[Preparation of paraoxonase liposomes and their pharmacokinetics in rats].

OBJECTIVE: To prepare paraoxonase 1 (PON1) liposomes, and investigate pharmacokinetics of common PON1 liposomes (L-PON1) and polyethylene glycol-modified PON1 long circulating liposomes (PEG-PON1-LCL) in rats after intravenous administration. METHODS: L-PON1 and PEG-PON1-LCL were prepared by film dispersion method. The entrapment efficiency, mean diameter and Zeta potential of the liposomes were measured, and the stability was evaluated. Thirty-six Wistar rats were divided into three groups according to random number table, with 12 rats in each group. The rats were intravenously administrated with PON1, L-PON1 or PEG-PON1-LCL 700 U/kg, respectively. The activity of PON1 in serum was determined by phenyl acetate method, the activity of PON1 at different time points after drug administration was compared with that before drug administration, and the difference value was considered as the activity of exogenous PON1, and PON1 activity-time curve was plotted. The pharmacokinetic parameters were calculated and analyzed by DAS 2.0 pharmacokinetic program and SPSS 17.0. RESULTS: The entrapment efficiencies of L-PON1 and PEG-PON1-LCL were above 85%, the mean diameter was about 126 nm, and Zeta potential was -14.35 mV. After 2 weeks of preservation, the above parameters showed no obvious change, indicating that liposomes had good stability and the properties of preparations were basically stable. Compared with purified PON1 administration, after L-PON1 and PEG-PON1-LCL administration, the activity of PON1 was increased, the half-life of PON1 activity in rats was significantly prolonged [the half-life of distribution (T1/2α, hours): 0.142±0.018, 0.147±0.021 vs. 0.126±0.022; the half-life of clearance (T1/2ß, hours): 3.877±1.010, 4.520±1.117 vs. 1.226±0.422], the area under PON1 activity-time curve (AUC) was significantly increased [AUC from 0 hour to 24 hours (AUC0-24, U×h-1×L-1): 499.305±64.710, 563.576±70.450 vs. 18.053±2.190; AUC from the immediate injection to the disappearance of PON1 activity (AUC0-∞, U×h-1×L-1): 516.256±60.940, 587.801±76.210 vs. 21.044±3.250], the apparent volume of distribution (Vd) and clearance (CL) were significantly decreased [Vd (L): 0.140±0.065, 0.144±0.064 vs. 0.493±0.032, CL (L/h): 0.039±0.008, 0.034±0.006 vs. 0.952±0.082, all P < 0.05]. There was no significant difference in pharmacokinetics between L-PON1 and PEG-PON1-LCL. CONCLUSIONS: The film dispersion method prepared PON1 liposomes have high entrapment efficiency and small particle size with a good stability. Both liposomes can raise PON1 activity in vivo, change the pharmacokinetics of PON1 in vivo, prolong the resident time of PON1 in the blood circulating system, and compensate for the short half-life of PON1 in vivo.

A simple and highly effective catalytic nanozyme scavenger for organophosphorus neurotoxins.

A simple and highly efficient catalytic scavenger of poisonous organophosphorus compounds, based on organophosphorus hydrolase (OPH, EC 3.1.8.1), is produced in aqueous solution by electrostatic coupling of the hexahistidine tagged OPH (His6-OPH) and poly(ethylene glycol)-b-poly(l-glutamic acid) diblock copolymer. The resulting polyion complex, termed nano-OPH, has a spherical morphology and a diameter from 25nm to 100nm. Incorporation of His6-OPH in nano-OPH preserves catalytic activity and increases stability of the enzyme allowing its storage in aqueous solution for over a year. It also decreases the immune and inflammatory responses to His6-OPH in vivo as determined by anti-OPH IgG and cytokines formation in Sprague Dawley rats and Balb/c mice, respectively. The nano-OPH pharmacokinetic parameters are improved compared to the naked enzyme suggesting longer blood circulation after intravenous (iv) administrations in rats. Moreover, nano-OPH is bioavailable after intramuscular (im), intraperitoneal (ip) and even transbuccal (tb) administration, and has shown ability to protect animals from exposure to a pesticide, paraoxon and a warfare agent, VX. In particular, a complete protection against the lethal doses of paraoxon was observed with nano-OPH administered iv and ip as much as 17h, im 5.5h and tb 2h before the intoxication. Further evaluation of nano-OPH as a catalytic bioscavenger countermeasure against organophosphorus chemical warfare agents and pesticides is warranted.

Epigenetic patterns of two gene promoters (TNF-alpha and PON) in stroke considering obesity condition and dietary intake

Some causal bases of stroke remain unclear, but the nutritional effects on the epigenetic regulation of different genes may be involved. The aim was to assess the impact of epigenetic processes of human tumor necrosis factor (TNF-alfa) and paraoxonase (PON) promoters in the susceptibility to stroke when considering body composition and dietary intake. Twenty-four patients (12 non-stroke/12 stroke) were matched by sex (12 male/12 female), age (mean 70 ± 12 years old), and BMI (12 normal-weight/12 obese; mean 28.1 ± 6.7 kg/m2). Blood cell DNA was isolated and DNA methylation levels of TNF-alfa (-186 to +349 bp) and PON (-231 to +250 bp) promoters were analyzed by the Sequenom EpiTYPER approach. Histone modifications (H3K9ac and H3K4me3) were analyzed also by chromatin immunoprecipitation in a region of TNF-α (-297 to -185). Total TNF-α promoter methylation was lower in stroke patients (p < 0.001) and showed no interaction with body composition (p = 0.807). TNF-α and PON total methylation levels correlated each other (r = 0.44; p = 0.031), especially in stroke patients (r = 0.72; p = 0.008). The +309 CpG methylation site from TNF-α promoter was related to body weight (p = 0.027) and the region containing three CpGs (from −170 to -162 bp) to the percentage of lipid intake and dietary indexes (p < 0.05) in non-stroke patients. The methylation of PON +15 and +241 CpGs was related to body weight (p = 0.021), waist circumference (p = 0.020), and energy intake (p = 0.018), whereas +214 was associated to the quality of the diet (p < 0.05) in non-stroke patients. When comparing stroke vs non-stroke patients regarding the histone modifications analyzed at TNF-α promoter, no changes were found, although a significant association was identified between circulating TNF-alfa level and H3K9ac with H3K4me3. TNF-alfa and PON promoter methylation levels could be involved in the susceptibility to stroke and obesity outcome, respectively. The dietary intake and body composition may influence this epigenetic regulation in non-stroke patients

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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.

Organophosphorus hydrolase as an in vivo catalytic nerve agent bioscavenger.

The use of proteins as a treatment for organophosphorus intoxication has been investigated since A. R. Main demonstrated protective efficacy against parathion with an exogenously administered arylesterase in the late 1950s. His experiments spurred over 60 years of research and progress in the development of enzymes as potential bioscavengers of nerve agents and pesticides. Efforts have been made to broaden the specificity of enzymes to make a universal scavenger that would protect against multiple compounds, and an understanding of the differential isomer toxicity of these compounds has provided the impetus for rational and random mutagenic approaches in the stereospecific design of enzymes. As improved candidate enzymes are continually developed, our understanding of the contributions of the catalytic parameters (k(cat) , K(M) and catalytic efficiency) to efficacy expands. In addition to the scavenging properties of the proteins, another important aspect of development is the pharmacokinetic profile of the drug product. Immunogenicity, absorption, distribution and elimination contribute significantly to the level of protection afforded by the protein. A review of the development of organophosphorus hydrolase (OPH) for use as in vivo catalytic bioscavengers is presented here.

Crossroads in the evaluation of paraoxonase 1 for protection against nerve agent and organophosphate toxicity.

Human paraoxonase 1 (PON1), a 45kDa arylesterase associated with circulating high density lipoproteins (HDL), has been described as an anti-atherogenic element in cardiovascular disorders. The efficacy of PON1 as a catalytic bioscavenger against OP and CWNA toxicity has been on debate for the last few decades. Hydrolysis of various organophosphates (OPs) and chemical warfare nerve agents (CWNAs) by PON1 has been demonstrated in both in vitro and in vivo experiments. Recently, we established the protective efficacy of human and rabbit serum purified PON1 as well as human recombinant PON1 expressed in Trichoplusia ni larvae against nerve agent toxicity in guinea pigs. Exogenous administration of purified PON1 was effective in protecting against 1.2 X LCt(50) of sarin and soman administered endotracheally with microinstillation technology. However, the short half-life of exogenously administered PON1, probably due to poor association with circulating HDL, warrant alternative approaches for successful utility of PON1 in the treatment of OP/CWNA toxicity. In this mini review, we address the pros and cons of current PON1 prophylaxis and propose potential solutions for successful development of PON1 as an effective catalytic bioscavenger.

Injection of paraoxonase 1 (PON1) to mice stimulates their HDL and macrophage antiatherogenicity.

We analyzed, for the first time, the effects of recombinant PON1 (rePON1) intraperitoneal injection to C57BL/6 mice on their HDL and macrophage antiatherogenic properties. Thioglycolate-treated mice were injected with either saline (Control), or rePON1 (50 µg/mouse), and 20 H post injection, their blood samples and peritoneal macrophages (MPM) were collected. A significant increase in serum and HDL-PON1 arylesterase and lactonase activities was noted. Similarly, a significant increment, by 3.8 and 2.8 fold, in MPM-PON1 arylesterase and lactonase activities, respectively, as compared to the activities in control MPM was observed. The HDL from rePON1-injected mice was resistant to oxidation by copper ions as compared to control HDL. Furthermore, enrichment of the mouse HDL with rePON1 increased its ability to induce cholesterol efflux from J774A.1 macrophage cell line, and to inhibit macrophage-mediated LDL oxidation. In MPM from rePON1-injected mice vs. control MPM, there was a significant reduction in cholesterol mass, by 42%, in association with inhibition in cellular cholesterol biosynthesis rate, by 33%, and with significant stimulation, by 65%, of human HDL-mediated cholesterol efflux from the cells. We conclude that rePON1 injection to mice improved the mice HDL and MPM antiatherogenic properties, and these effects could probably lead to attenuation of atherosclerosis development.

CHO cell expression, long-term stability, and primate pharmacokinetics and brain uptake of an IgG-paroxonase-1 fusion protein.

Paraoxonase (PON)-1 is the most potent human organophosphatase known, but recombinant forms of human PON1 have been difficult to produce owing to poor secretion by host cells. In the present investigation, human PON1 is re-engineered as an IgG-PON1 fusion protein. The 355 amino acid human PON1 is fused to the carboxyl terminus of the heavy chain of a chimeric monoclonal antibody (MAb) against the human insulin receptor (HIR), and this fusion protein is designated HIRMAb-PON1. The HIRMAb part of the fusion protein enables brain penetration of the PON1, which was considered important, because organophosphate toxicity causes death via a central nervous system site of action. A high producing line of stably transfected Chinese hamster ovary (CHO) cells secreting the HIRMAb-PON1 fusion protein in the absence of serum or lipid acceptors was cloned. The bioreactor generated fusion protein was purified to homogeneity with low impurities by protein A affinity chromatography and anion exchange chromatography. The HIRMAb-PON1 fusion protein was stable as a sterile liquid formulation stored at 4°C for at least 1 year. The plasma pharmacokinetics (PK) of the HIRMAb-PON1 fusion protein was evaluated in Rhesus monkeys, which is the first PK evaluation of a recombinant PON1 protein. The fusion protein was rapidly removed from blood, primarily by the liver. The blood-brain barrier permeation of the HIRMAb-PON1 fusion protein was high and comparable to other HIRMAb fusion proteins. Re-engineering human PON1 as the HIRMAb fusion protein allows for production of a stable, field-deployable formulation of the enzyme that is brain-penetrating.

Pharmacokinetics of OpdA, an organophosphorus hydrolase, in the African green monkey.

Organophosphorus (OP) pesticides are a broad class of acetylcholinesterase inhibitors that are responsible for tremendous morbidity and mortality worldwide, contributing to an estimated 300,000 deaths annually. Current pharmacotherapy for acute OP poisoning includes the use of atropine, an oxime, and benzodiazepines. However, even with such therapy, the mortality from these agents are as high as 40%. Enzymatic hydrolysis of OPs is an attractive new potential therapy for acute OP poisoning. A number of bacterial OP hydrolases have been isolated. A promising OP hydrolase is an enzyme isolated from Agrobacterium radiobacter, named OpdA. OpdA has been shown to decrease lethality in rodent models of parathion and dichlorvos poisoning. However, pharmacokinetic data have not been obtained. In this study, we examined the pharmacokinetics of OpdA in an African Green Monkey model. At a dose of 1.2mg/kg the half-life of OpdA was approximately 40 min, with a mean residence time of 57 min. As expected, the half-life did not change with the dose of OpdA given: at doses of 0.15 and 0.45 mg/kg, the half-life of OpdA was 43.1 and 38.9 min, respectively. In animals subjected to 5 daily doses of OpdA, the residual activity that was measured 24h after each OpdA dose increased 5-fold for the 0.45 mg/kg dose and 11-fold for the 1.2mg/kg dose. OpdA exhibits pharmacokinetics favorable for the further development as a therapy for acute OP poisoning, particularly for hydrophilic OP pesticides. Future work to increase the half-life of OpdA may be beneficial.

Kinetics and efficacy of an organophosphorus hydrolase in a rodent model of methyl-parathion poisoning.

OBJECTIVES: Organophosphorus (OP) pesticides exert a tremendous health burden, particularly in the developing world. Limited resources, the severity of intentional OP ingestions, and a paucity of beneficial therapies all contribute to the morbidity and mortality of this broad class of chemicals. A novel theoretical treatment for OP poisoning is the use of an enzyme to degrade the parent OP in the circulation after poisoning. The aims of this study were to determine the pharmacokinetics and efficacy of an OP hydrolase (OpdA) in a rodent model of severe methyl-parathion poisoning. METHODS: Two animal models were used. First, Wistar rats were administered two different doses of the hydrolase (0.15 and 1.5 mg/kg), and the ex vivo hydrolytic activity of plasma was determined by a fluorometric method. Second, an oral methyl-parathion animal poisoning model was developed to mimic severe human poisoning, and the efficacy of postpoisoning OpdA (as measured by survival to 4 and 24 hours) was determined. RESULTS: The half-life of OpdA in the Wistar rat was dependent on the dose administered and ranged between 45.0 and 57.9 minutes. The poisoning model of three times the lethal dose to 50% of the population (3 x LD(50)) of methyl-parathion resulted in 88% lethality at 4 and 24 hours. Using a single dose of 0.15 mg/kg OpdA 10 minutes after poisoning resulted in 100% survival at 4 hours (p = 0.001 vs. placebo), but 0% at 24 hours postpoisoning (p = NS vs. placebo). CONCLUSIONS: The OP hydrolase OpdA exhibits pharmacokinetics suitable for repeated dosing and increases short-term survival after severe methyl-parathion poisoning.

Improved pharmacokinetics and immunogenicity profile of organophosphorus hydrolase by chemical modification with polyethylene glycol.

A catalytic bioscavenger with broad substrate specificity for the therapeutic and prophylactic defense against recognized chemical threat agents has been a long standing objective of civilian and military research. A catalytic bioscavenger utilizing the bacterial enzyme organophosphorus hydrolase (OPH) is characterized in these studies, and has potential application for both military and civilian personnel in threat scenarios involving either nerve agents or OP pesticides. The present study examines the effects of PEGylation on the biochemical and pharmacological characteristics of OPH. The enzyme was conjugated with linear and branched methyl-PEO(n)-NHS esters of relatively small molecular mass from 333 to 2420Da. PEGylated OPH displayed a decreased maximal catalytic rate, though substantial activity was maintained against two tested substrates: up to 30% with paraoxon and up to 50-60% with demeton-S. The thermostability of the PEGylated enzymes ranged between 60 and 64 degrees C, compared to the unmodified OPH, which is approximately 67 degrees C. The enzyme conjugates revealed a significant improvement of pharmacokinetic properties in animal studies. The clearance from a guinea pig's blood stream significantly decreased relative to unmodified OPH, resulting in an increase of residence time and systemic availability. Evaluation of the humoral immune response indicated that the branched PEG-OPH conjugate significantly reduced production of anti-OPH antibodies, compared to the unmodified enzyme. The OPH-PEG conjugates with improved pharmacokinetic and immunogenicity properties, considerable catalytic activity and thermal stability provide a new opportunity for the in vivo detoxification of the neurotoxic OP compounds.

Genetic variability in the cytochrome P450-paraoxonase 1 (PON1) pathway for detoxication of organophosphorus compounds.

Detoxication of organophosphorus (OP) compounds is affected by genetic and environmental modulation of a number of enzymes involved in the process. For organophosphorothioate insecticides, different P450 isozymes and variants carry out two reactions that have quite different consequences; (1) they bioactivate their parent compounds to highly toxic oxon forms that are many times more toxic than the parent compounds, and (2) concurrently, they dearylate the parent OP compounds, generating much less toxic metabolites. The ratios at which these different P450s carry out bioactivation versus dearylation differ among the P450 isozymes. The detoxication of the oxon forms of diazinon and chlorpyrifos is achieved by hydrolysis to the respective aromatic alcohols and diethyl phosphates primarily by paraoxonase 1 (PON1), a plasma enzyme tightly associated with high-density lipoprotein particles and also found in liver. Stoichiometric binding to other targets also contributes to the detoxication of these oxons. PON1 is polymorphically distributed in human populations with an amino acid substitution (Gln/Arg) at position 192 of this 354-amino acid protein (the initiator Met residue is cleaved on maturation) that determines the catalytic efficiency of hydrolysis of some substrates. In addition to the variable catalytic efficiency determined by the position 192 amino acid, protein levels of PON1 vary by as much as 15-fold among individuals with the same PON1(192) genotype (Q/Q; Q/R; R/R). The generation of PON1 null mice and transgenic mice, expressing each of the human PON1(192) alloforms in place of mouse PON1, has allowed for the examination of the physiological function of the PON1(192) alloforms in OP detoxication. Sensitivity to diazoxon exposure is primarily determined by the plasma level of PON1, whereas for chlorpyrifos oxon exposure, both the plasma PON1 level and the position 192 amino acid are important--PON1(R192) is more efficient in inactivating chlorpyrifos oxon than is PON1(Q192). The availability of PON1 null mice provides an opportunity to examine the contribution of other enzymes in the OP detoxication pathways without PON1 interference.