Long Circulating Liposomes Encapsulating Organophosphorus Acid Anhydrolase in Diisopropylfluorophosphate Antagonism

I. Petrikovics*, T.-C. Cheng{dagger}, D. Papahadjopoulos{ddagger}, K. Hong{ddagger}, R. Yin{dagger}, J. J. DeFrank{ddagger}, J. Jaing*, Z. H. Song*, W. D. McGuinn*, D. Sylvester*, L. Pei*, J. Madec*, C. Tamulinas*, J. C. Jaszberenyi§, T. Barcza§ and J. L. Way*,1

* Department of Medical Pharmacology and Toxicology, Texas A&M University, College of Medicine, College Station, Texas 77843-1114; {dagger} U.S. Army Chemical and Biological Defense Agency, Aberdeen Proving Ground, Maryland 21010-5423; {ddagger} Liposomal Research Laboratory, California Medical Center Research Institute, San Francisco, California 94115; and § Department of Organic Chemical Technology, Research Group of the Hungarian Academy of Sciences, Technical University of Budapest, Budapest, Hungary H-1521

Received January 14, 2000; accepted June 9, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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)*.

Key Words: phosphotriesterase; OPA anhydrase; OPAA; DFP antagonism; sterically stabilized liposomes; OP hydrolase (OPH); organophosphorus antagonism; stealth liposomes; long circulating liposomes.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This investigational effort was focused on antagonizing organophosphorous intoxication by employing a new conceptual approach, based on encapsulation of recombinant enzymes within biodegradable enzyme carrier systems.

Under the recent IUB classification of esterases that hydrolyze organophosphates, these enzymes (EC 3.1) would be classified as OPA anhydrolase or OPA anhydrase or OPA hydrolase (Webb, 1992Go).

Two names, A-esterase and B-esterase, have been widely used in the literature to differentiate between the enzymes that hydrolyze organophosphorus compounds (A-esterases) and the enzymes that are progressively inhibited by organophosphorus compounds (B-esterases; e.g., cholinesterases, carboxylesterases, trypsin, chymotrypsin) (Aldridge, 1989). Both names encompass groups of enzymes and not individual enzymes. Overlaps in the use of a given term are likely to cause ambiguity, particularly for the name A-esterase.

OP compounds are acetylcholinesterase (AChE) inhibitors and exert their toxicity by causing an excessive accumulation of the neurotransmitter, acetylcholine, and subsequent disruption of cholinergic nervous transmission. One of the fundamental mechanisms for the detoxication of organophosphorus compounds would involve their degradation by OP-hydrolyzing enzymes. Once the OP compounds are hydrolyzed they are unable to phosphorylate the AChE (Cohen and Warringa, 1957Go). The use of exogenous enzymes in OP antagonism was reported by Cohen and Warringa (1957) and Ashani et al. (1991). However, the injection of purified free enzyme preparations directly into the blood stream has serious limitations because of possible immunologic reactions and various adverse physiological disposition factors. Biodegradable liposome carriers, which are permeable to the toxin molecules, can provide sufficiently large amounts of the highly purified metabolizing enzymes to remain protected in the circulation for a long period of time. Resealed, annealed erythrocytes (CRBC) were first used as drug/enzyme carriers by Ihler (1973) and Deloach (1980). Drugs and enzymes were encapsulated into resealed, annealed red blood cells by hypotonic dialysis. Carrier erythrocytes encapsulating rhodanese were successfully employed in cyanide antagonism (Cannon et al., 1992Go; Leung et al., 1986Go, 1991Go; Petrikovics et al., 1994Go, 1995Go; Way et al., 1985Go) and in organophosphorus (OP) antagonism (Pei, et al., 1994Go, 1995Go). McGuinn et al. (1993) reported the encapsulation of squid-type diisopropylphosphorofluoridate-hydrolyzing enzyme (DFPase) into carrier red blood cells. Sterically stabilized liposomes have been widely used as targeted drug delivery systems in clinical therapy (Lasic and Papahadjopoulus, 1998). In the stealth liposome system, a drug is encapsulated in biocompatible carrier vesicles that can circumvent the body's immune defenses, thereby circumventing rapid uptake by the macrophage cells of the reticuloendothelial system (Allen, 1994Go; Papahadjopoulos et al., 1991Go; Szoka and Papahadjopoulos, 1980Go; Woodle and Lasic, 1992Go).

Agricultural OP insecticides caused over half a million poisoning cases each year in the United States (Savage et al., 1988Go). Paraoxon serves as a model OP compound for studying the toxicity and antagonism of parathion with recombinant OP hydrolase (OPH). The chemical structures of DFP and the chemical warfare agents, soman and sarin, are different from that of paraoxon (Fig. 1Go). OP hydrolase (OPH) is a good hydrolyzing enzyme for paraoxon and other OP agricultural insecticides. OPA anhydrolase (OPAA) has good substrate specificity towards DFP, sarin, and soman. For military purposes, DFP has a better substrate specificity to study with OPAA, since its chemical structure is similar to the chemical warfare agents, sarin and soman. These latter agents are rapidly hydrolyzed by OPAA. Sequence and biochemical analysis of OPAA have established that this enzyme is a prolidase (EC 3.4.13.9), a type of dipeptidase that hydrolyzes dipeptides with a prolyl residue at the carboxyl terminus. The nucleotide sequence of a gene encoding OP, a degrading enzyme from Alteromonas haloplanktis, was first reported by Cheng et al. (1997).



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FIG. 1. Chemical structures of organophosphorus agents.

 
Two other OP antagonists, 2-PAM and atropine, exert their pharmacological protective effects by different mechanisms: pralidoxime (2-PAM) reactivates the OP-inhibited (phosphorylated) AChE (Wilson et al., 1955), whereas atropine antagonizes the muscarinic effects of the acetylcholine by competing with the acetylcholine for the muscarinic receptor. The combination of 2-PAM and atropine with an OP-hydrolyzing enzyme, in an appropriate protective carrier system, should provide a more effective antidotal protection against the lethal effects of OP agents, as neither 2-PAM nor atropine degrades the OP agent, but the OP hydrolyzing enzyme does. The liposome carrier acts as a protective environment for the hydrolyzing enzymes, destroying the toxic effects of OP agents.

These studies represent the first conceptual approach to the use of enzymes encapsulated within liposomes, to protect against and treat chemical poisoning. Earlier studies (Pei et al., 1994Go, 1995Go; Petrikovics et al., 1999Go) reported the use of carrier red blood cells containing recombinant OPH to antagonize, quite strikingly, the lethal effects of paraoxon. This study is focused on the application of a different recombinant OP hydrolyzing enzyme, OPAA, whose substrate specificity is amenable to hydrolyzing DFP, sarin, and soman.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
DFP was purchased from Sigma (St. Louis, MO). DFP solution was prepared by diluting in a cold (0–2°C) solution of 0.9% sodium chloride and was used no more than 2 h after preparation. Atropine sulfate and 2-PAM solutions were prepared fresh daily. All other chemicals used were of the highest purity commercially available.

Enzyme.
Purification of OPAA (EC 3.1.8.2) from the Alteromonas strain, JD6, has been previously reported by De Frank, et al. (1991) and Cheng, et al. (1993).

Sterically stabilized liposomes without OPAA (SL) and with OPAA-containing sterically stabilized liposomes (SL)*.
The above were prepared as described by Allen (1994). 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. 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 a dry, thin lipid film on the flask. Purified OPAA in HEPES [N-[2–Hydroxyethyl] piperazine-N1-[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 a rotary evaporator at 37°C for one h. The milky liposome suspension was extruded sequentially through 0.2-µ and 0.1-µ polycarbonate membrane filters. Extrusion was repeated 5 times for each membrane, to obtain a homogeneous size distribution of liposomes. Free OPAA was separated from liposomes by centrifugation (100,000 x g for 80 min). Centrifugation was repeated twice, the 2 preparations were combined, and the liposome pellet was resuspended in 20 mM HEPES saline. The lipid composition was 90 µmol POPC/60 µmol cholesterol/9 µmol PEG-PE to make the final phospholipid concentration 60–75 mM. The percentage of encapsulation efficiency was calculated from the amount of encapsulated OPAA divided by the amount of OPAA added, multiplied by 100.

OPAA activity determination in sterically stabilized carrier liposomes.
OPAA activity in liposomes was measured by monitoring the production of fluoride from DFP with a fluoride ion-sensitive electrode (Orion Research Inc., Boston, MA) (Hoskin and Roush, 1982Go). The assay solution for the enzyme fractions contained NaCl (70 mM), KCl (280 mM), Tris (70 mM, pH 7.2), and the optimal concentration of DFP was 3.44 mM. The solution for determining enzyme activity of (SL)* was isotonic: It contained phosphate buffer (10 mM), NaCl (0.144 mM), MgCl2*6H2O (2.0 mM) dextrose (5 mM), and had the osmolarity of 290 mosM. The DFP solution was kept on ice before using, and the enzyme activity was determined at 25 ± 1°C in a titration flask (Brinkman Instrument, Inc.). The total volume of the solution was 5.00 ml. Electrode potential was recorded as a function of time on a strip chart recorder (Orion Research Inc., Boston, MA), and the potential values were converted to concentration using the Nernst equation. Results are expressed as the mean ± 1 standard deviation unless otherwise indicated. Protein assays were done by the Bradford method (Bradford, 1976Go) using BioRad (Richmond, CA) protein assay reagents. One unit of OPAA is defined as the amount of enzyme that hydrolyzes 1 µmol of DFP to fluoride and isopropyl phosphate per min.

Animals.
Male Balb/C mice (Charles River Breeding Laboratories, Inc., Wilmington, MA), weighing between 18 and 20 g, were housed in room temperature and light controlled rooms (21 ± 2°C, 12-h light/dark cycle) and animals were furnished with water and 4% fat-Rodent Chow (Teklad HSD, Inc., WI) ad libitum. All animal procedures were conducted in accordance with the guidelines in The Guide for the Care and Use of Laboratory Animals (National Academic Press, 1996), accredited by AAALAC (American Association for the Assessment and Accreditation of Laboratory Animal Care, International). At the termination of the experiments, surviving animals were euthanized with Aerrane (Isoflurane) from Fort Dodge Animal Heath, Iowa 50501, in accordance with the 1986 report of the AVMA Panel of Euthanasia.

In vivo experiments.
Male mice received 20–30 units of OPAA (free enzyme or encapsulated within SL*) in a maximum volume of 200 µl, intravenously (dorsal tail vein injection), 1 h prior to receiving DFP subcutaneously. No gross toxic or immunological effects were noted in mice receiving 2-PAM and/or liposomes with or without OPAA. Atropine elicits anticholinergic effects at the dose employed, and DFP elicits cholinergic pharmacologic effects. Atropine (10 mg/kg) and 2-PAM (90 mg/kg) were not encapsulated and were administered intraperitoneally to mice 45 and 15 min, respectively, prior to receiving OPAA intravenously. The LD50 value was obtained from 5 or more grade doses of DFP administered to 5 or more groups of 6 to 8 mice, based on 24-h mortality. The blood stability of OPAA was followed for 2 days, but surviving animals were observed for a week following the treatments to determine if there were signs of late-developing toxicity. Efficacies of the antagonists are expressed as the potency ratios. The LD50 values were determined by the method of Litchfield and Wilcoxon (1949), as determined by the computer program PHARM/PC Version 4.1 (Tallarida and Murray, 1987Go). All statistical procedures were performed at the 95% confidence level.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study is focused on DFP antagonism with recombinant OPAA enzyme. Purity of OPAA was 95% and purification of OPAA (EC 3.1.8.2) from the Alteromonas strain JD6 has been previously reported (Cheng, et al. 1993Go; De Frank et al., 1991). OPAA, encapsulated within carrier cells, rapidly hydrolyzes DFP to fluoride and diisopropylphosphate. Enzymatic hydrolysis of DFP by various amounts of liposomes encapsulated with OPAA (SL)* is shown in Figure 2Go. The reaction was linear at the concentration employed in these studies. Formation of fluoride ions was directly proportional to the amount of (SL)* containing OPAA. There was no OPAA activity noted with liposomes containing no enzyme. Under these conditions, increases in the amount of (SL)* are equivalent to increases in the amount of OPAA in the reaction. Maximal encapsulation efficiency with carrier RBCs prepared by hypotonic dialysis (Leung et al., 1986Go; Pei et al., 1994Go; Petrikovics et al., 1994Go) was about 30%. The sterically stabilized liposomes gave a maximum OPAA encapsulation efficiency of 80–90%, and the OPAA activity was stable over 2 days when incubated in blood at 37°C.



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FIG. 2. OPAA hydrolysis as a function of (SL)* volume. Liposomes with no OPAA encapsulated had no OPAA activity. OPAA activity was determined by measuring the production of fluoride anion from DFP by a fluoride-sensitive electrode. One unit of OPAA is defined as that amount of enzyme that hydrolyzed 1 µmol of DFP per min.

 
The effects of OPAA (free and encapsulated within liposomes) are shown, and are compared with and without atropine and 2-PAM, in Table 1Go. Results of atropine (Experiment 2) and 2-PAM (Experiment 3) showed minimal but significant protection against OP intoxication, and the antidotal combination of 2-PAM and atropine was potentiated when employed in combination, as previously reported.


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TABLE 1 DFP Antagonism on Swiss Webster Mice with OPAA-2
 
It is of interest to note that the free OPAA enzyme provided very little if any further protection. For example, although free OPAA does protect against OP lethality (Experiment 5), the addition of atropine did not significantly increase the protective effect, as no significant increase in protection was noted when free OPAA was combined with atropine (Experiment 6). Furthermore, the increase in protection of free OPAA with 2-PAM (Experiment 7) was also minimal, but it was significant with 2-PAM plus atropine (Experiment 8).

The encapsulations of OPAA within liposomes clearly demonstrated the enhanced protective effects. The encapsulated OPAA (Experiment 9) was more effective than atropine or 2-PAM alone, and it tripled the protective effect with atropine (Experiment 10) or 2-PAM (Experiment 11) and the 2-PAM + atropine combination (Experiment 12).

Statistical calculation of the slope of the regression line in all experiments indicated that the slopes of these log dose-response curves are not significantly different from each other. Therefore, the statistical procedure employed to obtain the potency ratios was valid.

The overall importance of liposomal encapsulation of the OPAA enzyme was best illustrated when the protective effect of antagonized OP experiments were compared to unantagonized OP and expressed as the potency ratio (Fig. 3Go). It should be emphasized that Figure 3Go permitted the comparison of each antagonized OP experiment with unantagonized OP only. Also, Figure 3Go shows the comparison of the enhanced protection by sterically stabilized liposomal studies when compared with the non-liposomal studies. In order to obtain statistically valid cross comparison of relative potency between all experiments, every possible combination of these experimental groups was calculated and expressed as a potency ratio. Under these circumstances, OPAA-containing liposomes were effective in protecting against OP poisoning (potency ratio = 2.3) and more effective than atropine (potency ratio = 1.3) or 2-PAM (potency ratio 1.8). However, when the (SL)* was given in combination with atropine (potency ratio = 4.5) or with 2-PAM (potency ratio = 5.0) the protective effect was enhanced. The most effective protection was afforded when (SL)* was given with the 2-PAM and atropine combination; the potency ratio was 23.2.



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FIG. 3. Potency ratios for DFP with and without antagonists (potency ratio = LD50 of DFP with antagonist/LD50 of DFP without antagonists). The LD50 values were determined by the method of Litchfield and Wilcoxon (1949), and determined by the program PHARM/PC Version 4.1 (Tallarida and Murray, 1987Go). All statistical procedures were performed at the 95% confidence level.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
These results suggest a potentially new conceptual approach for the rational design of therapy against chemical poisoning. Presently, there are less than 6 specific antidotes that exceed the LD50 values by 5 times when protecting against the lethality of chemical poisoning. There is a need for a more successful approach for treating chemical poisoning. Recent developments in liposomal technology allow preparation of a liposomal delivery system with minimal macrophage recognition, thereby extending the half-life of conventional liposomes from a few hours to a few days by using sterically stabilized, long circulating liposomes. This technology, coupled with the rapid preparation of fast catalytic recombinant enzymes, permits the development of effective prophylactic and therapeutic agents. These results represent an application of sterically stabilized liposomes as a biodegradable enzyme carrier system for the OP hydrolyzing enzyme, OPAA, to degrade OP agents and to antagonize the lethal effects of various OP agents including DFP. Since the Vmax and Km data of paraoxon and OPH could explain its superior protection compared to DFP and OPAA (Table 3), it may be possible that extrapolations can be taken from chemical kinetic data with other OP agents such as soman and sarin.

These experimental procedures presently are strictly prophylactic studies. This is of importance in chemical defense prophylactic protection. In separate therapeutic studies (unpublished), when 2-PAM and/or atropine was administered after OP poisoning had occurred, the recombinant enzyme gave protection superior to that in the prophylactic experiments. In liposome and carrier RBC studies, no, or only minor, immune reaction was elicited. The reason for this is not clear, but in inborn errors of metabolism lifetime studies in humans, using carrier erythrocytes containing the deficient enzyme also caused no immunological problems (Beutler et al., 1997Go).

Earlier studies (Petrikovics et al., 1999Go) indicated that sterically stabilized liposomes encapsulating other OP-degrading enzymes (OPH) provide considerable protection, either alone or in a combination with 2-PAM and/or atropine. There are major differences in efficacy with different recombinant enzymes and substrates. In paraoxon antagonism studies, OPH-SL alone was superior to the atropine-2-PAM combination alone, and the OPH-SL with atropine-2-PAM combination increased the protection by over 1000 LD50 doses. The OPAA-SL combined with 2-PAM and atropine also enhanced the protection against DFP. Should extrapolation from these enzyme kinetic data of Km and Vmax be considered, protection against sarin should be superior to DFP, and against soman, should approximate that of DFP. Enzyme kinetic data indicate that paraoxon has a high affinity for OP hydrolase (OPH), but this enzyme is less active for DFP, sarin, and soman (Table 2Go). On extrapolations from these enzyme kinetic data, OPAA seems to be a more appropriate enzyme for studies on DFP, sarin, and soman (Fig. 1Go and Table 2Go).


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TABLE 2 Kinetic and Toxicologic Data of OP Compounds
 
We earlier reported the dramatic protective effect from this new approach with carrier RBCs (Pei et al., 1995Go) and sterically stabilized liposomes (Petrikovics et al, 1999Go) containing recombinant OPH. OPAA has moderate in vivo protection in DFP antagonism, and by projection of these chemical kinetic data compared to DFP, it may be found that OPAA mayprovide a similar in vivo protection to soman, and better protection against sarin. These studies indicate that improved delivery systems with longer t1/2 and less macrophage recognition provide a basis to enhance the application of fast catalytic enzymes. These studies reaffirm that highly catalytic enzymes with appropriate delivery systems may serve as new general conceptual approaches for protection from and treatment of various chemical intoxications.


    ACKNOWLEDGMENTS
 
This study was supported by research funds from Texas A&M University, USAMRMC, NATO, and NIEHS. The authors are grateful to Ms. Rebecca Magee, Ms. Lorena Taylor, and Mrs. Stephanie Zimmerman for their assistance in the preparation of this manuscript.


    NOTES
 
1 To whom correspondence should be addressed. Fax: (409) 845-0699. E-mail: jlway{at}medicine.tamu.edu. Back


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