aqueous sodium bicarbonate extraction (Pei et al., 1993; Petrikovics et al., 1999). Paraoxon solution was prepared immediately before use and was kept no longer than two h before administration. Atropine sulfate and 2-PAM solutions were prepared daily. All other chemicals used were of the highest purity commercially available. Enzyme. A recombinant organophosphorus hydrolase (OPH; EC 3.1.8) was puried from an Escherichia coli clone containing the plasmid expression vector pjK33, which was isolated from Flavobacteium sp. (McDaniel et al., 1988; Serdar and Gibson, 1985). This enzyme was obtained in preparative amounts and puried over 1600-fold by the method of Omburo et al. (1992) with minor modications (Pei et al., 1994, 1995). Sterically stabilized liposomes (SL) and SL-OPH preparation. SL and SL-OPH were prepared as previously described (Petrikovics et al., 1999) with minor modications. Palmitoyloleoylphosphatidylcholine (POPC) and dipalmitoyl phosphatidylethanolamine-N-[poly(ethylene glycol) 2000] (PEG-PE) were purchased from Avanti Polar Lipid (Alabaster, AL). Puried cholesterol was obtained from Calbiochem (San Diego, CA). Lipids were stored in chloroform under argon at 70C. Sepharose 4B was purchased from Pharmacia Fine Chemicals (Uppsala, Sweden). Chloroform solutions of POPC (60 mol), cholesterol (40 mol) and PEG-PE (5.4 mol) were mixed in a round-bottomed ask, and the solvent was removed slowly on a rotary evaporator at 37C to obtain dry thin lipids lm on the ask. Puried OPH in HEPES [N-(2 hydroxyethyl) piperazine-N -(2-ethanesulfonic acid) sodium salt] buffer (concentrated on Amicon Concentrator and claried by centrifugation if necessary) was added to the dry lipids. The lipid lm was hydrated slowly under argon by continuously rotating the ask on a rotary evaporator at 37C for 1 h. Milky liposome suspension was extruded sequentially through 0.2 and 0.1 polycarbonate membrane lter. Extrusion was repeated ve times for each membrane to obtain a homogeneous size distribution of liposomes. Unencapsulated OPH was separated from liposomes by gel ltration on sepharose 4B column. Encapsulation efciency was calculated from the amount of encapsulated OPH divided by the amount added times 100. Determination of OPH activity in sterically stabilized carrier liposomes. OPH activity in liposomes was measured at room temperature, by determining the increase of p-nitrophenol concentration in the presence of excess paraoxon. The standard solution used to determine OPH activity within carrier liposomes contained 1.0 ml of 15 mM phosphate buffer system containing 216.0 mM NaCl, 0.08 mM ZnCl 2; 3.0 mM MgCl 2, (pH 7.8, osmolality 290 mOsm); 0.2 ml of paraoxon standard (6.0 mM); and varying amount of sterically stabilized liposomes encapsulating OPH. Water was added to obtain the nal volume of 1.5 ml. The reaction was initiated with the addition of encapsulated OPH. Absorbance of the solution was determined at 400 nm with a Shimadzu UV 2101 PC spectrophotometer. The molecular extinction coefcient of pnitrophenol in phosphate buffer at pH 7.8 was determined using various dilutions of gravimetric standard solutions of p-nitrophenol. One unit of OPH

is dened as that amount of enzyme which hydrolyzed one mol of paraoxon to p-nitrophenol per min. Protein assays were performed by the Bradford method (Bradford, 1976) using the Bio-Rad protein assay reagent (Bio-Rad, Richmond, CA). Animals. Male Balb/C mice (Charles River Breeding Laboratories, Inc., Wilmington, MA) weighing 18 20 g were housed at 21 2C and in light-controlled rooms (12-h light/dark, full-spectrum lighting cycle with no twilling), and were furnished with water and 4% Rodent Chow (Teklad HSD, Inc., WI) ad libitum. All animal procedures were conducted in accordance with the guidelines by The Guide for the Care and Use of Laboratory Animals (National Academic Press, 1996), credited by AAALAC (American Association for the Assessment and Accreditation of Laboratory Animal Care, International). At the termination of the experiments, surviving animals were euthanized in accordance with the 1986 report of the AVMA Panel of Euthansia. Prophylactic in vivo experiments. Male mice received 510 units of OPH (SL-OPH) iv one h prior to receiving paraoxon (in 6% cyclodextrin and propylene glycol solvent system) sc. The propylene glycol solvent system consisted of 40% propylene glycol, 10 % ethanol, and 50% water (v/v). Animals exposed to paraoxon with antagonists (atropine and/or 2-PAM and/or OPH) were determined by 24-h mortality. Surviving animals were observed for an additional week for late-developing toxicity. No gross toxic effects were observed in mice caused by encapsulated OPH, atropine, and 2-PAM, when they were administered without paraoxon either alone or in various combinations. Atropine and 2-PAM were administered intraperitoneally to mice 45 and 15 min, respectively, prior to receiving paraoxon. LD 50 values were determined by the up-and-down method (simulated up-and-down study, Dixon, 1965). For each experiment, 6 to 10 mice were used. The LD 50 values were calculated from the equation of log(LD 50) log(dose nal) k log(d), where dose nal is the nal dose administered, k is the tabular value from table (Dixon, 1965), and d is the interval between doses. Therapeutic in vivo experiments. Paraoxon was administered sc, sterically stabilized liposomes encapsulating 10 units of OPH (SL-OPH) were administered iv, and 2-PAM (90 mg/kg) and/or atropine (10 mg/kg) were administered ip. The antagonists were administered 1 min after the paraoxon administration. When 2-PAM and atropine were used in a combination, they were mixed before they were administered ip. LD 50 values were determined by the up-and-down method (simulated up-and-down study, Dixon, 1965). For each experiment, 6 10 mice were used.

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RESULTS

The organophosphorus antidotes, 2-PAM and atropine, were employed to detoxify the lethal effects of paraoxon poisoning, and their prophylactic and therapeutic efcacies were compared with stealth liposomes containing recombinant OPH (SL-OPH). The prophylactic and therapeutic protections with these antidotal systems are summarized and expressed as LD 50 values in Table 1. The 2-PAM or atropine showed only a slight protection in the prophylactic and therapeutic experiments when they were used alone. The 2-PAM atropine combination resulted in a more potent protection in the prophylactic experiments, but there was only a slight increase in the therapeutic experiments. The SL-OPH alone protected better than the 2-PAM atropine combination. The highest protection was observed when SL-OPH was employed together with 2-PAM atropine. The overall importance of encapsulated OPH in protecting against paraoxon intoxication was best illustrated when the protection of antagonized paraoxon experiments were com-

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TABLE 1 Prophylactic and Therapeutic Protective Effects with Sterically Stabilized Liposomes Encapsulating Recombinant OPH (SL-OPH)
Paraoxon* LD 50 (mg/kg) Exp. no. 1 2 3 4 5 6 7 8 Treatment Control (paraoxon) Atropine 2-PAM 2-PAM atropine SL-OPH SL-OPH atropine SL-OPH 2-PAM SL-OPH 2-PAM Prophylactic** 0.95 2.08 4.50 55.00 125.20 540.30 550.20 920.00 Therapeutic*** 0.90 2.05 4.10 11.02 65.10 110.20 115.10 140.25

atropine

*Paraoxon was dissolved in 6% -cyclodextrin and/or propylene glycol solvent solutions. The propylene glycol solvent consisted of 40% propylene glycol, 10% ethanol, and 50% water. LD 50 values were determined by the up-and-down method (Dixon, 1965). **In the prophylactic experiments, SL-OPH (iv) (10 units OPH), atropine (ip) (10 mg/kg), and 2-PAM (ip) (90 mg/kg) were administered 60 min, 45 min, and 15 min, respectively, prior to the administration of paraoxon (sc). ***In the therapeutic experiments paraoxon was administered sc, and the antidotes (10 units of OPH) and/or 2-PAM (90 mg/kg) and/or atropine (10 mg/kg) were administered 1 min after the paraoxon administrations.

lower than their prophylactic effects (P-T ratios are 1.01 and 1.10, respectively). This difference is much higher when they are used in a combination (Exp. 4, P-T ratio 4.99). The 2-PAM and atropine have only moderate antidotal effects when they are used alone as OP antagonist (Exps. 2 and 3). Their combination provides a far better protection, especially when they are used prophylactically (Exp. 4). Employing sterically stabilized liposomes containing recombinant OPH alone provides a much better antidotal protection than the 2-PAM atropine combination (Table 1 and 2, Exp. 4 and Exp. 5, respectively). In the prophylactic experiments, the antagonists were administered prior to the paraoxon (atropine 45 min, 2-PAM 15 min, respectively, before paraoxon administration). In the therapeutic experiments, the antagonists were injected 1 min after paraoxon administration. At lower doses of paraoxon, none of the animals showed any sign or symptoms of being poisoned, but at higher paraoxon doses they were shivering and shaking, just shortly after paraoxon administration. Figure 1 demonstrates the correlation of paraoxon dose with the onset of the toxic signs. When the toxic signs appeared before the administration of the antidotes, all the mice died within 24 h. As it is shown in Table 2, the tendency of changes of antidotal potency ratios in the prophylactic experiments and in the

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pared to the unantagonized experiments and expressed as antidotal potency ratios (Table 2). Antidotal potency ratios LD 50 of paraoxon with antagonists/LD 50 of paraoxon without antagonists. The prophylactic-therapeutic (P-T) ratios are expressed as the ratio of the LD 50 of paraoxon in prophylactic experiments to the LD 50 of paraoxon in therapeutic experiments. In the case of atropine or 2-PAM (Exps. 2 or 3), the therapeutic antidotal effects are about the same or somewhat
TABLE 2 Prophylactic and Therapeutic Antidotal Potency Ratio with Sterically Stabilized Liposomes Encapsulating Recombinant OPH (SL-OPH)
Antidotal potency ratio* Exp. no. 1 2 3 4 5 6 7 8 Treatment Control (paraoxon) Atropine 2-PAM 2-PAM atropine SL-OPH SL-OPH atropine SL-OPH 2-PAM SL-OPH 2-PAM Prophylactic 1.00 2.31 5.00 61.10 139.00 600.30 611.30 1022.00 Therapeutic 1.00 2.27 4.55 12.24 72.33 122.44 127.88 155.83 P-T ratio** 1.00 1.01 1.10 4.99 1.92 4.90 4.70 6.56

atropine

*Antidotal potency ratio LD 50 of paraoxon with antagonist(s)/LD 50 of paraoxon without antagonist(s). **P-T Ratio LD 50 of paraoxon in prophylactic experiments/LD 50 of paraoxon in the therapeutic experiments.

FIG. 1. Appearance of toxic symptoms after paraoxon administration (sc).

COMPARING PROTECTION AGAINST THE LETHAL EFFECT OF PARAOXON

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therapeutic experiments are similar. However, the magnitude of the increase is much less in the therapeutic experiments. The encapsulated OPH (SL-OPH) alone was more effective (Exp. 5) than the classic antidotal treatment of atropine and 2-PAM. The therapeutic antidotal protection was dramatically increased when animals received both the encapsulated OPH and either 2-PAM or atropine (Exps. 6 and 7). The therapeutic antidotal protection was the highest when the encapsulated OPH was used in a combination with atropine and 2-PAM (Exp. 8). The LD 50 value of paraoxon of control animals (Exp. 1) was dened as 1.0. In therapeutic experiments, the encapsulated OPH alone (Exp. 5) caused an increase in the potency ratio to 72.3. When 2-PAM (potency ratio 4.55, Exp. 3) was employed in a combination with SL-OPH, the potency ratio was increased to 127.9 (Exp. 7). When atropine (potency ratio 2.27, Exp. 2) was used in a combination with SP-OPH, the potency ratio increased to 122.4 (Exp. 6). When both atropine and 2-PAM (potency ratio 12.4, Exp. 4) were tested in a combination of SL-OPH, the potency ratio was elevated to 155.8 (Exp. 8). The use of relative potency ratios (RPR LD 50 of paraoxon with SL-OPH/LD 50 of paraoxon without SL-OPH) provides an expression of the overall efcacy of the encapsulated OPH in the antagonism of the lethal effects of paraoxon. Table 3 shows the therapeutic protective effects of liposomes encapsulating OPH (SL-OPH). The SL-OPH alone gave a 72.3 LD 50 protection. The SL-OPH enhanced the protection of both atropine (53.8 times), and 2-PAM (28.1 times). The increase in the protection of atropine and 2-PAM, 12.7 times, was the most moderate (12.7). It should be noted that the relative potency ratios calculated in this way may lead to erroneous interpretation: The SL-OPH alone (Exp. 5) gave protection of 65.1 mg/kg (Table 1), and the highest relative potency ratio, 72.3 (Table 3), whereas the SL-OPH with atropine and 2-PAM (Exp. 8) showed the highest protection (140.2 mg/kg, Table 1) with an antidotal potency ratio of 155.8 (Table 2), but the lowest relative potency ratio (12.7, Table 3) in the therapeutic experiments.
TABLE 3 Relative Potency Ratio with and Without SL-OPH in Therapeutic Experiments
Relative potency ratio (RPR)*

DISCUSSION

Exp. no. 1 5 2 6 3 7 4 8

Treatment Control SL-OPH Atropine SL-OPH atropine 2-PAM SL-OPH 2-PAM 2-PAM atropine SL-OPH 2-PAM

72.3 53.8 28.1 atropine 12.7

*Relative potency ratio (RPR) LD 50 of paraoxon with SL-OPH / LD 50 of paraoxon without SL-OPH in therapeutic experiments.

These studies represent an attempt to study OP intoxication after signs of intoxication have occurred, or therapeutic antagonism. This is in contrast to most antagonism studies, where the antagonists are usually administered prior to the toxicant. This would be an attempt to develop an antagonist, which would reect real, actual conditions rather than convenient laboratory procedures. With some toxicants, such variations of experimental conditions are of minor consequence, whereas with other toxicants, the changes may be considerable. The development of an antidote under prophylactic conditions may reect therapeutic efcacy, but it is possible that this assumption may not always be totally valid. For example, it appears that 2-PAM and atropine are considerably less effective under therapeutic conditions, as is the liposomal recombinant enzyme. These results represent the rst therapeutic application of SL-OPH as a biodegradable enzyme carrier system to detoxify a toxicant. Earlier studies indicated that sterically stabilized liposomes encapsulating OPH show striking prophylactic protection alone or in a combination of 2-PAM and/or atropine (Petrikovics et al., 1999). Presently, 2-PAM and atropine are used to treat OP poisoning. It should be noted that atropine or 2-PAM are only effective OP antidotes when they are used together prophylactically or therapeutically. They protect prophylactically much better than therapeutically. This has importance in military situations, in eld conditions when soldiers would need immediate medical treatment after OP poisoning, and also, for agricultural workers who are exposed to the OP pesticides. Ashani et al. (1991) and Broomeld (1992) reported the antidotal protective effects of free OP hydrolyzing enzyme in soman poisoning. Recent studies (Pei et al., 1995; Petrikovics et al., 1999) have indicated that the limitations of the applications of free OP hydrolyzing enzymes can be minimized by encapsulating the enzyme within biodegradable microcapsules. Present studies indicate that sterically stabilized liposomes encapsulating OPH (SP-OPH) alone provides better protection than the 2-PAM and atropine combination either prophylactically or therapeutically. The protection was even more remarkable when the sterically stabilized liposomes encapsulating OPH (SL-OPH) were combined with 2-PAM atropine (Exp. 8): the prophylactic antidotal potency ratio was 1022, and the therapeutic antidotal potency ratio was 155.8. It should be noted that, in the therapeutic experiments, at extremely high doses of paraoxon, the mice were moribund before the antidotes could exert their protective effects. Therefore, these antidotes were less effective therapeutically than prophylactically. When the toxic signs (shivering, shaking) appeared before the administration of the antidotes, all the mice died. This suggests, that there was not sufcient time for the antidotes to exert their protective effects (OPH hydrolysis of paraoxon; 2-PAM reactivation of phosphorylated acetylcholinesterase;

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PETRIKOVICS ET AL. DeLoach, J. R., Harris, R. L., and Ihler, G. M. (1980). An erythrocyte encapsulation dialyzer used in preparing large quantities of erythrocyte ghosts and encapsulation of pesticide in erythrocyte ghosts. Anal. Biochem. 102, 220 227. Dixon, W. J. (1965). The up-and-down method for small animal samples. Amer. Statistic Ass. Journal 12, 967978. Ihler, G. M., Glew, R. H. and Schnure, F. W. (1973). Enzyme loading erythrocytes. Proc. Natl. Acad. Sci. U.S.A. 70, 26632666. Leung, P., David, W. D., Yao, C. C., Cannon, E. P. and Way, J. L. (1991). Rhodanese and sodium thiosulfate encapsulated in mouse carrier erythrocytes. Fundam. Appl. Toxicol. 16, 559 661. Leung, P., Ray, L. E., Sander, C. and Way, J. L. (1986). Encapsulation of thiosulfate cyanide sulfurtransferase by mouse erythrocytes. Toxicol. Appl. Pharmacol. 83, 101110. McDaniel, C. S., Happer, L. L., and Wild, J. R. (1988). Cloning and sequencing of a plasmid-borne gen (opd) encoding a phosphotriesterase. J. Bact. 170, 2306 2311. Oku, N., and Namba, Y. (1994). Long circulating liposomes. Crit. Rev. Drug Carrier Syst. 11, 321270. Omburo, G. A., Kuo, J. M., Mullins, L. S., and Raushel, F. M. (1992). Characterization of the zinc binding site of bacterial phosphotriesterase. J. Biol. Chem. 267, 13278 13283. Papahadjopoulos, D., Allen, T. M., Gabizon, A., Mayhew, K., Matthay, K., Huang, S. K, Lee, K. D., Woodle, M. C., Lasic, D. D., Redemann, C., et al. (1991). Sterically stabilized liposomes: Improvements in pharmacokinetics and antitumor therapeutic efcacy. Proc. Natl. Acad. Sci. U.S.A. 88, 11460 11464. Pei, L., McGuinn, W. D., Petrikovics, I., Pu, L., Cannon, E.P., and Way, J. L. (1993). Determination of phosphotriesterase in blood. Toxicol. Methods 4, 261264. Pei, L., Omburo, G., McGuinn, W. D., Petrikovics, I., Dave, K., Raushel, J. L., Wild, J. R., DeLoach, J. L., and Way, J. L. (1994). Encapsulation of phosphothiesterase within murine erythrocytes. Toxicol. Appl. Pharmacol. 124, 296 301. Pei, L., Petrikovics, I., and Way, J. L. (1995). Antagonism of the lethal effect of paraoxon by carrier erythrocytes containing organophosphorous acid anhydrase. Fundam. Appl. Toxicol. 28, 209 214. Petrikovics, I., Cannon, E. P., McGuinn, W. D., and Way, J. L. (1995). Cyanide antagonism with organic thiosulfonates and carrier red blood cells containing rhodanese. Fundam. Appl. Toxicol. 24, 1 8. Petrikovics, I. Hong, K., Omburo, G., Hu., Q., Pei, L., McQuinn, W. D., Sylvester, D., Tamulinas, C., Papahadjopoulos, D., Jaszberenyi, J. C., et al. (1999). Antagonism of Paraoxon intoxication by recombinant phosphotriesterase encapsulated within sterically stabilized liposomes. Toxicol. Appl. Pharmacol. 156, 56 63. Serdar, C.M., and Gibson, D. T. (1985). Enzymatic hydrolysis of organophosphates: Cloning and expression of parathion hydrolase gene from Pseudomonas diminuta. Biotechnology 3, 567571. Way, J. L., Leung, P., Ray, L., and Saunder, C. (1985). Erythrocyte encapsulated thiosulfate sulfurtransferase. Bibl. Haematol. 51, 75 81. Woodle, M. C., and Lasic, D. D. (1992). Sterically Stabilized Liposomes. Biochim. Biophys. Acta 1113, 171199.

atropine occupation of OP receptor sites) on the mice. As shown in Figure 1, the higher were the paraoxon doses, the sooner the toxic symptoms appeared. This suggests, that these OP antidotal systems may be useful therapeutically at lower doses of the toxicants ( 10 LD 50 paraoxon doses). At higher doses, the treatment should be available right after the intoxication to be able to achieve signicant antidotal protection. The exact mechanism of the difference in the prophylactic and therapeutic antidotal protection is neither clearly understood nor predictable. Pharmacokinetic and pharmacodinamic parameters are probably the determining factors. The 150 times LD 50 therapeutic antidotal protection with the combination of 2-PAM atropine SL-OPH (Table 2) suggests that these antidotal systems may serve as an effective antidote in some military or agricultural situations. The prophylactic protection with this antidotal system was so striking (over 1000 LD 50, Table 2), that at these very high doses of paraoxon, a solubility problem occurred because of the high (100 mg/ml) concentration of the paraoxon solutions necessarily used in the experiments. This means, that prophylactically the 2-PAM atropine SL-OPH system provided over 1000-fold protection against paraoxon poisoning. The therapeutic protective effects of this antidotal system are also very remarkable, since they are far superior to the clinically used antidotal combination of 2-PAM and atropine.
ACKNOWLEDGMENTS
This study was supported by research funds from Texas A&M University, USAMRMC, NATO, and NIEHS.

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