Gene therapy delivering a paraoxonase 1 variant offers long-term prophylactic protection against nerve agents in mice.

Chemical warfare nerve agents are organophosphorus chemical compounds that induce cholinergic crisis, leaving little or no time for medical intervention to prevent death. The current chemical treatment regimen may prevent death but does not prevent postexposure complications such as brain damage and permanent behavioral abnormalities. In the present study, we have demonstrated an adeno-associated virus 8 (AAV8)-mediated paraoxonase 1 variant IF-11 (PON1-IF11) gene therapy that offers asymptomatic prophylactic protection to mice against multiple lethal doses of G-type chemical warfare nerve agents, namely, tabun, sarin, cyclosarin, and soman, for up to 5 months in mice. A single injection of liver-specific adeno-associated viral particles loaded with PON1-IF11 gene resulted in expression and secretion of recombinant PON1-IF11 in milligram quantities, which has the catalytic power to break down G-type chemical warfare nerve agents into biologically inactive products in vitro and in vivo in rodents. Mice containing milligram concentrations of recombinant PON1-IF11 in their blood displayed no clinical signs of toxicity, as judged by their hematological parameters and serum chemistry profiles. Our study unfolds avenues to develop a one-time application of gene therapy to express a near-natural and circulating therapeutic PON1-IF11 protein that can potentially protect humans against G-type chemical warfare nerve agents for several weeks to months.

Gene Therapy Leaves a Vicious Cycle.

The human genetic code encrypted in thousands of genes holds the secret for synthesis of proteins that drive all biological processes necessary for normal life and death. Though the genetic ciphering remains unchanged through generations, some genes get disrupted, deleted and or mutated, manifesting diseases, and or disorders. Current treatment options-chemotherapy, protein therapy, radiotherapy, and surgery available for no more than 500 diseases-neither cure nor prevent genetic errors but often cause many side effects. However, gene therapy, colloquially called "living drug," provides a one-time treatment option by rewriting or fixing errors in the natural genetic ciphering. Since gene therapy is predominantly a viral vector-based medicine, it has met with a fair bit of skepticism from both the science fraternity and patients. Now, thanks to advancements in gene editing and recombinant viral vector development, the interest of clinicians and pharmaceutical industries has been rekindled. With the advent of more than 12 different gene therapy drugs for curing cancer, blindness, immune, and neuronal disorders, this emerging experimental medicine has yet again come in the limelight. The present review article delves into the popular viral vectors used in gene therapy, advances, challenges, and perspectives.

Assessing the stoichiometric efficacy of mammalian expressed paraoxonase-1 variant I-F11 to afford protection against G-type nerve agents.

We evaluated the ability of evolved paraoxonase-1 (PON1) to afford broad spectrum protection against G-type nerve agents when produced in mammalian cells via an adenovirus expression system. The PON1 variants G3C9, VII-D11, I-F11, VII-D2 and II-G1 were screened in vitro for their ability to hydrolyze G-agents, as well as for their preference towards hydrolysis of the more toxic P(-) isomer. I-F11, with catalytic efficiencies of (1.1 ± 0.1) × 106 M-1 min-1, (2.5 ± 0.1) × 106 M-1 min-1, (2.3 ± 0.5) × 107 M-1 min-1and (9.2 ± 0.1) × 106 M-1 min-1 against tabun (GA), sarin (GB), soman (GD) and cyclosarin (GF), respectively, was found to be a leading candidate for further evaluation. To demonstrate the broad spectrum efficacy of I-F11 against G-agents, a sequential 5 × LD50 dose of GD, GF, GB and GA was administered to ten mice expressing I-F11 on days 3, 4, 5 and 6 following virus injection, respectively. At the conclusion of the experiment, 80% of the animals survived exposure to all four G-agents. Using the concept of stoichiometric efficacy, we determined that I-F11 affords protection from lethality against an administered dose of 10, 15, 90 and 80 molar equivalents of GA, GB, GD and GF, respectively, relative to the molar equivalents of I-F11 in circulation. It also appears that I-F11 can associate with high density lipoprotein in circulation, suggesting that I-F11 retained this function of native PON1. This combination of attractive attributes demonstrates that I-F11 is an attractive candidate for development as a broad-therapeutic against G-type nerve agent exposure.

An in vitro and in vivo evaluation of the efficacy of recombinant human liver prolidase as a catalytic bioscavenger of chemical warfare nerve agents.

In this study, we determined the ability of recombinant human liver prolidase to hydrolyze nerve agents in vitro and its ability to afford protection in vivo in mice. Using adenovirus containing the human liver prolidase gene, the enzyme was over expressed by 200- to 300-fold in mouse liver and purified to homogeneity by affinity and gel filtration chromatography. The purified enzyme hydrolyzed sarin, cyclosarin and soman with varying rates of hydrolysis. The most efficient hydrolysis was with sarin, followed by soman and by cyclosarin {apparent kcat/Km [(1.9 ± 0.3), (1.7 ± 0.2), and (0.45 ± 0.04)] × 10(5 )M(-1 )min(-1), respectively}; VX and tabun were not hydrolyzed by the recombinant enzyme. The enzyme hydrolyzed P (+) isomers faster than the P (-) isomers. The ability of recombinant human liver prolidase to afford 24 hour survival against a cumulative dose of 2 × LD50 of each nerve agent was investigated in mice. Compared to mice injected with a control virus, mice injected with the prolidase expressing virus contained (29 ± 7)-fold higher levels of the enzyme in their blood on day 5. Challenging these mice with two consecutive 1 × LD50 doses of sarin, cyclosarin, and soman resulted in the death of all animals within 5 to 8 min from nerve agent toxicity. In contrast, mice injected with the adenovirus expressing mouse butyrylcholinesterase, an enzyme which is known to afford protection in vivo, survived multiple 1 × LD50 challenges of these nerve agents and displayed no signs of toxicity. These results suggest that, while prolidase can hydrolyze certain G-type nerve agents in vitro, the enzyme does not offer 24 hour protection against a cumulative dose of 2 × LD50 of G-agents in mice in vivo.

Investigation of evolved paraoxonase-1 variants for prevention of organophosphorous pesticide compound intoxication.

We investigated the ability of the engineered paraoxonase-1 variants G3C9, VII-D11, I-F11, and VII-D2 to afford protection against paraoxon intoxication. Paraoxon is the toxic metabolite of parathion, a common pesticide still in use in many developing countries. An in vitro investigation showed that VII-D11 is the most efficient variant at hydrolyzing paraoxon with a kcat/Km of 2.1 × 10(6) M(-1) min(-1) and 1.6 × 10(6) M(-1) min(-1) for the enzyme expressed via adenovirus infection of 293A cells and mice, respectively. Compared with the G3C9 parent scaffold, VII-D11 is 15- to 20-fold more efficacious at hydrolyzing paraoxon. Coinciding with these results, mice expressing VII-D11 in their blood survived and showed no symptoms against a cumulative 6.3 × LD50 dose of paraoxon, whereas mice expressing G3C9 experienced tremors and only 50% survival. We then determined whether VII-D11 can offer protection against paraoxon when present at substoichiometric concentrations. Mice containing varying concentrations of VII-D11 in their blood (0.2-4.1 mg/ml) were challenged with doses of paraoxon at fixed stoichiometric ratios that constitute up to a 10-fold molar excess of paraoxon to enzyme (1.4-27 × LD50 doses) and were assessed for tremors and mortality. Mice were afforded complete asymptomatic protection below a paraoxon-to-enzyme ratio of 8:1, whereas higher ratios produced tremors and/or mortality. VII-D11 in mouse blood coeluted with high-density lipoprotein, suggesting an association between the two entities. Collectively, these results demonstrate that VII-D11 is a promising candidate for development as a prophylactic catalytic bioscavenger against organophosphorous pesticide toxicity.

Persistent and high-level expression of human liver prolidase in vivo in mice using adenovirus.

Human liver prolidase, a metal-dependent dipeptidase, is being tested as a potential catalytic bioscavenger against organophosphorus (OP) chemical warfare nerve agents. The purpose of this study was to determine whether persistent and high-levels of biologically active and intact recombinant human (rHu) prolidase could be introduced in vivo in mice using adenovirus (Ad). Here, we report that a single intravenous injection of Ad containing the prolidase gene with a 6× histidine-tag (Ad-prolidase) introduced high-levels of rHu prolidase in the circulation of mice which peaked on days 5-7 at 159 ± 129 U/mL. This level of prolidase is ~120 times greater than that of the enzyme level in mice injected with Ad-null virus. To determine if all of Ad-prolidase-produced rHu prolidase was exported into the circulation, enzyme activity was measured in a variety of tissues. Liver contained the highest levels of rHu prolidase on day 7 (5647 ± 454 U/g) compared to blood or any other tissue. Recombinant Hu prolidase hydrolyzed DFP, a simulant of OP nerve agents, in vitro. In vivo, prolidase overexpression extended the survival of 4 out of 6 mice by 4-8h against exposure to two 1× LD(50) doses of DFP. In contrast, overexpression of mouse butyrylcholinesterase (BChE), a proven stoichiometric bioscavenger of OP compounds, protected 5 out of 6 mice from DFP lethality and surviving mice showed no symptoms of DFP toxicity. In conclusion, the results suggest that gene delivery using Ad is capable of introducing persistent and high levels of human liver prolidase in vivo. The gene-delivered prolidase hydrolyzed DFP in vitro but provided only modest protection in vivo in mice, delaying the death of the animals by only 4-8h.

Assessing protection against OP pesticides and nerve agents provided by wild-type HuPON1 purified from Trichoplusia ni larvae or induced via adenoviral infection.

Human paraoxonase-1 (HuPON1) has been proposed as a catalytic bioscavenger of organophosphorus (OP) pesticides and nerve agents. We assessed the potential of this enzyme to protect against OP poisoning using two different paradigms. First, recombinant HuPON1 purified from cabbage loopers (iPON1; Trichoplusia ni) was administered to guinea pigs, followed by exposure to at least 2 times the median lethal dose (LD(50)) of the OP nerve agents tabun (GA), sarin (GB), soman (GD), and cyclosarin (GF), or chlorpyrifos oxon, the toxic metabolite of the OP pesticide chlorpyrifos. In the second model, mice were infected with an adenovirus that induced expression of HuPON1 and then exposed to sequential doses of GD, VX, or (as reported previously) diazoxon, the toxic metabolite of the OP pesticide diazinon. In both animal models, the exogenously added HuPON1 protected animals against otherwise lethal doses of the OP pesticides but not against the nerve agents. Together, the results support prior modeling and in vitro activity data which suggest that wild-type HuPON1 does not have sufficient catalytic activity to provide in vivo protection against nerve agents.

Senescence marker protein 30 (SMP30) expression in eukaryotic cells: existence of multiple species and membrane localization.

Senescence marker protein (SMP30), also known as regucalcin, is a 34 kDa cytosolic marker protein of aging which plays an important role in intracellular Ca(2+) homeostasis, ascorbic acid biosynthesis, oxidative stress, and detoxification of chemical warfare nerve agents. In our goal to investigate the activity of SMP30 for the detoxification of nerve agents, we have produced a recombinant adenovirus expressing human SMP30 as a fusion protein with a hemaglutinin tag (Ad-SMP30-HA). Ad-SMP30-HA transduced the expression of SMP30-HA and two additional forms of SMP30 with molecular sizes ∼28 kDa and 24 kDa in HEK-293A and C3A liver cells in a dose and time-dependent manner. Intravenous administration of Ad-SMP30-HA in mice results in the expression of all the three forms of SMP30 in the liver and diaphragm. LC-MS/MS results confirmed that the lower molecular weight 28 kDa and 24 kDa proteins are related to the 34 kDa SMP30. The 28 kDa and 24 kDa SMP30 forms were also detected in normal rat liver and mice injected with Ad-SMP30-HA suggesting that SMP30 does exist in multiple forms under physiological conditions. Time course experiments in both cell lines suggest that the 28 kDa and 24 kDa SMP30 forms are likely generated from the 34 kDa SMP30. Interestingly, the 28 kDa and 24 kDa SMP30 forms appeared initially in the cytosol and shifted to the particulate fraction. Studies using small molecule inhibitors of proteolytic pathways revealed the potential involvement of ß and γ-secretases but not calpains, lysosomal proteases, proteasome and caspases. This is the first report describing the existence of multiple forms of SMP30, their preferential distribution to membranes and their generation through proteolysis possibly mediated by secretase enzymes.

Gene-delivered butyrylcholinesterase is prophylactic against the toxicity of chemical warfare nerve agents and organophosphorus compounds.

Gene delivery using an adenoviral system has been effective in introducing therapeutic proteins in vitro and in vivo. This study tested the feasibility of using adenovirus to deliver clinically relevant amounts of butyrylcholinesterase (BChE), a proven bioscavenger of nerve agents. The adenovirus construct expressed full-length mouse BChE. Mice were injected with a single dose of adenovirus (1.5 × 10(10) infectious units) in the tail vein; plasma was collected through day 11 and assayed for BChE activity. Maximum activity, representing a 300- to 3400-fold increase over baseline, was found on day 4. Expression levels returned to baseline by day 10. Nondenaturing gel electrophoresis showed the recombinant BChE was a dimer that could be converted to tetramers by addition of polyproline. The toxic compounds chosen for protection studies were positively charged organophosphorus agents, echothiophate, and O-ethyl-S-2-N,N-diisopropylaminoethyl methylphosphonothiolate (VX). Mice containing elevated blood levels of BChE (300- to 3,000-fold over the control mice) were challenged with incremental doses of echothiophate or VX. Mice showed no signs of toxicity and were protected from up to 30× LD(50) dose of echothiophate and 5× LD(50) dose of VX. A good correlation was observed between tolerated echothiophate dose and plasma BChE levels at time of challenge. The absolute increases in levels of circulating BChE and the sustained nature of the response resulted in a very high enzyme concentration, deemed critical in acute toxicity (5× LD(50) or more) scenarios. These results suggest that gene-delivered BChE is a prophylactic and affords protection equivalent to that of a multimilligram injection of the same.

Demonstration of in vivo stability and lack of immunogenicity of a polyethyleneglycol-conjugated recombinant CHO-derived butyrylcholinesterase bioscavenger using a homologous macaque model.

Human serum and recombinant butyrylcholinesterase (rHuBChE) are the most advanced prophylactics against organophosphate (OP) toxicity due to nerve agent or insecticide exposure. For ethical reasons, such potential multi-use treatments cannot be tested in humans and will require extensive testing in animal models and the "Animal Rule" 21 (21 CFR 601.90) for regulatory approval. This will involve multiple injections of rHuBChE into heterologous animals, e.g. macaques, rodents with inevitable immunogenicity and subsequent elimination of the enzyme on repeat injections. In order to accurately assess pharmacokinetics, efficacy and safety of a candidate rBChE in an "antibody free" system, a homologous macaque (Ma) model has been developed. In these studies, macaques received single or multiple intravenous injections of native MaBChE as well as unmodified or PEG-conjugated forms of rMaBChE produced in CHO cells. Compared to the poor plasma retention of unmodified rBChE (MRT: <10h), three injections of 1.5-2.3mg/kg of PEG-conjugated tetrameric rBChE resulted in high circulatory stability (MRT: >134h) and lack of immunogenicity similar to native MaBChE. PEG-conjugation of the monomeric rMaBChE form also exhibited pharmacokinetic profiles comparable to the tetrameric form (MRT: >113h). However, despite the increased bioavailability of PEG-rBChE, antigenicity studies using sandwich ELISA showed that while macaque BChE was not immunogenic in macaques, PEGylation of rMaBChE did not prevent binding to anti-BChE antibodies, suggesting PEGylation may not be sufficient to mask non-human epitopes on rBChE. This homologous model can provide necessary preclinical protection data for the use of PEG-rHuBChE in humans and bodes well for a safe and efficacious CHO-derived rHuBChE therapeutic.

A strategy for the production of soluble human senescence marker protein-30 in Escherichia coli.

Senescence marker protein-30 (SMP30) has been reported to hydrolyze diisopropyl fluorophosphate (DFP), a surrogate compound of chemical warfare nerve agents. Thus, SMP30 has the potential to be useful as a prophylactic against chemical warfare nerve agent toxicity. Our efforts to generate human SMP30 in bacteria using a variety of expression vectors invariably resulted in insoluble and inactive preparations. In this study, properly folded and active recombinant human SMP30 (rHuSMP30) was produced in Escherichia coli by coexpressing it with molecular chaperones in a combined strategy. The coexpression of rHuSMP30 with GroES/GroEL/Tf at 15 degrees C, combined with the addition of a membrane fluidizer, increased osmolytes, and a two-step expression resulted in the highest enhancement of solubility and DFPase activity. Our results pave the way for exploring the use of rHuSMP30 against organophosphate and nerve agent toxicity.

Adenovirus-transduced human butyrylcholinesterase in mouse blood functions as a bioscavenger of chemical warfare nerve agents.

Human serum butyrylcholinesterase (Hu BChE) is a promising therapeutic against the toxicity of chemical warfare nerve agents. We have showed previously that recombinant (r) Hu BChE can be expressed at very high levels, 400 to 600 U/ml in mouse blood, by delivering the Hu BChE gene using adenovirus (Ad). Here, we report the biochemical properties of the Ad-expressed full-length and truncated rHu BChE in mouse blood. The molecular sizes of the full-length rHu BChE subunit and its oligomers were similar to those of native Hu BChE, although only a small portion of the full-length rHu BChE subunit underwent assembly into dimers and tetramers. As expected, Ad containing the truncated Hu BChE gene transduced the expression of monomeric rHu BChE only. Compared with 415 U of rHu BChE per milliliter in blood, tissues including liver, lung, heart, brain, kidney, muscle, intestine, diaphragm, salivary gland, and fat expressed <10 U/g of rHu BChE activity. Ad-expressed rHu BChE in mouse blood neutralized soman and O-ethyl S-2-N,N-diisopropylaminoethyl methylphosphonothiolate at rates similar to those of native Hu BChE and rHu BChE expressed in vitro. Because the expression of rHu BChE rapidly decreased 6 days after virus administration, sera were assayed for the presence of anti-Hu BChE antibodies. Anti-Hu BChE antibodies were detected on day 7 and in increased amounts thereafter, which coincided with the loss of Hu BChE expression in sera. In conclusion, the delivery of Hu BChE gene using Ad can be a promising strategy that can provide protection against multiple lethal doses of chemical warfare nerve agents in vivo.

Effect of polyethylene glycol modification on the circulatory stability and immunogenicity of recombinant human butyrylcholinesterase.

The therapeutic value of human serum butyrylcholinesterase (Hu BChE) as a bioscavenger of chemical warfare agents is due to its high reactivity with organophosphorus compounds and prolonged circulatory stability. Native Hu BChE is mostly tetrameric in form while the enzyme produced using molecular cloning technology is a mixture of tetramers, dimers, and monomers. Previous studies revealed that monomers and dimers of recombinant human (rHu) BChE cleared rapidly from the circulation of mice compared to tetrameric rHu BChE and native Hu BChE, which have mean residence times (MRTs) of 18h and 45h, respectively. It was also shown that polyethylene glycol-20K (PEG) modification of tetrameric rHu BChE prolonged its circulatory stability and bioavailability in vivo. The goal of this study was to determine if modification with PEG could prolong the circulatory stability and eliminate the immunogenicity of monomeric rHu BChE. Monomeric rHu BChE was expressed in human 293A cells using a cDNA lacking the 45 amino acid tetramerization domain from the carboxyl terminus and the adenovirus expression system. The catalytic and inhibitory properties of purified monomeric rHu BChE were similar to those for native Hu BChE and were not affected by PEG modification. As expected, monomeric rHu BChE rapidly cleared from the circulation of mice (MRT=3.2+/-0.3h) while monomeric PEG-rHu BChE demonstrated significant improvement in its bioavailability and circulatory stability in blood (MRT=31.4+/-5.4h). However, a second injection of monomeric PEG-rHu BChE, 28 days after the first, displayed a much shorter MRT=11.6+/-0.4h, and circulating anti-monomeric PEG-rHu BChE antibodies were detected in the blood of mice. These results suggest that PEG modification increased the circulatory stability of monomeric rHu BChE but failed to reduce or eliminate its immunogenicity.

A repeated injection of polyethyleneglycol-conjugated recombinant human butyrylcholinesterase elicits immune response in mice.

Human serum butyrylcholinesterase (Hu BChE) serves as an efficacious bioscavenger of highly toxic organophosphorus (OP) compounds. Since there is a concern that the supply of native Hu BChE may be limited, monomeric and tetrameric forms of recombinant Hu BChE (rHu BChE) were evaluated as replacements and found that they lacked sufficient stability in vivo. However, their in vivo stability could be significantly prolonged by conjugation with polyethyleneglycol-20K (PEG) suggesting that monomeric and tetrameric PEG-rHu BChE could function as bioscavengers. Here, the immunogenicity of PEG-rHu BChE was evaluated in mice following two injections given four weeks apart. In addition to pharmacokinetic parameters, such as mean residence time, maximal concentration, time to reach the maximal concentration, elimination half-life and area under the plasma concentration-time curve extrapolated to infinity, the presence of circulating anti-rHu BChE antibodies was also determined. Although the pharmacokinetic parameters were significantly improved for the first injection of monomeric and tetrameric PEG-rHu BChEs, they were much lower for the second injection. Anti-rHu BChE antibodies were detected in the blood of mice following the first and second enzyme injections and their levels were approximately higher by 5-fold and 2-fold in mice injected with monomeric and tetrameric PEG-rHu BChEs as compared to mice injected with unconjugated enzymes. The findings that the rapid clearance of a repeat injection of PEG-rHu BChEs in mice which coincides with the presence of circulating anti-rHu BChE antibodies suggest that PEG conjugation prolonged the circulatory stability of rHu BChE but failed to eliminate its immunogenicity in mice.

Adenovirus-mediated gene transfer of human butyrylcholinesterase results in persistent high-level transgene expression in vivo.

Human serum butyrylcholinesterase (Hu BChE) is a promising therapeutic against the toxicity of chemical warfare nerve agents, pesticide intoxication, and cocaine overdose. However, its widespread application is hampered by difficulties in large-scale production of the native protein from human plasma and/or availability as a recombinant protein suitable for use in vivo. This limitation may be resolved by in vivo delivery and expression of the Hu BChE gene. In this study, recombinant (r) adenoviruses (Ads) encoding full-length and truncated rHu BChEs were tested for in vivo expression in mice. Mice injected with these rAds intraperitoneally failed to express rHu BChE. However, a single tail vein injection of both rAds resulted in persistent high serum levels of rHu BChE in BChE knockout mice, which peaked on days 4/5 at 377+/-162U/ml for full-length rHu BChE and 574+/-143U/ml for truncated rHu BChE. These activity levels are orders of magnitude higher than 1.9U/ml of mouse BChE present in wild-type mouse serum. Thereafter, rHu BChE levels dropped rapidly and very little or no activity was detected in the serum 10 days post-virus administration. In conclusion, the present study demonstrates the potential of rAd-mediated Hu BChE gene therapy to counteract multiple lethal doses of chemical warfare nerve agent toxicity.

An in vitro comparative study on the reactivation of nerve agent-inhibited guinea pig and human acetylcholinesterases by oximes.

The reactivation of nerve agent-inhibited acetylcholinesterase (AChE) by oxime is the most important step in the treatment of nerve agent poisoning. Since the evaluation of nerve agent antidotes cannot be conducted in humans, results from animal experiments are extrapolated to humans. Guinea pig is one of the animal models that is frequently used for conducting nerve agent antidote evaluations. Several investigations have demonstrated that the efficacy of an oxime primarily depends on its ability to reactivate nerve agent-inhibited AChE. If the in vitro oxime reactivation of nerve agent-inhibited animal AChE is similar to that of human AChE, it is likely that the results of an in vivo animal study will reliably extrapolate to humans. Therefore, the goal of this study was to compare the reactivation of guinea pig and human AChEs inhibited by six different G and V type nerve agents. Reactivation kinetic studies with five mono- and bis-pyridinium oximes showed that oxime reactivation of nerve agent-inhibited human AChE in most cases was faster than guinea pig AChE. The most significant enhancement was observed in the reactivation of human AChE inhibited by nerve agents containing bulky side chains GF, GD, and VR, by H-series oximes HLo-7, HI-6, and ICD-585. In these cases, species-related differences observed between the two AChEs, based on the second-order reactivation rate constants, were 90- to over 400-fold. On the other hand, less than 3-fold differences were observed in the rates of aging of nerve agent-inhibited guinea pig and human AChEs. These results suggest that the remarkable species-related differences observed in the reactivation of nerve agent-inhibited guinea pig and human AChEs were not due to differences in the rates of aging. These results also suggest that guinea pig may not be an appropriate animal model for the in vivo evaluation of oxime therapy.

Protective effects of recombinant kunitz-domain 1 of human tissue factor pathway inhibitor-2 against 2-chloroethyl ethyl sulfide toxicity in vitro.

OBJECTIVE: Sulfur mustard is a well-known blistering chemical warfare agent that has been investigated for its toxicological mechanisms and an efficacious antidote. Since sulfur mustard injury involves dermal:epidermal separation, proteolytic enzymes were suspected to be involved for this separation and eventual blister development. Therefore, protease inhibitors could be of therapeutic utility against sulfur mustard injury. In this study, the effects of Kunitz-domain 1 of human tissue factor pathway inhibitor-2 were evaluated against the toxic effects of 2-chloroethyl ethyl sulfide, a surrogate agent of sulfur mustard. Tissue factor pathway inhibitor-2 is a 32-kDa serine protease inhibitor produced by a variety of cell types including human epidermal keratinocytes, fibroblasts, and endothelial cells. It consists of 3 Kunitz-domains and the first Kunitz-domain contains the putative P(1) residue (arginine at position 24) responsible for protease inhibitory activity. METHODS: Recombinant wild-type and R24Q mutant Kunitz-domain 1s were expressed in Escherichia coli and purified. The purified proteins were refolded, and their effects were tested in an in vitro human epidermal keratinocyte cell wounding assay. RESULTS: Wild-type but not R24Q Kunitz-domain 1 inhibited the amidolytic activity of trypsin and plasmin. Wild-type Kunitz-domain1 was stable for 4 weeks at 42 degrees C and for more than 8 weeks at room temperature. Wild-type Kunitz-domain 1 significantly improved wound healing of unexposed and 2-chloroethyl ethyl sulfide-exposed cells without influencing cell proliferation. Although R24Q Kunitz-domain 1 lacked trypsin and plasmin inhibitory activity, it promoted wound closure of untreated and 2-chloroethyl ethyl sulfide-treated cells but to a much lesser degree. CONCLUSION: These data suggest that wild-type Kunitz-domain 1 of human tissue factor pathway inhibitor-2 can be developed as a medical countermeasure against sulfur mustard cutaneous injury.

Protective effects of recombinant kunitz-domain 1 of human tissue factor pathway inhibitor-2 against 2-chloroethyl ethyl sulfide toxicity in vitro.

OBJECTIVE: Sulfur mustard is a well-known blistering chemical warfare agent that has been investigated for its toxicological mechanisms and an efficacious antidote. Since sulfur mustard injury involves dermal:epidermal separation, proteolytic enzymes were suspected to be involved for this separation and eventual blister development. Therefore, protease inhibitors could be of therapeutic utility against sulfur mustard injury. In this study, the effects of Kunitz-domain 1 of human tissue factor pathway inhibitor-2 were evaluated against the toxic effects of 2-chloroethyl ethyl sulfide, a surrogate agent of sulfur mustard. Tissue factor pathway inhibitor-2 is a 32-kDa serine protease inhibitor produced by a variety of cell types including human epidermal keratinocytes, fibroblasts, and endothelial cells. It consists of 3 Kunitz-domains and the first Kunitz-domain contains the putative P(1) residue (arginine at position 24) responsible for protease inhibitory activity. METHODS: Recombinant wild-type and R24Q mutant Kunitz-domain 1s were expressed in Escherichia coli and purified. The purified proteins were refolded, and their effects were tested in an in vitro human epidermal keratinocyte cell wounding assay. RESULTS: Wild-type but not R24Q Kunitz-domain 1 inhibited the amidolytic activity of trypsin and plasmin. Wild-type Kunitz-domain 1 was stable for 4 weeks at 42 degrees C and for more than 8 weeks at room temperature. Wild-type Kunitz-domain 1 significantly improved wound healing of unexposed and 2-chloroethyl ethyl sulfide-exposed cells without influencing cell proliferation. Although R24Q Kunitz-domain 1 lacked trypsin and plasmin inhibitory activity, it promoted wound closure of untreated and 2-chloroethyl ethyl sulfide-treated cells but to a much lesser degree. CONCLUSION: These data suggest that wild-type Kunitz-domain 1 of human tissue factor pathway inhibitor-2 can be developed as a medical countermeasure against sulfur mustard cutaneous injury.