Bis 4-nitrophenyl Phosphate Research Articles

Biotransformation and brain distribution of the anti-COVID-19 drug molnupiravir and herb-drug pharmacokinetic interactions between the herbal extract Scutellaria formula-NRICM101

The aim of this study was to explore the effects of herbal drug pharmacokinetic interactions on the biotransformation of molnupiravir and its metabolite β-D-N4-hydroxycytidine (NHC) in the blood and brain. To investigate the biotransformation mechanism, a carboxylesterase inhibitor, bis(4-nitrophenyl)phosphate (BNPP), was administered. Not only molnupiravir but also the herbal medicine Scutellaria formula-NRICM101 is potentially affected by coadministration with molnupiravir. However, the herb-drug interaction between molnupiravir and the Scutellaria formula-NRICM101 has not yet been investigated. We hypothesized that the complex bioactive herbal ingredients in the extract of the Scutellaria formula-NRICM101, the biotransformation and penetration of the bloodbrain barrier of molnupiravir are altered by inhibition of carboxylesterase. To monitor the analytes, ultrahigh-performance liquid chromatography tandem mass spectrometry (UHPLCMS/MS) coupled with the microdialysis method was developed. Based on the dose transfer from humans to rats, a dose of molnupiravir (100 mg/kg, i.v.), molnupiravir (100 mg/kg, i.v.) + BNPP (50 mg/kg, i.v.), and molnupiravir (100 mg/kg, i.v.) + the Scutellaria formula-NRICM101 extract (1.27 g/kg, per day, for 5 consecutive days) were administered. The results showed that molnupiravir was rapidly metabolized to NHC and penetrated into the brain striatum. However, when concomitant with BNPP, NHC was suppressed, and molnupiravir was enhanced. The blood-to-brain penetration ratios were 2% and 6%, respectively. In summary, the extract of the Scutellaria formula-NRICM101 provides a pharmacological effect similar to that of the carboxylesterase inhibitor to suppress NHC in the blood, and the brain penetration ratio was increased, but the concentration is also higher than the effective concentration in the blood and brain.

Hydrolysis of dibutyl phthalate and di(2-ethylhexyl) phthalate in human liver, small intestine, kidney, and lung: An in vitro analysis using organ subcellular fractions and recombinant carboxylesterases

Phthalates are widely used plasticizers that are primarily and rapidly metabolized to monoester phthalates in mammals. In the present study, the hydrolysis of dibutyl phthalate (DBP) and di(2-ethylhexyl) phthalate (DEHP) in the human liver, small intestine, kidney, and lung was examined by the catalytic, kinetic, and inhibition analyses using organ microsomal and cytosolic fractions and recombinant carboxylesterases (CESs). The Vmax (y-intercept) values based on the Eadie-Hofstee plots of DBP hydrolysis were liver > small intestine > kidney > lung in microsomes, and liver > small intestine > lung > kidney in cytosol, respectively. The CLint values (x-intercept) were small intestine > liver > kidney > lung in both microsomes and cytosol. The Vmax and CLint or CLmax values of DEHP hydrolysis were small intestine > liver > kidney > lung in both microsomes and cytosol. Bis(4-nitrophenyl) phosphate (BNPP) effectively inhibited the activities of DBP and DEHP hydrolysis in the microsomes and cytosol of liver, small intestine, kidney, and lung. Although physostigmine also potently inhibited DBP and DEHP hydrolysis activities in both the microsomes and cytosol of the small intestine and kidney, the inhibitory effects in the liver and lung were weak. In recombinant CESs, the Vmax values of DBP hydrolysis were CES1 (CES1b, CES1c) > CES2, whereas the CLmax values were CES2 > CES1 (CES1b, CES1c). On the other hand, the Vmax and CLmax values of DEHP hydrolysis were CES2 > CES1 (CES1b, CES1c). These results suggest an extensive organ-dependence of DBP and DEHP hydrolysis due to CES expression, and that CESs are responsible for the metabolic activation of phthalates.

A UHPLC-QTOF-MS/MS method with a superimposed multiple product ion strategy and esterase inhibitor improved sensitivity for the determination of xylocarpin H in rat plasma

Xylocarpin H is a natural limonoid that has the potential to be a viable therapeutic candidate whether used alone or in combination with other medications. However, the pharmacokinetic properties of xylocarpin H are still not well understood and require further investigation. Sensitive analysis of xylocarpin H using liquid chromatography tandem triple-quadrupole mass spectrometry is challenging due to its structural feature. In the present research, a sensitive UHPLC-QTOF-MS/MS method was developed and validated to determine xylocarpin H in rat plasma. After simple methanol protein precipitation pretreatment with 50 mM esterase inhibitor bis(4-nitrophenyl)-phosphate (BNPP), xylocarpin H and Schisandrin (internal standard) were separated on a Synergi 4 u fusion-RP C18 column (50 mm × 3.0 mm, 4 µm) using isocratic elution with a mobile phase consisting of formic acid (0.1%) and acetonitrile, which were detected only within 3 min. The QTOF-MS/MS detection, which is a high-resolution mode, can accurately quantify analytes (ppm<5). The peak area of xylocarpin H was determined by adding the single peak areas of m/z 483.2013, m/z 465.1930 and m/z 345.1333 as the superimposed multiple product ions (SMPI) mode for data processing with optimized the high-resolution extraction widths of fragment ions at ± 0.05 Da. The S/N of the SMPI mode was found to be approximately two to seven times greater than the independent product ion mode. The linearity, precision, accuracy, and stability were completely validated for the method. Finally, the validated method was employed for the pharmacokinetic study of xylocarpin H in rats. The study indicated that the optimized high-resolution extraction widths of fragment ions and SMPI mode for data processing significantly improve the quantitative sensitivity of QTOF-MS/MS. In addition, esterase inhibitor BNPP was added in the process of sample treatment to inhibit the degradation of xylocarpin H in rat blood, which further improved the sensitivity of the quantitative method.

Promiscuous Catalytic Activity of a Binuclear Metallohydrolase: Peptide and Phosphoester Hydrolyses.

In this study, chemical promiscuity of a binuclear metallohydrolase Streptomyces griseus aminopeptidase (SgAP) has been investigated using DFT calculations. SgAP catalyzes two diverse reactions, peptide and phosphoester hydrolyses, using its binuclear (Zn-Zn) core. On the basis of the experimental information, mechanisms of these reactions have been investigated utilizing leucine p-nitro aniline (Leu-pNA) and bis(4-nitrophenyl) phosphate (BNPP) as the substrates. The computed barriers of 16.5 and 16.8 kcal/mol for the most plausible mechanisms proposed by the DFT calculations are in good agreement with the measured values of 13.9 and 18.3 kcal/mol for the Leu-pNA and BNPP hydrolyses, respectively. The former was found to occur through the transfer of two protons, while the latter with only one proton transfer. They are in line with the experimental observations. The cleavage of the peptide bond was the rate-determining process for the Leu-pNA hydrolysis. However, the creation of the nucleophile and its attack on the electrophile phosphorus atom was the rate-determining step for the BNPP hydrolysis. These calculations showed that the chemical nature of the substrate and its binding mode influence the nucleophilicity of the metal bound hydroxyl nucleophile. Additionally, the nucleophilicity was found to be critical for the Leu-pNA hydrolysis, whereas double Lewis acid activation was needed for the BNPP hydrolysis. That could be one of the reasons why peptide hydrolysis can be catalyzed by both mononuclear and binuclear metal cofactors containing hydrolases, while phosphoester hydrolysis is almost exclusively by binuclear metallohydrolases. These results will be helpful in the development of versatile catalysts for chemically distinct hydrolytic reactions.

Metal-free synthesis and self-assembly of poly(ethylene glycol) methyl ether-block-poly(ε-decalactone)-block-poly(methyl methacrylate) triblock terpolymers

Derived from sustainable sources, ε-decalactone (ε-DL) emerges as a renewable alternative to synthetic cyclic esters. However, its copolymerization with different monomers often requires using metal complexes potentially toxic to humans and the environment. Here, we report the synthesis and self-assembly of poly(ethylene glycol) methyl ether-block-poly(ε-decalactone)-block-poly(methyl methacrylate) (mPEG-block-PDL-block-PMMA) triblock terpolymers via two synthetic pathways using a simple phosphazene- or phosphate-based metal-free catalytic system. Our size exclusion chromatography (SEC) results showed that the two-step synthetic mechanism combining bis(4-nitrophenyl) phosphate (BNPP) and 1-tert-butyl-4,4,4-tris(dimethylamino)-2,2-bis[tris(dimethylamino)-phosphoranylidenamino]-2λ5,4λ5-catenadi(phosphazene) (t-BuP4) provided a better polymerization control than sequential copolymerization. In addition, our SAXS and AFM analysis also demonstrated that the blocks are incompatible in the terpolymers, further promoting their self-assembly into multicompartment micelles in water, as confirmed by cryo-TEM. Therefore, mPEG-block-PDL-block-PMMA triblock terpolymers may be used as both environmentally friendly alternatives to metal-catalyzed polymers and drug delivery systems.

Pig Liver Esterases Hydrolyze Endocannabinoids and Promote Inflammatory Response.

Endocannabinoids are endogenous ligands of cannabinoid receptors and activation of these receptors has strong physiological and pathological significance. Structurally, endocannabinoids are esters (e.g., 2-arachidonoylglycerol, 2-AG) or amides (e.g., N-arachidonoylethanolamine, AEA). Hydrolysis of these compounds yields arachidonic acid (AA), a major precursor of proinflammatory mediators such as prostaglandin E2. Carboxylesterases are known to hydrolyze esters and amides with high efficiency. CES1, a human carboxylesterase, has been shown to hydrolyze 2-AG, and shares a high sequence identity with pig carboxylesterases: PLE1 and PLE6 (pig liver esterase). The present study was designed to test the hypothesis that PLE1 and PLE6 hydrolyze endocannabinoids and promote inflammatory response. Consistent with the hypothesis, purified PLE1 and PLE6 efficaciously hydrolyzed 2-AG and AEA. PLE6 was 40-fold and 3-fold as active as PLE1 towards 2-AG and AEA, respectively. In addition, both PLE1 and PLE6 were highly sensitive to bis(4-nitrophenyl) phosphate (BNPP), an aryl phosphodiester known to predominately inhibit carboxylesterases. Based on the study with BNPP, PLEs contributed to the hydrolysis of 2-AG by 53.4 to 88.4% among various organs and cells. Critically, exogenous addition or transfection of PLE6 increased the expression and secretion of proinflammatory cytokines in response to the immunostimulant lipopolysaccharide (LPS). This increase was recapitulated in cocultured alveolar macrophages and PLE6 transfected cells in transwells. Finally, BNPP reduced inflammation trigged by LPS accompanied by reduced formation of AA and proinflammatory mediators. These findings define an innovative connection: PLE-endocannabinoid-inflammation. This mechanistic connection signifies critical roles of carboxylesterases in pathophysiological processes related to the metabolism of endocannabinoids.

Phosphoester hydrolysis promoted by quinoline functionalized Ni(II) and Zn(II) complexes

A quinoline functionalized nitrogen-based bidentate ligand (L) was synthesized and used for the preparation of mononuclear Ni(II) and Zn(II) complexes which differ only by the halide ion attached to the respective metal center. All complexes of this series show closely related crystal packing regardless of the halide ion and metal center (Ni/Zn). All the complexes were characterized by different spectroscopic techniques such as UV-vis, emission, IR and NMR spectroscopies, ESI-MS, single-crystal XRD and thermogravimetry. All the complexes were examined for hydrolysis of a phosphoester bond in the DNA-model substrate 4-nitrophenyl phosphate (NPP) and bis(4-nitrophenyl)phosphate (BNPP). All complexes were able to hydrolyze BNPP and NPP and showed better rate of hydrolysis with diesters as compared to monoesters.

Biomarker responses and metabolism in Lumbricus terrestris exposed to drugs of environmental concern, an in vivo and in vitro approach

The earthworm Lumbricus terrestris is an anecic species living in natural soils but it is also a sentinel in pollution monitoring. Specimens of L.terrestris were exposed for 48 h though the filter paper contact test at 1 mg/mL of the chemicals: Lamotrigine (LMG), Cocaine (COC), Fipronil (FIP) and the pesticide bis-4-nitrophenyl phosphate (BNPP). After that period, the activities of Acetylcholinesterase, Glutathione S-transferase, Carboxylesterase (CE) using different substrates, and lipid peroxidation levels were evaluated in the exposed whole tissue earthworms. The results revealed differences only in CE activity, with 4-nitrophenyl butyrate (4NPB) and 1-naphthyl butyrate (1NB) the most responsive substrates to COC. The kinetic parameters of CE were characterized, for the first time, in whole tissue of this species. The chemical analysis by LC-MS/MS, confirmed the exposure to the parent compounds, identified metabolites and evidenced biotransformation pathways in earthworms. Metabolic reactions included oxidation (LMG and FIP), hydrolysis (COC and FIP) as well as glycosylation (LMG, COC and FIP). A hitherto unknown metabolite of LMG due to the conjugation with phenylalanine glutamine was formed. The in vivo results on CE activity with the specific inhibitor, BNPP, were confirmed in vitro. Moreover, in the in vitro approach, the inclusion of other contaminants of environmental concern supports the potential of CE as biomarker. This study identifies the main metabolites formed by earthworms for further in vivo exposures under more realistic conditions and the potential use of CE measures as biomarker of emerging contaminants.

Copper(II) Complexes with Tetradentate Piperazine-Based Ligands: DNA Cleavage and Cytotoxicity

Five-coordinate Cu(II) complexes, [Cu(Ln)X]ClO4/PF6, where Ln = piperazine ligands bearing two pyridyl arms and X = ClO4− for Ln = L1 (1-ClO4), L2 (2-ClO4), L3 (3-ClO4), and L6 (6-ClO4) as well as [Cu(Ln)Cl]PF6 for Ln = L1 (1-Cl), L4 (4-Cl), and L5 (5-Cl) have been synthesized and characterized by spectroscopic techniques. The molecular structures of the last two complexes were determined by X-ray crystallography. In aqueous acetonitrile solutions, molar conductivity measurements and UV-VIS spectrophotometric titrations of the complexes revealed the hydrolysis of the complexes to [Cu(Ln)(H2O)]2+ species. The biological activity of the Cu(II) complexes with respect to DNA cleavage and cytotoxicity was investigated. At micromolar concentration within 2 h and pH 7.4, DNA cleavage rate decreased in the order: 1-Cl ≈ 1-ClO4 &gt; 3-ClO4 ≥ 2-ClO4 with cleavage enhancements of up to 23 million. Complexes 4-Cl, 5-Cl, and 6-ClO4 were inactive. In order to elucidate the cleavage mechanism, the cleavage of bis(4-nitrophenyl)phosphate (BNPP) and reactive oxygen species (ROS) quenching studies were conducted. The mechanistic pathway of DNA cleavage depends on the ligand’s skeleton: while an oxidative pathway was preferable for 1-Cl/1-ClO4, DNA cleavage by 2-ClO4 and 3-ClO4 predominantly proceeds via a hydrolytic mechanism. Complexes 1-ClO4, 3-ClO4, and 5-Cl were found to be cytotoxic against A2780 cells (IC50 30–40 µM). In fibroblasts, the IC50 value was much higher for 3-ClO4 with no toxic effect.

Efficient Catalysis of Phosphate Ester Hydrolysis by Bare Silica

This work reports the catalysis of bare silica materials (MCM-41, SBA-15, and silica gel) for the complete hydrolyses of bis(2,4-nitrophenyl)phosphate (BDNPP) and bis(4-nitrophenyl)phosphate (BNPP)...

Flame retardancy and smoke suppression effect of bis(4-nitrophenyl) phosphate modified layered double hydroxides derived from red mud in thermoplastic polyurethanes

Red mud-based layered double hydroxides (LRM) was synthesized by a co-precipitation route, and then a hybrid with bis(4-nitrophenyl) phosphate (BNPP)-modified LRM material (BRL) was prepared in this paper. Through X-ray diffraction(XRD), Fourier transform infrared spectra (FTIR), nuclear magnetic resonance spectra (NMR), transmission electron microscope (TEM) and energy dispersive spectrometer (EDS) analysis, the results show that BRL is better dispersed than LRM. Then, thermoplastics polyurethanes (TPU) was used as the base material. TPU/LRM and TPU/BRL composites were prepared by a melt-blending method. Cone calorimeter test (CCT) was used to investigate the effect on flame retardancy and smoke suppression. Specifically, the PHRR values of TPU/LRM and TPU/BRL composites were reduced by 41.6% and 52.5% compared with pure TPU sample. The main factor of improved flame retardancy and smoke suppression can be explained by the catalytic carbonization of LRM or BRL in solid phase and the P-radicals (such as PO· and HPO·) in gas phase which demonstrated by thermo-gravimetric analyzer test (TGA) and char analysis.

Effect of Chemically Distinct Substrates on the Mechanism and Reactivity of a Highly Promiscuous Metallohydrolase

In this DFT study, the substrate promiscuity of the binuclear [Fe(II)-Zn(II)] core containing glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes has been investigated through the hydrolysis of three chemically diverse groups of substrates: i.e., phosphomono-, phosphodi-, and phosphotriesters. The hydrolysis of these substrates is studied by comparing stepwise, concerted, and substrate-assisted mechanisms. Both the stepwise and concerted mechanisms occur with similar barriers, while the energetics for the substrate-assisted mechanism are significantly less favorable. Irrespective of the mechanism, active site residue His217 plays a critical role, in agreement with structural, kinetics, and spectroscopic data, but the transition state of the reaction depends on the identity of the substrate (dissociative for the triester paraoxon, associative for the monoester 4-nitrophenyl phosphate (NPP), and in-between for the diesters glycerol-3-phosphoethanolamine (GPE) and bis(4-nitrophenyl)phosphate (BNPP)). In good agreement with available kinetic and spectrophotometric data, the calculations highlight the preference of GpdQ for diester substrates, followed by tri- and monoesters. For substrates with two different types of scissile bonds (paraoxon and GPE) a clear preference for the bond with the stronger electron withdrawing leaving group was observed. The extensive agreement between experimental data and DFT calculations enhances the understanding of the mechanism of GpdQ-catalyzed hydrolysis and paves the way for the rational design of optimized catalysts for the hydrolysis of different types of phosphoesters.

Fabrication of highly active phosphatase-like fluorescent cerium-doped carbon dots for in situ monitoring the hydrolysis of phosphate diesters.

Ce-Doped carbon dots (CeCDs) were fabricated via a one-step hydrothermal carbonization using Ce(NO3)3·6H2O and EDTA·2H2O as precursors, and various experimental techniques were employed to characterize the morphology, structure and composition of the as-obtained CeCDs. Using the disodium salt of bis(4-nitrophenyl)phosphate (BNPP) as a DNA model substrate, mimetic phosphatase activity toward phosphate ester hydrolysis cleavage was investigated. It was found that similar to the catalytic character of the natural enzyme, CeCDs as a hydrolase mimic can exhibit a good catalytic activity for promoting BNPP hydrolysis, in which metal Ce(iii) acts as a center for binding and activation of the metal-bound hydroxide complex as well as a source of nucleophilic metal hydroxides. Additionally, based on the Inner Filter Effect (IFE), fluorescence spectra can be used to monitor the hydrolysis of BNPP using CeCDs, which mimic phosphatase. Thus, the unique properties of the CeCD mimetic phosphatases as well as the IFE sensing strategy would provide an ideal platform to monitor the catalytic phosphate ester hydrolysis processes. Finally, to validate the availability of the established catalytic systems, the CeCDs were further applied for degradation of organophosphorus pesticide chlorpyrifos. The degradation efficiency was estimated to be a satisfactory value of 74.50%, exhibiting a potential application prospect for treatment of the pollutants in the soil and water.

Role of esterase mediated hydrolysis of simvastatin in human and rat blood and its impact on pharmacokinetic profiles of simvastatin and its active metabolite in rat

Simvastatin is known as a pro-drug, which could be hydrolyzed by esterases to its active form, simvastatin acid. Although pharmacokinetics of simvastatin and simvastatin acid have been widely studied, hydrolysis of simvastatin to simvastatin acid during blood sampling and plasma preparation has been overlooked in the previous studies, leading to underestimation of simvastatin concentration and overestimation of simvastatin acid concentration in plasma. Since both efficacy and adverse drug reaction of simvastatin are highly dependent on simvastatin and simvastatin acid concentrations in vivo, accurate assessment of the two compounds are critical in their pharmacokinetic and pharmacodynamic studies. The current study was proposed aiming to investigate the esterase mediated hydrolysis of simvastatin in human and rat blood and its impact on the pharmacokinetic study of simvastatin and simvastatin acid. Using various esterase inhibitors including potassium florid (KF), bis(4-nitrophenyl) phosphate (BNPP), and ethylenediaminetetraacetic acid (EDTA), carboxylesterase was found to be the major esterase that hydrolyzed simvastatin in rat blood, while carboxylesterase and paraoxonase were the major esterases mediating the hydrolysis of simvastatin in human blood. Further studies using human recombinant enzymes identified simvastatin as substrates of PON1, CES1b, PON3 and CES1c with Clint of 8.75, 5.77, 3.93, and 2.45 μL/min/mg protein. Therefore, inhibition treatments with 20 mM BNPP and 50 mM KF/ 10 mM EDTA were developed to efficiently prevent the hydrolysis of simvastatin during blood sampling and plasma preparation in rat/human. The subsequent pharmacokinetics of orally administered simvastatin at 8.66 mg/kg in rats found that the Cmax and AUC0-∞ of simvastatin in absence of such esterase inhibitors in the blood sampling process were only 17.04 ± 6.60% and 15.30 ± 6.76% of those in presence of the inhibitors, whereas the Cmax and AUC0-∞ of simvastatin acid were 1.60 ± 0.30 and 1.80 ± 0.22 times of that obtained in presence of the inhibitors. Nevertheless, T1/2 of simvastatin and simvastatin acid remained the same regardless of the blood sampling method. Our current study for the first time demonstrated the importance for assessment of simvastatin stability during the blood sampling and plasma preparation process, which may be applicable to therapeutic drug monitoring of not only simvastatin but also other pro-drugs/compounds sharing similar metabolic properties.

123I-iomazenil whole-body imaging to detect hepatic carboxylesterase drug-metabolizing enzyme activity.

Drugs are mainly metabolized by hepatic enzymes, the activity of which can differ between individuals. Although it is ideal to measure the hepatic clearance of liver-targeted drugs in individualized medicine, blood enzyme tests typically measure metabolic drug clearance in the entire body, and not just in the liver. We investigated whether I-iomazenil imaging can directly assess and quantify the activity of hepatic drug-metabolizing enzymes. Hepatic enzymes that metabolize I-iomazenil were identified by thin-layer chromatography in mouse liver homogenates with bis(4-nitrophenyl) phosphate (BNPP) inhibitor for carboxylesterase enzymes and nicotinamide adenine dinucleotide phosphate (NADPH) generator for cytochrome P450 enzymes. Whole-body images of mice were acquired using I-iomazenil with and without BNPP, and the distribution was also obtained. The metabolism of I-iomazenil in the blood, liver, gall bladder, and bladder was investigated by thin-layer chromatography. From the in-vitro metabolism of I-iomazenil using BNPP, the enzyme converting I-iomazenil to I-R-COOH was identified as carboxylesterase, and that converting I-iomazenil to M2 was identified as cytochrome P450 in experiments with and without an NADPH generator. The biological distribution and whole-body imaging showed increased accumulation in the liver of mice administered BNPP compared with normal mice, but decreased levels in the gall bladder and small intestine. The main fraction in bile and urine was I-R-COOH, with two unknown metabolites (M1 and M2), I, and I-iomazenil also being present. I-iomazenil whole-body imaging has good possibility of direct measurement of hepatic carboxylesterase activity as accumulation of I-R-COOH in the gall bladder through bile and in the bladder through urine.

New Zn(II) complexes based on biologically active substituted acetic acid and nitrogen donor ligands: synthesis, crystal structure and biological applications

The complexes [Zn(phenylacetato)2(2-aminopyridin)2] (3), [Zn(phenylacetato)2(1,10-phenanthroline)]·H2O (4), and [Zn(phenylacetato)2(2,9-dimethyl-1,10-phenanthroline)]·0.5 H2O (5) were prepared and characterized by IR-, UV–Visible, 1H and 13C NMR spectroscopy, and single crystal X-ray diffraction. BNPP hydrolysis of the complexes and their parent nitrogen ligands showed that the hydrolysis rate of bis-(4-nitrophenyl) phosphate (BNPP) was 1.7 × 105 L mol−1 s−1 for 3, 3.1 × 105 L mol−1 s−1 for 4 and 4.3 × 104 L mol−1 s−1 for 5. Antibacterial activities show the effect of complexation on activity against Gram-positive (S. epidermidis, S. aureus, E. faecalis, M. luteus and B. subtilis) and Gram-negative (K. pneumonia, E. coli, P. mirabilis and P. aeruginosa) bacteria using the agar well diffusion method. Complex 4 showed good activity against G− bacteria except P. aeruginosa, and against G+ bacteria except E. ferabis. Complex 5 showed no activity against G− bacteria, low activity against M. luteus and B. subtilis bacteria and high activity against S. epidemidis and S. aureus. Complex 3 did not show any activity against G− or G+ bacteria.

Degradation of bis-p-nitrophenyl phosphate using zero-valent iron nanoparticles

Phosphate esters are employed in some agrochemical formulations and have long life time in the Environment. They are neurotoxic to mammals and it is very difficult to hydrolyze them. It is easy to find papers in the literature dealing with transition metal complexes used in the hydrolysis processes of organophosphorous compounds. However, there are few reports related with degradation of phosphate esters with inorganic nanoparticles. In this work bis-4-nitrophenyl phosphate (BNPP) was used as an agrochemical agent model. The BNPP interaction with zero-valent iron nanoparticles (ZVI NPs), in aqueous media, was searched. The concentration of BNPP was 1000 times higher than the ZVI NPs concentration. The average size of the used iron nanoparticles was 10.2 ± 3.2 nm. The BNPP degradation process was monitored by means of UV-visible method. Initially, the BNPP hydrolysis happens through the P-O bonds breaking-off under the action of the ZVI NPs. Subsequently, the nitro groups were reduced to amine groups. The overall process takes place in 10 minutes. The reaction products were identified employing standard substances in adequate concentrations. The iron by-products were isolated and characterized by X-RD. These iron derivatives were identified as magnetite (Fe3O4) and/or maghemite (γ-Fe2O3) and lepidocrocite (γ-FeOOH). A suggested BNPP degradation mechanism will be discussed.

Simultaneous determination of curcumin diethyl disuccinate and its active metabolite curcumin in rat plasma by LC–MS/MS: Application of esterase inhibitors in the stabilization of an ester-containing prodrug

Four esterase inhibitors, ethylenediamine tetraacetic acid disodium (Na2EDTA), sodium fluoride (NaF), bis(4-nitrophenyl) phosphate (BNPP) and phenylmethanesulfonyl fluoride (PMSF), were evaluated for their inhibitory effects on enzymatic hydrolysis of labile phenolate esters in curcumin diethyl disuccinate (CDD), a prodrug of curcumin (CUR), in rat plasma. BNPP and PMSF at 10mM exhibited stabilization by preventing degradation of CDD. BNPP at a final concentration of 10mM was subsequently selected to prevent ex vivo metabolism of CDD throughout LC-MS/MS analysis of CDD and CUR in rat plasma. A simple protein precipitation technique using acetonitrile as a precipitating agent was used to extract CDD, CUR and dimethylcurcumin (DMC), an internal standard, from rat plasma. Chromatographic separation was performed on a Halo C8 column (4.6×50mm, 2.7μm) using an isocratic mobile phase containing acetonitrile-0.2% formic acid in water (73:27v/v) with a flow rate of 0.4mLmin(-1). An AB SCIEX QTRAP(®) 6500 mass spectrometer was operated using a positive ion electrospray mode for ionization and detection of analytes and internal standard. Calibration curves for CDD and CUR were established using 50μL of rat plasma over the concentration range of 1-500ngmL(-1). The developed method was fully validated according to US Food and Drug Administration (FDA) guidelines for selectivity, sensitivity, linearity, accuracy, precision, dilution integrity, recovery, matrix effect, and stability. The validated method was applied to evaluate the pharmacokinetics of CDD and CUR in rats after a single intravenous dose of 40mgkg(-1). The method using BNPP as an esterase inhibitor was successful in determining the remaining CDD in rat plasma. The pharmacokinetic results indicate that CDD in rats is converted instantaneously to CUR after intravenous administration and a higher CUR plasma concentration at 5min is achieved in comparison with direct intravenous injection of CUR.

Effective cleavage of phosphodiester promoted by the zinc(II) and copper(II) inclusion complexes of β-cyclodextrin

To construct the model of metallohydrolase, two inclusion complexes [MLCl2(β-CD)] (1, M=Zn(II); 2, M=Cu(II); L=N,N′-bis(2-pyridylmethyl)amantadine; β-CD=β-cyclodextrin) were synthesized by mixing β-CDs with the pre-synthesized complexes G1, [ZnLCl2] and G2, [CuLCl2]. Structures of G1, G2, 1 and 2 were characterized by X-ray crystallography, respectively. In solution, two chloride anions of G1 and G2 underwent ligand exchange with solvent molecules according to ESI-MS analysis. The chemical equilibrium constants were determined by potentiometric pH titration. The kinetics of bis(4-nitrophenyl) phosphate (BNPP) hydrolysis catalyzed by G1, G2, 1 and 2 were examined at pHs ranging from 7.50 to 10.50 at 308±0.1K. The pH profile of rate constant of BNPP hydrolysis catalyzed by 1 exhibited an exponential increase with the second-order rate constant of 2.68×10−3M−1s−1 assigned to the di-hydroxo species, which was approximately an order of magnitude higher than those of reported mono-Zn(II)-hydroxo species. The high reactivity was presumably hydroxyl-rich microenvironment provided by β-CDs, which might effect in stabilizing either the labile zinc-hydroxo species or the catalytic transition state.

Abstract 4806: Enhancing the efficacy of tosedostat through carboxylesterase induction

Abstract Malignant cells, including acute myeloid leukaemia (AML), have high protein turnover to support their accelerated cell growth. Aminopeptidases regulate proteolysis, supplying free amino acids for new protein synthesis. Inhibition of aminopeptidases depletes free amino acids, impairing new protein production, leading to impaired cell growth and proliferation. Hence, aminopeptidase inhibition is of particular interest as a therapeutic strategy for AML. Tosedostat (CHR-2797) is an aminopeptidase inhibitor which is converted to its active metabolite, CHR-79888, by intracellular carboxylesterases (CES). This active metabolite is poorly membrane permeable, resulting in intracellular accumulation. Tosedostat has shown promise as a potential therapeutic strategy for AML and is well-tolerated in adult patients. However, its efficacy in paediatric AML has not been established. We set out to investigate the use of tosedostat for paediatric AML and enhance its efficacy through CES1 induction. It is hypothesised that inducing CES will lead to increased CHR-79888 production and subsequently, increased tosedostat efficacy. The IC50 of tosedostat across 11 AML cell lines (6 paediatric and 5 adult), determined by resazurin reduction assay, was approximately 13μM. Biochemical assays demonstrated that tosedostat inhibited cellular aminopeptidase activity in a concentration-dependent manner. However, this inhibitory effect was reversible with rapid, but incomplete, enzyme activity recovery upon drug withdrawal, irrespective of initial concentration or treatment duration. Quantitative PCR and immunoblotting revealed no correlation between CES expression and sensitivity to tosedostat. Cellular CES1 activity was also measured. Benzil and Bis(4-nitrophenyl)phosphate (BNPP) were used to modulate cellular CES1 activity. Assays of CES1 enzyme kinetics demonstrated that following temporary exposure to the reversible CES inhibitor benzil, there is a rebound in CES1 activity in AML cells. Consistent with this observation, pre-treatment by benzil increased CES1 activity, especially in MV4-11 cells. Moreover, reduced cell viability with tosedostat treatment following benzil pre-treatment correlated with the induction of CES1 activity in AML cell lines. Together, these data suggest that enhanced tosedostat efficacy through induction of CES activity is a novel therapeutic approach to treat AML. Citation Format: Priscilla Wei Ling Hong, Amanda Melanie Smith, Lambro Angelo Johnson, Dorothy Loo, Thomas Stoll, Michelle M. Hill, Andrew S. Moore. Enhancing the efficacy of tosedostat through carboxylesterase induction. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4806.