* 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
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ABSTRACT |
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Key Words: organophosphorus hydrolase (OPH); OPA anhydrase; paraoxonase; paraoxon antagonism; sterically stabilized liposomes; stealth liposomes; long circulating liposomes; organophorus antagonism; prophylactic; therapeutic.
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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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., 1988; Serdar and Gibson, 1985
). This enzyme was obtained in preparative amounts and purified over 1600-fold by the method of Omburo et al.(1992)
with minor modifications (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 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-(2hydroxyethyl) 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, 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 1820 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 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. LD50 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 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, 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. LD50 values were determined by the up-and-down method (simulated up-and-down study, Dixon, 1965). For each experiment, 610 mice were used.
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RESULTS |
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DISCUSSION |
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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., 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 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) and Broomfield (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 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 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 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 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 LD50, 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.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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Ashani, Y., Rothshild, N., Segall, Y., Levanon, D., and Raveh, L. (1991). Prophylaxis against organophosphate poisoning by an enzyme hydrolysing organophosphorus compound in mice. Life Sci. 49, 367374.[CrossRef][ISI][Medline]
Bradford, M. (1976). A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248254.[CrossRef][ISI][Medline]
Broomfield, C. A. (1992). A purified recombinant organophosphorus acid anhydrase protects mice against soman. Physiol. Toxicol. 70, 6566.
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, 187190.[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, 220227.[ISI][Medline]
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.[Abstract]
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, 559661.[ISI][Medline]
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.[ISI][Medline]
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, 23062311.[ISI][Medline]
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, 1327813283.
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 efficacy. Proc. Natl. Acad. Sci. U.S.A. 88, 1146011464.[Abstract]
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, 296301.[CrossRef][ISI][Medline]
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, 209214.[CrossRef][ISI][Medline]
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, 18.[CrossRef][ISI][Medline]
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, 5663.[CrossRef][ISI][Medline]
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.[ISI]
Way, J. L., Leung, P., Ray, L., and Saunder, C. (1985). Erythrocyte encapsulated thiosulfate sulfurtransferase. Bibl. Haematol. 51, 7581.[Medline]
Woodle, M. C., and Lasic, D. D. (1992). Sterically Stabilized Liposomes. Biochim. Biophys. Acta 1113, 171199.
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