Association of PON1, APOE and SDF-1 Gene Polymorphisms with Treatment Response to Intravitreal Anti-VEGF Treatment in Patients with Retinal Vein Occlusion.

PURPOSE: The purpose of this study was to determine whether specific genetic polymorphisms affect the response to intravitreal anti-vascular endothelial growth factor (anti-VEGF) treatment in patients with macular oedema secondary to retinal vein occlusion (RVO). METHODS: Participants in this prospective study were 50 patients with macular oedema secondary to RVO, who were treated with intravitreal ranibizumab or aflibercept, and were followed-up for 12 months after initiation of treatment. Five single nucleotide polymorphisms (SNPs) from three different genes (APOE, PON1, SDF-1) were examined as potential predictors for treatment response to intravitreal anti-VEGF agents. RESULTS: Patients with the LL genotype of the PON1 L55M SNP had significantly higher reduction in central subfield thickness (CST) at month 12 after initiation of intravitreal anti-VEGF treatment (101.63 ± 56.80 µm in LL vs. 72.44 ± 39.41 µm in LM vs. 40.25 ± 19.33 µm in MM, p = .026). Patients with the M allele of the PON1 L55M SNP were significantly associated with lower reduction in CST compared to non-carriers (68.29 ± 38.77 µm in LM + MM vs. 101.63 ± 56.80 µm in LL, p = .032). CONCLUSION: PON1 L55M SNP may serve as a promising genetic biomarker for predicting response to intravitreal anti-VEGF treatment in patients with macular oedema due to RVO.

Proteomic analysis of tears in dry eye disease: A prospective, double-blind multicenter study.

PURPOSE: To identify specific dry eye disease (DED) tear biomarker(s) using tear proteomic analysis, clinical parameters, and their correlations before and after DED treatment. METHODS: A prospective, double-blinded, national multicenter clinical study was performed using data from 80 DED patients. The patients were treated with 0.1% cyclosporine (CsA, n = 28), 0.05% CsA (n = 26), or 3% diquafosol (DQS, n = 26) eye drops, and tear proteome changes and clinical outcomes (tear break-up time [TBUT], corneal erosion [Cor-Er], conjunctival erosion [Conj-Er], and symptom assessment in dry eye [SANDE] scores) were observed at 4, 8, and 12 weeks. For all clinical parameters, correlation analysis was performed between the three drug conditions and the differentially expressed proteins (DEPs) from the proteomic analysis. RESULTS: AFM, ALCAM, CFB, H1-4, PON1, RAP1B, and RBP4 were identified in all treatment groups and were downregulated after treatment. All clinical parameters significantly improved at 12 weeks than at baseline (p-value <0.0001); however, their values were not significantly different among groups, except for Cor-Er (p-value = 0.007). Compared with the DQS group, Cor-Er score significantly improved after treatment with 0.1% and 0.05% CsA. The seven DEPs identified in all groups were not consistently correlated with the clinical parameters (p-value >0.05). CONCLUSIONS: Despite differences in drug concentration and action mechanisms, the improvement levels of TBUT, Cor-Er, and SANDE scores were clinically adequate. However, useful tear protein biomarkers, clinically acceptable biomarker combinations correlating with clinical parameters, and clinically acceptable levels of specificity and sensitivity were not identified.

Power doppler ultrasound signal predicts abnormal HDL function in patients with rheumatoid arthritis.

Active rheumatoid arthritis (RA) is associated with increased cardiovascular risk and impaired function of high-density lipoprotein (HDL). Previous work suggests that HDL may become dysfunctional through oxidative modifications within the RA joint. The current work evaluates an association of synovial power doppler ultrasound signal (PDUS) with HDL function and structure. Two open-label clinical therapeutic studies using PDUS as a disease outcome measure were included in this analysis, including a 12-month trial of subcutaneous abatacept in 24 RA patients and a 6-month trial of IV tocilizumab in 46 RA patients. Laboratory assays included assessments of HDL function and structure, HDL and total cholesterol levels, and a cytokine/chemokine panel. Patients with the highest baseline PDUS scores in both clinical studies, had worse HDL function, including suppression of paraoxonase 1 (PON1) activity as well as lower HDL-C levels. Associations between other disease assessments (DAS28 and CDAI) and HDL function/structure were noted but were generally of lesser magnitude and consistency than PDUS across the HDL profile. Treatment with tocilizumab for 6 months was associated with increases in cholesterol levels and improvements in the HDL function profile, which correlated with greater decreases in PDUS scores. Similar trends were noted following treatment with abatacept for 3 months. Higher baseline PDUS scores identified patients with worse HDL function. This data supports previous work suggesting a direct association of joint inflammation with abnormal HDL function.

STAT5 inhibitor Pimozide as a probable therapeutic option in overcoming Ponatinib resistance in K562 leukemic cells.

Signal Transducer and Activator of Transcription 5 (STAT5) is a transcription factor that plays a key role in neoplasia, triggered by the fusion oncogene BCR-ABL1; it is not only an essential protein for the pathogenesis of chronic myeloid leukemia (CML), but also its overexpression is associated with drug resistance developed toward various generations of Tyrosine Kinase Inhibitors (TKIs); these are still accepted as gold standard therapeutics for the treatment of CML. In this study, it was investigated whether suppression of STAT5 via a "STAT5 inhibitor" Pimozide resulted in any regain of chemosensitivity to third-generation TKI Ponatinib. Accordingly, the experimental work was designed on both parental CML cell line K562WT and its 1 nM Ponatinib-resistant counterpart, indicated as K562-Pon1. Based on the experimental results, Pimozide was more effective in resistant cells compared to wild-type cells for inducing apoptosis and block cell arrest. Combination therapy of Pimozide and Ponatinib demonstrated that STAT5 was a significant protein for regaining chemosensitivity to Ponatinib when its expression was suppressed both at mRNA and protein level. In conclusion, we consider that STAT5 inhibitor Pimozide can be a good alternative or combination therapy with TKIs for patients suffering from chemotherapeutic drug resistance. Communicated by Ramaswamy H. Sarma.

Effects of Combined Garcinia kola and Kigelia africana on Insulin and Paraoxonase 1 (PON1) Levels in Type 2 Diabetic Rats.

BACKGROUND: Individual extracts of Garcinia kola and Kigelia africana have been shown to have therapeutic effects against a variety of variables linked to the development of diabetes mellitus. However, there is still a lack of information about the combined effects of these extracts on Insulin and Paraoxonase 1 (PON-1) in Streptozotocin-Nicotinamide-induced type-2 diabetic Wistar rats. METHODS: Forty-two young male rats (180-200g) were randomly divided into six groups (n = 7/group). Diabetes was intraperitoneally induced with 110 mg/kg of nicotinamide constituted in distilled water and fifteen minutes later with 65 mg/kg of streptozocin freshly prepared in 0.1M citrate buffer (pH of 4.5) and treated for six weeks as follows: the control rats received either 0.9% normal saline (NS) or 250 mg/kg extract by gavage. The remaining animals were diabetes induced and subsequently treated with either NS, graded doses of the extract (250 mg/kg and 500 mg/kg), or 5 mg/kg Glibenclamide + 100mg/kg Metformin. Gas chromatography-mass spectrometry (GCMS) of the combined extracts was also analyzed to identify the bioactive compounds present in it. Insulin, PON-1 levels, lipid profiles, and atherogenic index were assessed. RESULTS: Our findings show that Insulin and PON-1 levels in the plasma of diabetic rats treated with the combined extracts were significantly increased when compared to the control rats. Moreover, the GCMS of the extract shows the presence of both monounsaturated (oleic acid) and polyunsaturated (linoleic acid) fatty acids. CONCLUSION: The current findings suggest that the extract may help improve glucose homeostasis and prevent atherosclerosis through the established mechanism of the identified bioactive compounds.

Network pharmacology analysis reveals neuroprotection of Gynostemma pentaphyllum (Thunb.) Makino in Alzheimer' disease.

BACKGROUND: Alzheimer's disease (AD) is one of the most common neurodegenerative disorders in the world, but still lack of effective drug treatment. Gynostemma Pentaphyllum (Thunb.) Makino (GpM), a Chinese medicinal herb, plays important roles in anti-inflammation, anti-oxidative stress and anti-tumor, which has been reported to ameliorate cognitive impairment of AD. However, the neuroprotective mechanism of GpM remains unclear. This study aims to investigate the targets and possible signaling pathways of GpM in the treatment of AD. METHODS: Active compounds of GpM and their putative target proteins were selected from Traditional Chinese Medicine Systems Pharmacology (TCMSP) Database and Analysis Platform. AD-associated targets were identified from GeneCards, the Online Mendelian Inheritance in Man (OMIM) database and the Therapeutic Target Database (TTD). The intersecting targets of GpM and AD were identified and Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were carried out to analyze the mechanism of them. Compound-target-pathway (CTP) network and protein-protein interaction (PPI) network were constructed and analyzed to elucidate the correlation between compounds, proteins and pathways. Molecular docking was performed to further demonstrate the possibility of GpM for AD. RESULTS: A total of 13 active compounds of GpM, 168 putative target proteins of compounds and 722 AD-associated targets were identified. Eighteen intersecting targets of GpM and AD were found and the epidermal growth factor receptor (EGFR), interleukin-1 beta (IL-1ß), interleukin-6 (IL-6), nitric oxide synthase in endothelial (NOS3) and serum paraoxonase/arylesterase 1 (PON1) were selected as the primary targets of GpM in the treatment of AD. The neuroprotective effect of GPM was related to a variety of pathways, including amoebiasis, HIF-1 signaling pathway, cytokine-cytokine receptor interaction and so on. CONCLUSIONS: Our findings elucidate the active compounds, targets and pathways of GpM involved in effects of anti-AD. The novel mechanism of GpM against AD provides more treatment options for AD.

Influence of Clinically Significant Genes on Antiplatelet Effect of Clopidogrel and Clinical Outcomes in Patients with Acute Coronary Syndrome and Atrial Fibrillation.

INTRODUCTION: The interindividual variability of the antiplatelet effect of clopidogrel is determined by multiple clinical and genetic factors. A lot of genotype-oriented studies have concentrated on the impact of CYP2C19 gene polymorphisms on platelet aggregation in patients receiving clopidogrel. However, the influence of this polymorphism may be only 12-20%, so other genetic markers should also be investigated. The aim of this work was to study the impact of carriage of CES1, PON1, ABCG2, CYP4F2, CYP3A4, IGTB3, P2Y12, PEAR1, and B4GALT2 polymorphisms on antiplatelet effect of clopidogrel and clinical outcomes in patients with acute coronary syndrome (ACS) and atrial fibrillation (AF). METHODS: 103 patients who underwent ACS with or without percutaneous coronary intervention and concomitant nonvalvular AF were included in an open multicenter prospective study to assess efficacy and safety of combined antithrombotic therapy. The study assessed the frequency of different primary clinical outcomes (incidence of major bleeding, hospital mortality, cardiovascular mortality, stroke and transient ischemic attacks (TIAs), renal mortality) and secondary outcomes (resistance to therapy - high residual platelet reactivity, excessive platelet suppression). Residual platelet reactivity was examined using the VerifyNow system (Accumetrics, Latham, NY, USA). RESULTS: None of the studied genetic markers had no statistically significant effect on the antiaggregant response to clopidogrel in patients with ACS and AF. However, CYP4F2 C(Val433Met) T, PEAR1 rs41273215 C>T were statistically significantly associated with an increased frequency of bleeding on antithrombotic therapy. B4GALT2 rs1061781 was statistically significantly associated with increased frequency of strokes and TIA. CONCLUSION: In our study, we determined that carriers of CYP4F2 gene polymorphisms C(Val433Met)T, PEAR1 rs41273215 C>T (CT+TT) were associated with lower safety of antithrombotic therapy in patients with ACS and AF. And, the B4GALT2 rs1061781 gene polymorphism was associated with a greater risk of insufficient efficacy of the therapy. The data obtained in our study may improve the understanding of the effect of less studied genetic markers on the efficacy and safety of antithrombotic therapy in patients with ACS and AF.

Importance of paraoxonase 1 (PON1) as an antioxidant and antiatherogenic enzyme in the cardiovascular complications of type 2 diabetes: Genotypic and phenotypic evaluation.

Oxidant-antioxidant imbalance is involved in the etiology of different diseases, including cardiovascular diseases (CVDs), liver disorders, kidney diseases, cancers and diabetes mellitus. Antioxidant enzymes play a key role in striking an oxidant-antioxidant balance. Moreover, paraoxonase 1 (PON1) is an antioxidant enzyme that binds with high-density lipoprotein (HDL) in the circulation, and antioxidant and antiaterogenic properties of this lipoprotein are significantly associated with PON1. Research suggests PON1 contributes to the pathogenesis of certain human diseases such as type 2 diabetes (T2D). The association between PON1 and T2D appear to be reciprocal so that the disease significantly decreases PON1 levels and in turn, the genetics of PON1 may have a role the risk of susceptibility to T2D. Several factors that reduce the activity and concentration of PON1 in patients with T2D include increased glycation and loss-of-function polymorphisms. The genotypic and phenotypic evaluations of PON1 are therefore crucial for assessing the risk of cardiovascular complications in these patients, and strategies for increasing or restoring PON1 levels are useful for reducing or preventing their cardiovascular complications as their main cause of mortality. The present review aimed at discussing and emphasizing the key role of PON1 in T2D as a silent and dangerous disease.

Efficacy of an organophosphorus hydrolase enzyme (OpdA) in human serum and minipig models of organophosphorus insecticide poisoning.

Objectives: Current therapeutic options for organophosphorus (OP) insecticide self-poisoning including atropine and oximes are inadequate and case fatality may exceed 20%. An OP hydrolase enzyme, OpdA, has been used for environmental cleansing of OP insecticides and prevented death in rat and non-human primate models of OP insecticide poisoning if given very quickly after exposure. We here tested OpdA's ability to break down OP insecticides in human serum and in clinically relevant minipig models of OP insecticide poisoning.Methods: Human serum was spiked with seven diverse WHO Class II OP insecticides (chlorpyrifos, quinalphos, diazinon, dimethoate, fenthion, phenthoate, and profenofos) and the effect of OpdA on degradation measured. The pharmacodynamic and clinical effects of OpdA treatment were studied in Gottingen minipigs orally poisoned with agricultural formulations of dimethoate EC40 or methyl parathion EC60; pharmacodynamic effects were also assessed in profenofos EC50-poisoned pigs.Results: OpdA effectively hydrolysed OP insecticides in human serum, with rates varying from 856 (SD 44) down to 0.107 (SD 0.01) moles of substrate hydrolysed/mole of enzyme/sec (kcat) for quinalphos and phenthoate, respectively, although at rates 2-3 log orders less than found in vitro in buffered solution. It showed clinical benefit in minipig models, reducing the dose of noradrenaline required to sustain an adequate mean arterial pressure after dimethoate (mean 0.149 [SD 0.10] µg/kg/h vs. 1.07 [SD 0.77] µg/kg/h, p < .0001) and methyl parathion (mean 0.077 [SD 0.08] µg/kg/h vs. 0.707 [SD 0.49] µg/kg/h, p < .0001) poisoning. OpdA reduced blood OP insecticide concentration and acetylcholinesterase inhibition after poisoning by dimethoate, methyl parathion, and profenofos insecticides.Conclusions:In vitro incubation of OpdA in human serum showed hydrolysis of diverse OP insecticides, although at lower rates than found in buffer solutions. This activity results in clinical and pharmacodynamic efficacy in vivo against several OP insecticides. These results support the testing of OpdA in further animal models before considering human trials to determine whether it may become an urgently required novel therapeutic agent for OP insecticide self-poisoning.

Is Human Paraoxonase 1 the Saviour Against the Persistent Threat of Organophosphorus Nerve Agents?

Nerve agents have been used extensively in chemical warfare in the past. However, recent use of Novichok agents have reignited the debate on the threat posed by Organophosphorus Nerve Agents (OPNAs). The currently available therapy for OPNA toxicity is only symptomatic and is potentially ineffective in neutralizing OPNAs. Hence, there is a dire need to develop a prophylactic therapy for counteracting OPNA toxicity. In this regard, human paraoxonase 1 has emerged as the enzyme of choice. In this review, we have focussed upon the recent and past events of OPNA use, their mechanism of action and toxicity. Further, we have emphasized upon the potential of enzyme based therapy and the various advances in the development of paraoxonase 1 as a countermeasure for OPNA poisoning. Finally, we have elaborated the shortcomings of paraoxonase 1 and the work that needs to be undertaken in order to develop human paraoxonase 1 as a prophylactic against OPNA poisoning.

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.

Refolded Recombinant Human Paraoxonase 1 Variant Exhibits Prophylactic Activity Against Organophosphate Poisoning.

Organophosphate (OP) compounds are neurotoxic chemicals, and current treatments available for OP-poisoning are considered as unsatisfactory and inadequate. There is an urgent need for the development of more effective treatment(s) for OP-poisoning. Human paraoxonase 1 (h-PON1) is known to hydrolyze a variety of OP-compounds and is a leading candidate for the development of prophylactic and therapeutic agent against OP-poisoning in humans. Non-availability of effective system(s) for the production of recombinant h-PON1 (rh-PON1) makes it hard to produce improved variant(s) of this enzyme and analyze their in vivo efficacy in animal models. Production of recombinant h-PON1 (rh-PON1) using an Escherichia coli expression system is a key to develop variant(s) of h-PON1. Recently, we have developed a procedure to produce active rh-PON1 enzymes by using E. coli expression system. In this study, we have characterized the OP-hydrolyzing properties of refolded rh-PON1(wt) and rh-PON1(H115W;R192K) variant. Our results show that refolded rh-PON1(H115W;R192K) variant exhibit enhanced OP-hydrolyzing activity in in vitro and ex vivo assays and exhibited prophylactic activity in mouse model of OP-poisoning, suggesting that refolded rh-PON1 can be developed as a therapeutic candidate.

Transduced PEP-1-PON1 proteins regulate microglial activation and dopaminergic neuronal death in a Parkinson's disease model.

Parkinson's disease (PD) is an oxidative stress-mediated neurodegenerative disorder caused by selective dopaminergic neuronal death in the midbrain substantia nigra. Paraoxonase 1 (PON1) is a potent inhibitor of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) against oxidation by destroying biologically active phospholipids with potential protective effects against oxidative stress-induced inflammatory disorders. In a previous study, we constructed protein transduction domain (PTD) fusion PEP-1-PON1 protein to transduce PON1 into cells and tissue. In this study, we examined the role of transduced PEP-1-PON1 protein in repressing oxidative stress-mediated inflammatory response in microglial BV2 cells after exposure to lipopolysaccharide (LPS). Moreover, we identified the functions of transduced PEP-1-PON1 proteins which include, mitigating mitochondrial damage, decreasing reactive oxidative species (ROS) production, matrix metalloproteinase-9 (MMP-9) expression and protecting against 1-methyl-4-phenylpyridinium (MPP(+))-induced neurotoxicity in SH-SY5Y cells. Furthermore, transduced PEP-1-PON1 protein reduced MMP-9 expression and protected against dopaminergic neuronal cell death in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mice model. Taken together, these results suggest a promising therapeutic application of PEP-1-PON1 proteins against PD and other inflammation and oxidative stress-related neuronal diseases.

Serum concentration of high density lipoproteins (HDLs) in leishmaniotic dogs.

In order to assess whether the concentration of high density lipoproteins (HDLs) changes in leishmaniotic dogs before and after treatment, HDL cholesterol (HDL-Chol and HDL%), C reactive protein (CRP) and activity of the antioxidant enzyme paraoxonase (PON-1) were measured in sera from 10 controls and 10 leishmaniotic dogs. Seven of these latter were sampled also 3, 7, 14, 21 and 28 days after treatment with antimonials and allopurinol. HDL-chol, and PON-1 were low in leishmaniotic dogs at admission and increased after treatment. HDL-chol and HDL% correlated positively with PON-1 and negatively with CRP suggesting that HDLs decrease through an oxidative mechanism. Therefore, HDLs may be used to monitor the magnitude of oxidation associated with inflammation in leishmaniotic dogs.

An alternative pseudolikelihood method for multivariate random-effects meta-analysis.

Recently, multivariate random-effects meta-analysis models have received a great deal of attention, despite its greater complexity compared to univariate meta-analyses. One of its advantages is its ability to account for the within-study and between-study correlations. However, the standard inference procedures, such as the maximum likelihood or maximum restricted likelihood inference, require the within-study correlations, which are usually unavailable. In addition, the standard inference procedures suffer from the problem of singular estimated covariance matrix. In this paper, we propose a pseudolikelihood method to overcome the aforementioned problems. The pseudolikelihood method does not require within-study correlations and is not prone to singular covariance matrix problem. In addition, it can properly estimate the covariance between pooled estimates for different outcomes, which enables valid inference on functions of pooled estimates, and can be applied to meta-analysis where some studies have outcomes missing completely at random. Simulation studies show that the pseudolikelihood method provides unbiased estimates for functions of pooled estimates, well-estimated standard errors, and confidence intervals with good coverage probability. Furthermore, the pseudolikelihood method is found to maintain high relative efficiency compared to that of the standard inferences with known within-study correlations. We illustrate the proposed method through three meta-analyses for comparison of prostate cancer treatment, for the association between paraoxonase 1 activities and coronary heart disease, and for the association between homocysteine level and coronary heart disease.

Paraoxonase-2 (PON2) in brain and its potential role in neuroprotection.

Paraoxonase 2 (PON2) is a member of a gene family which also includes the more studied PON1, as well as PON3. PON2 is unique among the three PONs, as it is expressed in brain tissue. PON2 is a lactonase and displays anti-oxidant and anti-inflammatory properties. PON2 levels are highest in dopaminergic regions (e.g. striatum), are higher in astrocytes than in neurons, and are higher in brain and peripheral tissues of female mice than male mice. At the sub-cellular level, PON2 localizes primarily in mitochondria, where it scavenges superoxides. Lack of PON2 (as in PON2(-/-) mice), or lower levels of PON2 (as in male mice compared to females) increases susceptibility to oxidative stress-induced toxicity. Estradiol increases PON2 expression in vitro and in vivo, and provides neuroprotection against oxidative stress. Such neuroprotection is not present in CNS cells from PON2(-/-) mice. Similar results are also found with the polyphenol quercetin. PON2, given its cellular localization and antioxidant and anti-inflammatory actions, may represent a relevant enzyme involved in neuroprotection, and may represent a novel target for neuroprotective strategies. Its differential expression in males and females may explain gender differences in the incidence of various diseases, including neurodevelopmental, neurological, and neurodegenerative diseases.

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.

Human PON1, a biomarker of risk of disease and exposure.

Human paraoxonase 1 (PON1) is a high-density lipoprotein (HDL)-associated serum enzyme that exhibits a broad substrate specificity. In addition to protecting against exposure to some organophosphorus (OP) pesticides by hydrolyzing their toxic oxon metabolites, PON1 is important in protecting against vascular disease by metabolizing oxidized lipids. Recently, PON1 has also been shown to play a role in inactivating the quorum sensing factor N-(3-oxododecanoyl)-l-homoserine lactone (3OC12-HSL) of Pseudomonas aeruginosa. Native, untagged engineered recombinant human PON1 (rHuPON1) expressed in Escherichia coli and purified by conventional column chromatographic purification is stable, active, and capable of protecting PON1 knockout mice (PON1(-/-)) from exposure to high levels of the OP compound diazoxon. The bacterially derived rHuPON1 can be produced in large quantities and lacks the glycosylation of eukaryotic systems that can produce immunogenic complications when inappropriately glycosylated recombinant proteins are used as therapeutics. Previous studies have shown that the determination of PON1 status, which reveals both PON1(192) functional genotype and serum enzyme activity level, is required for a meaningful evaluation of PON1's role in risk of disease or exposure. We have developed a new two-substrate assay/analysis protocol that provides PON1 status without use of toxic OP substrates, allowing for use of this protocol in non-specialized laboratories. Factors were also determined for inter-converting rates of hydrolysis of different substrates. PON1 status also plays an important role in revealing changes in HDL-associated PON1 activities in male patients with Parkinson disease (PD). Immunolocalization studies of PONs 1, 2 and 3 in nearly all mouse tissues suggest that the functions of PONs 1 and 3 extend beyond the plasma and the HDL particle.

Effect of efavirenz on high-density lipoprotein antioxidant properties in HIV-infected patients.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT: * In previous work, we showed a long-term and concentration-dependent beneficial effect of the non-nucleoside reverse transcriptase inhibitor efavirenz (EFV) on high-density lipoproteins (HDL) in human immunodeficiency virus (HIV)-infected patients. * Furthermore, it has been suggested that instead of the current practice of only measuring HDL-chelesterol values, the evaluation of HDL function, namely its antioxidant properties, might be an improved tool for identifying subjects at increased risk for cardiovascular events. * Paraoxonase-1 (PON-1) is an enzyme associated with HDL that is responsible for HDL antioxidant function. WHAT THIS STUDY ADDS: * In the present work, we studied the effect of EFV on the activity of PON-1 and showed, for the first time, that EFV-based antiretroviral therapy is associated with a better antioxidant function, i.e. with a higher PON-1 activity. AIMS: A long-term and concentration-dependent beneficial effect of efavirenz (EFV) on cholesterol associated with high-density lipoprotein (HDL-c) in human immunodeficiency virus (HIV)-infected patients has been documented. Furthermore, it has been suggested that, instead of the current practice of only measuring HDL-c values, the evaluation of HDL quality might be an improved tool for identifying subjects at increased risk of cardiovascular events. Paraoxonase-1 (PON-1) is an enzyme associated with HDL that is involved in the onset of cardiovascular disease and responsible for HDL antioxidant function. The aim of the present study was to investigate the effect of EFV on the circulating activity of PON-1 in HIV-infected patients. METHODS: The patients included were adults with a documented HIV-1 infection, nontreated or treated with antiretroviral regimens including EFV 600 mg once daily as first therapeutic regimen for at least 3 months. The influence of treatment with EFV, HDL-c and CD4 cell count on PON-1 activity was analysed. RESULTS: HIV-infected White patients treated with EFV had higher PON-1 activity [77.35 U l(-1) (65.66, 89.04)] (P < 0.05) and higher PON-1 activity : HDL-c ratio [1.88 (1.49, 2.28)] (P < 0.01) than untreated patients. PON-1 activity was higher in Black patients (P < 0.001) and in patients with a CD4 cell count >500 cells ml(-1) (P= 0.0120). CONCLUSIONS: EFV-based antiretroviral regimens are associated with HDL particles with a better antioxidant function, i.e. with a higher PON-1 activity. The PON-1 activity of Black patients is higher than that found in Whites regardless of treatment. Ethnicity should be taken into consideration when studying drug effects on PON-1 activity.