Comparing Therapeutic and Prophylactic Protection against the Lethal Effect of Paraoxon

I. Petrikovics*, D. Papahadjopoulos{dagger}, K. Hong{dagger}, T.-C. Cheng*, S. I. Baskin*,1, J. Jiang§, J. C. Jaszberenyi{ddagger}, 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; {dagger} Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, California 94143; {ddagger} 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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 LD50 [920 mg/kg (LD50 of paraoxon with antagonists)/ 0.95 mg/kg (LD50 of control paraoxon)], and the therapeutic antidotal protection was 156 LD50 [140 mg/kg (LD50 of paraoxon with antagonists)/0.9 mg/kg (LD50 of control paraoxon)]. The current study firmly 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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 hydrolysis of the OP molecules in the body. Injection of purified 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 efficiently provide large amounts of the metabolizing enzymes in the circulation with minimal leakage and few immunologic reactions. Ihler et al.(1973)Go first reported that enzymes and drugs could be successfully encapsulated within carrier red blood cells. DeLoach et al.(1980)Go 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., 1992Go; Leung et al., 1986Go, 1991Go; Petrikovics et al., 1995Go; Way et al., 1985Go) and in OP antagonism (Pei et al., 1994Go, 1995Go). 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, 1994Go; Oku and Namba, 1994Go; Papahadjopoulos et al., 1991Go; Woodle and Lasic, 1992Go). 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., 1999Go). 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 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
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
Paraoxon (ca. 90%) was purchased from Sigma (St Louis, MO), and was further purified by aqueous sodium bicarbonate extraction (Pei et al., 1993Go; Petrikovics et al., 1999Go). 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 purified from an Escherichia coli clone containing the plasmid expression vector pjK33, which was isolated from Flavobacteium sp. (McDaniel et al., 1988Go; Serdar and Gibson, 1985Go). This enzyme was obtained in preparative amounts and purified over 1600-fold by the method of Omburo et al.(1992)Go with minor modifications (Pei et al., 1994Go, 1995Go).

Sterically stabilized liposomes (SL) and SL-OPH preparation.
SL and SL-OPH were prepared as previously described (Petrikovics et al., 1999Go) with minor modifications.

Palmitoyloleoylphosphatidylcholine (POPC) and dipalmitoyl phosphatidylethanolamine-N-[poly(ethylene glycol)2000] (PEG-PE) were purchased from Avanti Polar Lipid (Alabaster, AL). Purified cholesterol was obtained from Calbiochem (San Diego, CA). Lipids were stored in chloroform under argon at -70°C. Sepharose 4B was purchased from Pharmacia Fine Chemicals (Uppsala, Sweden). Chloroform solutions of POPC (60 µmol), cholesterol (40 µmol) and PEG-PE (5.4 µmol) were mixed in a round-bottomed flask, and the solvent was removed slowly on a rotary evaporator at 37°C to obtain dry thin lipids film on the flask. Purified OPH in HEPES [N-(2–hydroxyethyl) piperazine-N'-(2-ethanesulfonic acid) sodium salt] buffer (concentrated on Amicon Concentrator and clarified by centrifugation if necessary) was added to the dry lipids. The lipid film was hydrated slowly under argon by continuously rotating the flask on a rotary evaporator at 37°C for 1 h. Milky liposome suspension was extruded sequentially through 0.2 µ and 0.1µ polycarbonate membrane filter. Extrusion was repeated five times for each membrane to obtain a homogeneous size distribution of liposomes. Unencapsulated OPH was separated from liposomes by gel filtration on sepharose 4B column. Encapsulation efficiency 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 ZnCl2; 3.0 mM MgCl2, (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 final 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 coefficient of p-nitrophenol in phosphate buffer at pH = 7.8 was determined using various dilutions of gravimetric standard solutions of p-nitrophenol. One unit of OPH is defined as that amount of enzyme which hydrolyzed one µmol of paraoxon to p-nitrophenol per min. Protein assays were performed by the Bradford method (Bradford, 1976Go) 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 ± 2°C 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 5–10 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. LD50 values were determined by the up-and-down method (simulated up-and-down study, Dixon, 1965Go). For each experiment, 6 to 10 mice were used. The LD50 values were calculated from the equation of log(LD50) = log(dosefinal) + k log(d), where dosefinal is the final dose administered, k is the tabular value from table (Dixon, 1965Go), 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. LD50 values were determined by the up-and-down method (simulated up-and-down study, Dixon, 1965Go). For each experiment, 6–10 mice were used.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The organophosphorus antidotes, 2-PAM and atropine, were employed to detoxify the lethal effects of paraoxon poisoning, and their prophylactic and therapeutic efficacies were compared with stealth liposomes containing recombinant OPH (SL-OPH). The prophylactic and therapeutic protections with these antidotal systems are summarized and expressed as LD50 values in Table 1Go. 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.


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TABLE 1 Prophylactic and Therapeutic Protective Effects with Sterically Stabilized Liposomes Encapsulating Recombinant OPH (SL-OPH)
 
The overall importance of encapsulated OPH in protecting against paraoxon intoxication was best illustrated when the protection of antagonized paraoxon experiments were compared to the unantagonized experiments and expressed as "antidotal potency ratios" (Table 2Go). Antidotal potency ratios = LD50 of paraoxon with antagonists/LD50 of paraoxon without antagonists. The prophylactic-therapeutic (P-T) ratios are expressed as the ratio of the LD50 of paraoxon in prophylactic experiments to the LD50 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 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 1Go and 2Go, 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 1Go 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 2Go, the tendency of changes of antidotal potency ratios in the prophylactic experiments and in the 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 LD50 value of paraoxon of control animals (Exp. 1) was defined 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).


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TABLE 2 Prophylactic and Therapeutic Antidotal Potency Ratio with Sterically Stabilized Liposomes Encapsulating Recombinant OPH (SL-OPH)
 


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

 
The use of relative potency ratios (RPR = LD50 of paraoxon with SL-OPH/LD50 of paraoxon without SL-OPH) provides an expression of the overall efficacy of the encapsulated OPH in the antagonism of the lethal effects of paraoxon. Table 3Go shows the therapeutic protective effects of liposomes encapsulating OPH (SL-OPH). The SL-OPH alone gave a 72.3 LD50 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 1Go), and the highest relative potency ratio, 72.3 (Table 3Go), whereas the SL-OPH with atropine and 2-PAM (Exp. 8) showed the highest protection (140.2 mg/kg, Table 1Go) with an antidotal potency ratio of 155.8 (Table 2Go), but the lowest relative potency ratio (12.7, Table 3Go) in the therapeutic experiments.


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TABLE 3 Relative Potency Ratio with and Without SL-OPH in Therapeutic Experiments
 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 reflect 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 reflect therapeutic efficacy, 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 first 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., 1999Go). 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 field 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)Go and Broomfield (1992)Go reported the antidotal protective effects of free OP hydrolyzing enzyme in soman poisoning. Recent studies (Pei et al., 1995Go; Petrikovics et al., 1999Go) 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 sufficient time for the antidotes to exert their protective effects (OPH = hydrolysis of paraoxon; 2-PAM = reactivation of phosphorylated acetylcholinesterase; atropine = occupation of OP receptor sites) on the mice. As shown in Figure 1Go, 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 LD50 paraoxon doses). At higher doses, the treatment should be available right after the intoxication to be able to achieve significant 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 LD50 therapeutic antidotal protection with the combination of 2-PAM + atropine + SL-OPH (Table 2Go) 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 LD50, Table 2Go), 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.


    NOTES
 
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{at}apg.amedd.army.mil. Back


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 DISCUSSION
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Cannon, E. P., Zitzer, A. H., McGuinn, W. D., Petrikovics, I., Leung, P., and Way, J. L. (1992). Antagonism of lethal effects of cyanide with rhodanese containing murine carrier erythrocytes. Proc. West. Pharmacol. Soc. 35, 187–190.[Medline]

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.[ISI][Medline]

Dixon, W. J. (1965). The up-and-down method for small animal samples. Amer. Statistic Ass. Journal 12, 967–978.

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