Free Inhibitor Research Articles

Targeted Proteomics for Multiplexed Verification of Markers of Colorectal Tumorigenesis

Targeted proteomic methods can accelerate the verification of multiple tumor marker candidates in large series of patient samples. We utilized the targeted approach known as selected/multiple reaction monitoring (S/MRM) to verify potential protein markers of colorectal adenoma identified by our group in previous transcriptomic and quantitative shotgun proteomic studies of a large cohort of precancerous colorectal lesions. We developed SRM assays to reproducibly detect and quantify 25 (62.5%) of the 40 selected proteins in an independent series of precancerous and cancerous tissue samples (19 adenoma/normal mucosa pairs; 17 adenocarcinoma/normal mucosa pairs). Twenty-three proteins were significantly up-regulated (n = 17) or downregulated (n = 6) in adenomas and/or adenocarcinomas, as compared with normal mucosa (linear fold changes ≥ ±1.3, adjusted p value <0.05). Most changes were observed in both tumor types (up-regulation of ANP32A, ANXA3, SORD, LDHA, LCN2, NCL, S100A11, SERPINB5, CDV3, OLFM4, and REG4; downregulation of ARF6 and PGM5), and a five-protein biomarker signature distinguished neoplastic tissue from normal mucosa with a maximum area under the receiver operating curve greater than 0.83. Other changes were specific for adenomas (PPA1 and PPA2 up-regulation; KCTD12 downregulation) or adenocarcinoma (ANP32B, G6PD, RCN1, and SET up-regulation; downregulated AKR1B1, APEX1, and PPA1). Some changes significantly correlated with a few patient- or tumor-related phenotypes. Twenty-two (96%) of the 23 proteins have a potential to be released from the tumors into the bloodstream, and their detectability in plasma has been previously reported. The proteins identified in this study expand the pool of biomarker candidates that can be used to develop a standardized precolonoscopy blood test for the early detection of colorectal tumors.

Homogeneous, unimolecular gas-phase pyrolysis kinetics of 4- and 2-hydroxyacetophenone

The pyrolyses kinetics of 4- and 2-hydroxyacetophenones in the gas phase were determined in a static system, where the reaction vessel was deactivated with allyl bromide, and in the presence of the free radical inhibitor propene when necessary. The working temperature range was 464–485°C, and the pressure range was 44–133Torr. The reactions were found to be homogeneous and unimolecular and to obey a first-order rate law. The products for 4-hydroxyacetophenone are phenol and ketene, while for 2-hydroxyacetophenone are phenol and ketene, with smaller amounts of phenyl acetate and benzofuran. The Arrhenius expression of the pyrolyses was found for 4-hydroxyacetophenone: logk1 (s−1)=(13.83±0.08)−(258.1±1.1)kJmol−1(2.303RT)−1(r=0.9999); for 2-hydroxyacetophenone: logk1 (s−1)=(13.81±0.43)−(251.9±6.2)kJmol−1(2.303RT)−1(r=0.9991). Deacetylation may be considered as the primary reasonable mechanism, and the process appears to proceed through a semi-polar concerted four-membered cyclic transition state.

Importance of Protein Vibration Directionality on Function

Global protein vibrations have long been associated with protein functionality. Long-range intramolecular vibrations have been measured in proteins using anisotropic absorption in the terahertz frequency range [1,2]. These measurements directly correspond to the directionality of vibrations and show large changes in the directionality of the vibrational displacements for free chicken egg white lysozyme (CEWL) and inhibitor bound CEWL. Normal mode ensemble analysis (NMEA) and quasiharmonic analysis (QHA) calculations of the free and tri-acetylglucosamine (3NAG) bound CEWL dynamics were performed and also show large changes in vibration directionality with binding. This in spite of the calculated energy distribution showing very little change, in agreement with neutron scattering measurements. We investigate the importance of the protein directionality by extending the calculations to a double deletion mutant (DD CEWL) with an activity rate 1.4 times that of WT [3]. The deletions are far from the active site, indicating that the increased activity arises from changes in the dynamics. The calculated anisotropic spectra show large changes with mutation, whereas the energy distribution show practically no change from the WT. Similarities in the bound WT, free DD, and bound DD CEWL NMEA spectra indicate the mutation may be changing the directionality of the vibrations toward more efficient motions. Projections of the low frequency vibrations, from QHA, on the functional displacement show higher overlap of residues around Glu35 and Asp52 in the mutant system. These residues are known to be critical in CEWL functionality. The results reveal that the mutation may be steering the protein toward more functional dynamics without significantly affecting the energy distribution. [1] K.A. Niessen, et al. (2015) DOI: 10.1007/s12551-015-0168-4[2] G. Acbas, et al. (2014) DOI: 10.1038/ncomms4076[3] S. Mine, et al. (1999) DOI: 10.1006/jmbi.1999.2572. Supported by NSF (DBI 1556359, MCB 1616529) and DOE (DE-SC0016317).

Kinetic and mechanism of the gas-phase pyrolysis of exo-2-norbornyl formate

The rate coefficient of the gas-phase pyrolysis or elimination of exo-2-norbornyl formate was determined in a deactivated, static reaction vessel over the temperature range 300–350°C and pressure range 41–105Torr. The substrate exo-2-norbornyl formate yielded surprisingly exo-2-norborneol and CO gas. This reaction, in the presence or absence of the free radical inhibitor toluene, proved to be homogeneous, unimolecular and follows a first-order rate law. The rate of equation is given by the following Arrhenius equation: log k1 (s−1)=(13.85±0.23)−(194.6±2.7)kJmol−1(2.303RT)−1, r=0.9998.Experimental data suggested a semi-polar concerted three-membered cyclic transition state type of mechanism.

Procoagulant activity in patients with combined type 2 diabetes and coronary artery disease: No effects of long-term exercise training.

We investigated the effects of 12-month exercise training on hypercoagulability in patients with combined type 2 diabetes mellitus and coronary artery disease. Associations with severity of disease were further explored. Patients ( n = 131) were randomized to exercise training or a control group. Blood was collected at inclusion and after 12 months. Tissue factor, free and total tissue factor pathway inhibitor, prothrombin fragment 1 + 2 (F1 + 2) and D-dimer were determined by enzyme-linked immunosorbent assay and ex vivo thrombin generation by the calibrated automated thrombogram assay. Tissue factor and ex vivo thrombin generation increased from baseline to 12 months ( p < 0.01, all), with no significant differences in changes between groups. At baseline, free and total tissue factor pathway inhibitor significantly correlated to fasting glucose ( p < 0.01, both) and HbA1c ( p < 0.05, both). In patients with albuminuria ( n = 34), these correlations were strengthened, and elevated levels of D-dimer, free and total tissue factor pathway inhibitor ( p < 0.01, all) and decreased ex vivo thrombin generation ( p < 0.05, all) were observed. These results show no effects of exercise training on markers of hypercoagulability in our population with combined type 2 diabetes mellitus and coronary artery disease. The association between poor glycaemic control and tissue factor pathway inhibitor might indicate increased endothelial activation. More pronounced hypercoagulability and increased tissue factor pathway inhibitor were demonstrated in patients with albuminuria.

Linear-dendritic block copolymers as a green scale inhibitor for calcium carbonate in cooling water systems

Water-soluble monomer APEG-PG-(OH)n were produced and the Structure of APEG-PG-(OH)5 were identified by 1H-NMR. APEG-PG-(OH)n were copolymerized with maleic anhydride (MA) to synthesize no phosphate and nitrogen free calcium carbonate inhibitor MA/APEG-PG-(OH)n. The structure and thermal property of MA/APEG-PG-(OH)5 were characterized and measured by 1H-NMR, GPC and TGA. The observation shows that the dosage and n value of MA/APEG-PG-(OH)n plays an important role on CaCO3 inhibition. MA/APEG-PG-(OH)5 displays superior ability to inhibit the precipitation of calcium carbonate, with approximately 97% inhibition at a level of 8 mg/L. The effect on formation of CaCO3 was investigated with combination of SEM and XRD analysis.

Naringenin prevents experimental liver fibrosis by blocking TGFβ-Smad3 and JNK-Smad3 pathways.

AIMTo study the molecular mechanisms involved in the hepatoprotective effects of naringenin (NAR) on carbon tetrachloride (CCl4)-induced liver fibrosis.METHODSThirty-two male Wistar rats (120-150 g) were randomly divided into four groups: (1) a control group (n = 8) that received 0.7% carboxy methyl-cellulose (NAR vehicle) 1 mL/daily p.o.; (2) a CCl4 group (n = 8) that received 400 mg of CCl4/kg body weight i.p. 3 times a week for 8 wk; (3) a CCl4 + NAR (n = 8) group that received 400 mg of CCl4/kg body weight i.p. 3 times a week for 8 wk and 100 mg of NAR/kg body weight daily for 8 wk p.o.; and (4) an NAR group (n = 8) that received 100 mg of NAR/kg body weight daily for 8 wk p.o. After the experimental period, animals were sacrificed under ketamine and xylazine anesthesia. Liver damage markers such as alanine aminotransferase (ALT), alkaline phosphatase (AP), γ-glutamyl transpeptidase (γ-GTP), reduced glutathione (GSH), glycogen content, lipid peroxidation (LPO) and collagen content were measured. The enzymatic activity of glutathione peroxidase (GPx) was assessed. Liver histopathology was performed utilizing Masson’s trichrome and hematoxylin-eosin stains. Zymography assays for MMP-9 and MMP-2 were carried out. Hepatic TGF-β, α-SMA, CTGF, Col-I, MMP-13, NF-κB, IL-1, IL-10, Smad7, Smad3, pSmad3 and pJNK proteins were detected via western blot.RESULTSNAR administration prevented increases in ALT, AP, γ-GTP, and GPx enzymatic activity; depletion of GSH and glycogen; and increases in LPO and collagen produced by chronic CCl4 intoxication (P < 0.05). Liver histopathology showed a decrease in collagen deposition when rats received NAR in addition to CCl4. Although zymography assays showed that CCl4 produced an increase in MMP-9 and MMP-2 gelatinase activity; interestingly, NAR administration was associated with normal MMP-9 and MMP-2 activity (P < 0.05). The anti-inflammatory, antinecrotic and antifibrotic effects of NAR may be attributed to its ability to prevent NF-κB activation and the subsequent production of IL-1 and IL-10 (P < 0.05). NAR completely prevented the increase in TGF-β, α-SMA, CTGF, Col-1, and MMP-13 proteins compared with the CCl4-treated group (P < 0.05). NAR prevented Smad3 phosphorylation in the linker region by JNK since this flavonoid blocked this kinase (P < 0.05).CONCLUSIONNAR prevents CCl4 induced liver inflammation, necrosis and fibrosis, due to its antioxidant capacity as a free radical inhibitor and by inhibiting the NF-κB, TGF-β-Smad3 and JNK-Smad3 pathways.

In vivo pharmacodynamics of piperacillin/tazobactam: implications for antimicrobial efficacy and resistance suppression with innovator and generic products

Recent studies have shown that the pharmacodynamic (PD) index driving the efficacy of β-lactam/β-lactamase inhibitor combinations such as ceftazidime/avibactam and ceftolozane/tazobactam is the percentage of time the free inhibitor concentration is above a threshold (fT>threshold). However, data with piperacillin/tazobactam (TZP) are scarce. Here we aimed to assess the relationship between fT>threshold and TZP antibacterial efficacy by a population pharmacokinetic study in mice and dose–effect experiments in a neutropenic murine thigh infection model with two isogenic strains of Escherichia coli differentially expressing TEM-1 β-lactamase. We also explored the dynamics of resistance selection with the innovator and a non-equivalent generic, extrapolated the results to the clinic by Monte Carlo simulation of standard TZP doses, and estimated the economic impact of generic-selected resistance. The fT>threshold index described well the efficacy of TZP versus E. coli, with threshold values from 0.5 mg/L to 2 mg/L and mean exposures of 42% for stasis and 56% for 1 log10 kill. The non-equivalent generic required a longer exposure (fT>threshold 33%) to suppress resistance compared with the innovator (fT>threshold 22%), leading to a higher frequency of resistance selection in the clinical simulation (16% of patients with the generic vs. 1% with the innovator). Finally, we estimated that use of TZP generics in a scenario of 25% therapeutic non-equivalence would result in extra expenses approaching US$1 billion per year in the USA owing to selection of resistant micro-organisms, greatly offsetting the savings gained from generic substitution and further emphasising the need for demonstrated and not assumed therapeutic equivalence.

Fluorescent-tagged block copolymer as an effective and green inhibitor for calcium sulfate scales

Allyloxy polyethoxy ether (APEG) and succinic anhydride were used to prepare allyloxy polyethoxy carboxylate (APEL). 8-Hydroxy-1,3,6-pyrene trisulfonic acid trisodium salt (PY) was reacted with allyl chloride to produce fluorescent monomer 8-allyloxy-1,3,6-pyrene trisulfonic acid trisodium salt (PA). APEL and PA were copolymerized with maleic anhydride (MA) to synthesize PA tagged no phosphate and nitrogen free CaSO4 inhibitor MA–APEL–PA. Structures of PA, APEG, APEL, and MA–APEL–PA were identified by 1H NMR. The observation shows that the dosage of MA–APEL–PA plays an important role on CaSO4 inhibition. The effect on formation of CaSO4 was investigated with scanning electron microscope (SEM) analysis. Relationship between MA–APEL–PA’s fluorescent intensity and its dosage was studied. Correlation coefficient R2 of MA–APEL–PA fluorescent intensity and MA–APEL–PA dosage is 0.9991.

Antcin H Protects Against Acute Liver Injury Through Disruption of the Interaction of c-Jun-N-Terminal Kinase with Mitochondria

Antrodia Camphorate (AC) is a mushroom that is widely used in Asian countries to prevent and treat various diseases, including liver diseases. However, the active ingredients that contribute to the biological functions remain elusive. The purpose of the present study is to test the hepatoprotective effect of Antcin H, a major triterpenoid chemical isolated from AC, in murine models of acute liver injury. We found that Antcin H pretreatment protected against liver injury in both acetaminophen (APAP) and galactosamine/tumor necrosis factor (TNF)α models. More importantly, Antcin H also offered a significant protection against acetaminophen-induced liver injury when it was given 1 h after acetaminophen. The protection was verified in primary mouse hepatocytes. Antcin H prevented sustained c-Jun-N-terminal kinase (JNK) activation in both models. We excluded an effect of Antcin H on acetaminophen metabolism and TNF receptor signaling and excluded a direct effect as a free radical scavenger or JNK inhibitor. Since the sustained JNK activation through its interaction with mitochondrial Sab, leading to increased mitochondrial reactive oxygen species (ROS), is pivotal in both models, we examined the effect of Antcin H on p-JNK binding to mitochondria and impairment of mitochondrial respiration. Antcin H inhibited the direct effect of p-JNK on isolated mitochondrial function and binding to isolated mitochondria. Innovation and Conclusion: Our study has identified Antcin H as a novel active ingredient that contributes to the hepatoprotective effect of AC, and Antcin H protects against liver injury through disruption of the binding of p-JNK to Sab, which interferes with the ROS-dependent self-sustaining activation of MAPK cascade. Antioxid. Redox Signal. 26, 207-220.

The Impact of Continuous Oxygen Flux in a Thin Film Photopolymerization Reaction with Peroxy‐Mediated Regeneration of Initiator

Eosin, a photosensitizer dye that can exist in multiple oxidation states, has been shown to initiate visible‐light photopolymerizations of acrylates in open air when used in combination with tertiary amines. The exact mechanism behind this reactivity with micromolar concentrations of eosin in aqueous monomer solutions initially containing millimolar concentrations of oxygen has not been conclusively established, although pathways for regeneration of eosin in the presence of oxygen certainly play a role. In this work, a reaction–diffusion model incorporating a peroxy‐mediated eosin‐regeneration mechanism is built to explore the effects of oxygen diffusing into the system as the light‐activated reaction proceeds. An oxygen concentration‐dependent flux boundary condition is used to model the continuous replenishment of oxygen at the surface open to air as it is consumed by reaction. The model predicts the formation of a free radical concentration front that initially forms closer to the open surface and gradually moves toward the closed surface, with polymer film thickness increasing and the time required for polymerization to begin decreasing as the initial eosin concentration is increased. These results suggest that oxygen's dual role as both a free radical inhibitor and a precursor of the oxidizing species required for regeneration of eosin brings about interesting spatial variations when the reaction is carried out in a thin film geometry that is exposed to open air. In this case, an assumption of a well‐mixed system is not appropriate and the kinetics of the reaction and conversion as a function of position are likely to depend on the geometry of the system. image

OC-06 - Pro-thrombotic biomarkers in pancreatic diseases: are they specific of cancer?

Venous thrombo-embolic events (VTE) occur frequently in patients with pancreatic cancer and contribute to elevated morbidity and mortality. Clinical risk factors for thrombosis such as cancer stage and tumor grade have been clearly identified. Recently, several biomarkers have been proposed which may help identifying cancer patients at high risk of thrombosis. Those biomarkers have been studied in heterogeneous cohorts of patients with different cancer types. To compare pro-thrombotic biomarkers in pancreatic cancer and in chronic pancreatitis to determine whether these biomarkers are related to cancer or inflammation and to validate their association with thrombotic risk in a pancreatic cancer population. 45 patients with pancreatic cancer, 49 with intraductal papillary mucinous tumor of the pancreas (IPMN), a precancerous lesion, and 50 with chronic pancreatitis were recruited. Plasma levels of factor VIII, D-dimers, thrombin-antithrombin complexes, soluble p-selectin, tissue factor-dependent procoagulant activity of MP (TF-MP), free Tissue Factor Pathway Inhibitor (TFPI) and extracellular DNA were measured. Thrombin generation triggered by 1pM of TF was evaluated with the Calibrated Automated Thrombogram assay. Levels of factor VIII, D-dimers, TF-MP, TFPI and extracellular DNA were significantly higher in cancer patients compared to IPMN or chronic pancreatitis (Table1). Patients with metastatic cancer (n=27) presented higher levels of D-dimers (mean±sd 1.77±1.28 vs 0.80±0.96 μg/ml, p=0.004) and MP-TF (54.3±53.2 vs 15.8±10.4 fM, p=0.02) compared to patients with localized lesions (n=18). Among cancer patients, 42 were followed for a median duration of 187 days (min 21-max 802 days). VTE occurred in 10 (23%) patients. All had metastatic cancer at the time of thrombosis. Only D-dimers were significantly elevated in cancer patients with VTE compared to patients without VTE (median 1.85 vs 0.7 μg/ml, p=0.02). Elevation of factor VIII, D-dimers, TF-MP, TFPI and extracellular DNA seems to be related to cancer process, not to local or systemic inflammation as these parameters differentiate cancer from chronic pancreatitis. Interestingly, D-dimers and TF-MP are related to the disseminated cancer stage, suggesting that vascular invasion is a prerequisite to the release of TF-MP from the primary tumor into the bloodstream and to coagulation activation. However, only D-dimers are associated with the occurrence of future VTE. We propose that D-dimers could be a useful tool to predict thrombotic events in pancreatic cancer patients. This should be confirmed in a larger population.

PO-24 - Determinants of thrombin generation in gynaecological malignancies

Increased thrombin production is associated with malignancy and is a marker for venous thromboembolism (VTE). Our group has shown that thrombin generation is increased in gynaecological malignancies. Although tumour derived Tissue Factor (TF) has been implicated, the precise mechanism by which thrombin production is increased is not fully understood. Our group has shown that gynaecological cancers can alter tumour expression of cogulation proteases. These changes may be implicated in the increased thrombin generation observed. Previous studies in normal control patients by other groups have shown that multiple coagulation factors are implicated in thrombin generation and that the effects depend on assay conditions however free Tissue Factor Pathway Inhibitor (TFPI), Factors V, VIIIc and protein S, were significant determinants of thrombin generation. The aim of this study was to determine the effect of factor V factor, VIIIc, TFPI and free protein S on the thrombin generation in patients with endometrial and ovarian cancer compared with benign controls. Patients with a gynaecological malignancy (n=43) (ovarian or endometrial) were matched with patients with a benign tumour (n=43) gave full and informed consent. Venous blood samples were obtained prior to surgery and chemotherapy. Thrombin generation was was measured using a fluorogenic assay. Lag time, peak thrombin and area under the thrombin generation curve (ETP) was determined and reported for each sample. Free protein S, free TFPI and factor V were determined using ELISA. Factor VIIIc was determined using a hromogenic assay. Factor V and factor VIIIc were significantly increased in the malignant group compared with the benign group (P<0.03; P<0.006). Increased free TFPI levels were also observed in the malignant group but this did not reach significance (P<0.06). There was no difference in free Protein S levels between the groups. Highest levels of peak thrombin generation were observed in the high grade serous and clear cell ovarian cancer patients. Changes were less marked in the endometrial patients. Free TFPI, factor V and factor VIIIc were important determinants of thrombin generation in the malignant group. Coagulation factors V and VIIIc and free TFPI are altered in patients with gynaecological malignancies and contribute to the increased thrombin generation found in these cancer patients. We have previously found increased expression of factor V in tumours from patients with ovarian cancer in addition to increased TF expression. These changes may explain the high rate of venous thromboembolism found in these patients.

Measuring benzohydroxamate complexation with Mg2+, Mn2+, Co2+, and Ni2+ using isothermal titration calorimetry

Isothermal titration calorimetry has been used to determine the stoichiometry and first (K1) and second (K2) conditional stability constants, where applicable, for the formation of complexes of benzohydroxamate (BzH) with Mg2+, Mn2+, Co2+, and Ni2+ ions, and benzoylphosphonate with Ni2+ ions. Binding determinations were conducted in HEPES buffer (0.1 mol L−1, pH 7.5) at 25.0 °C by titration of the ligand with metal ion. BzH and Mg2+, and benzoylphosphonate and Ni2+, formed complexes with a 1:1 stoichiometry; however, BzH formed complexes with Mn2+, Co2+, and Ni2+ ions in a step-wise binding model with a stoichiometry of 2:1 (BzH:M2+). In addition, the Gibbs energy change accompanying the binding of BzH to each of the divalent metal ions was found to follow the order of the Irving–Williams series (Mg2+ < Mn2+ < Co2+ < Ni2+) and to correlate with the electron affinities of the metal ions. Conditional stability constants, determined in this manner, may be useful for calculating the concentrations of free inhibitor when inhibition studies are conducted with metalloenzymes and enzyme inhibitors containing a hydroxamic acid group.

Abstract 212: Andexanet Alfa, a Universal Antidote for Reversal of Anticoagulation of Factor Xa Inhibitors in Healthy Human Volunteers

Introduction: Andexanet alfa (AnXa) is a recombinant modified fXa molecule that acts as a specific antidote for fXa inhibitors. We report clinical results in healthy subjects anticoagulated with apixaban (apix), rivaroxaban (riva), edoxaban (edox), or enoxaparin (enox), demonstrating rapid and sustained reversal of anticoagulation following administration of AnXa. Methods: These were Phase 2/3 randomized, double-blind, placebo-controlled studies in healthy subjects. In Phase 2, about153 subjects age18 - 45 were given one of the fXa inhibitors (apix 5 mg BID, riva 20 mg QD, edox 60 mg QD or enox 40 mg QD) for 6 days. AnXa or placebo (3:1 randomization) was given IV on Day 6, 3hrs after the last inhibitor dose (∼inhibitor Cmax). Safety was followed through Day 48. A range of AnXa doses (bolus or bolus+infusion) was evaluated by correction of biomarkers (anti-fXa activity, free inhibitor concentrations and thrombin generation (TG)). In Phase 3 (ANNEXA™), older subjects age 50 to 75 were dosed with apix (5 mg BID) or riva (20 mg QD) for 4 days. ANNEXA™-A had 63 subjects treated with apix. AnXa (400 mg bolus; 400 mg bolus plus 4 mg/min x 2hr infusion) or placebo (3:1 randomization) was given on Day 4, 3 hrs after the last apix dose. ANNEXA™-R had 82 subjects treated with riva. AnXa (800 mg bolus; 800 mg bolus plus 8 mg/min x 2hr infusion) or placebo (2:1) was given on Day 4, 4 hrs after the last riva dose. Safety was followed through Day 43. Results: About 298 healthy subjects were enrolled in the studies. AnXa demonstrated rapid and sustained reversal of both direct and indirect fXa inhibitors as measured by correction of biomarkers. The ANNEXA™ studies confirmed findings from Phase 2, and met all primary (reversal of anti-fXa) and secondary endpoints (reduction of free inhibitor concentration and restoration of TG) with high statistical significance. AnXa was well-tolerated with no serious adverse events, thrombotic events, or antibodies to fX or fXa reported. Conclusion: AnXa treatment results in rapid and sustained reversal of anticoagulation of fXa inhibitors. A Phase 3b/4 confirmatory study (ANNEXA-4) in patients with acute major bleeds is ongoing.

A new lysine derived glyoxal inhibitor of trypsin, its properties and utilization for studying the stabilization of tetrahedral adducts by trypsin

New trypsin inhibitors Z-Lys-COCHO and Z-Lys-H have been synthesised. Ki values for Z-Lys-COCHO, Z-Lys-COOH, Z-Lys-H and Z-Arg-COOH have been determined. The glyoxal group (–COCHO) of Z-Lys-COCHO increases binding ~300 fold compared to Z-Lys-H. The α-carboxylate of Z-Lys-COOH has no significant effect on inhibitor binding. Z-Arg-COOH is shown to bind ~2 times more tightly than Z-Lys-COOH. Both Z-Lys-13COCHO and Z-Lys-CO13CHO have been synthesized. Using Z-Lys-13COCHO we have observed a signal at 107.4ppm by 13C NMR which is assigned to a terahedral adduct formed between the hydroxyl group of the catalytic serine residue and the 13C-enriched keto-carbon of the inhibitor glyoxal group. Z-Lys-CO13CHO has been used to show that in this tetrahedral adduct the glyoxal aldehyde carbon is not hydrated and has a chemical shift of 205.3ppm. Hemiketal stabilization is similar for trypsin, chymotrypsin and subtilisin Carlsberg. For trypsin hemiketal formation is optimal at pH 7.2 but decreases at pHs 5.0 and 10.3. The effective molarity of the active site serine hydroxyl group of trypsin is shown to be 25300M. At pH 10.3 the free glyoxal inhibitor rapidly (t1/2=0.15h) forms a Schiff base while at pH 7 Schiff base formation is much slower (t1/2=23h). Subsequently a free enol species is formed which breaks down to form an alcohol product. These reactions are prevented in the presence of trypsin and when the inhibitor is bound to trypsin it undergoes an internal Cannizzaro reaction via a C2 to C1 alkyl shift producing an α-hydroxycarboxylic acid.

Purification Mechanism Analysis and performance Characterization of Mn-zeolite Catalyst

AbstractIn this study, the clinoptilolite is taken as the matrix, manganese nitrate as a modifier; through a medium temperature calcination pore making technique. Mostly, the micro pores in the matrix have been enlarged through the means of dipping and high temperature calcination modification. A small amount of active catalytic components are exchanged stably between the pores and surface. The mechanism of manufacturing new catalyst is analyzed using test methods of TEM, SEM, EDS, XRD and BET. These test methods testify the existence of active catalytic components of manganese in the Mn-Zeolite, while researching a new type of Mn-zeolite catalyst heterogeneous catalytic mechanism of Fenton and ozone oxidation process. The results show that added Mn-zeolite could improve the oxidation rate from 51% to 100%. Addition of free radical inhibitor, t-butanol, significantly inhibited the catalytic reaction of Fenton and ozone oxidation. This indicated that the mechanism of the reaction of Fenton and ozonation followed the hydroxyl radical.

The Presence of Thyroid-Stimulation Blocking Antibody Prevents High Bone Turnover in Untreated Premenopausal Patients with Graves’ Disease

Osteoporosis-related fractures are one of the complications of Graves’ disease. This study hypothesized that the different actions of thyroid-stimulating hormone receptor (TSHR) antibodies, both stimulating and blocking activities in Graves’ disease patients might oppositely impact bone turnover. Newly diagnosed premenopausal Graves’ disease patients were enrolled (n = 93) and divided into two groups: patients with TSHR antibodies with thyroid-stimulating activity (stimulating activity group, n = 83) and patients with TSHR antibodies with thyroid-stimulating activity combined with blocking activity (blocking activity group, n = 10). From the stimulating activity group, patients who had matched values for free T4 and TSH binding inhibitor immunoglobulin (TBII) to the blocking activity group were further classified as stimulating activity-matched control (n = 11). Bone turnover markers BS-ALP, Osteocalcin, and C-telopeptide were significantly lower in the blocking activity group than in the stimulating activity or stimulating activity-matched control groups. The TBII level showed positive correlations with BS-ALP and osteocalcin levels in the stimulating activity group, while it had a negative correlation with the osteocalcin level in the blocking activity group. In conclusion, the activation of TSHR antibody-activated TSH signaling contributes to high bone turnover, independent of the actions of thyroid hormone, and thyroid-stimulation blocking antibody has protective effects against bone metabolism in Graves’ disease.

Pharmacokinetic and Pharmacodynamic (PKPD) Modeling to Determine the Andexanet Alfa Dose to Reverse the Anticoagulant Activity of Direct Fxa Inhibitors

IntroductionAndexanet alfa (AnXa) is a specific antidote under development for direct and indirect factor Xa (fXa) inhibitors. AnXa is a recombinant, engineered version of human fXa that is catalytically inactive but retains high affinity for direct fXa inhibitors. AnXa binds fXa inhibitors, sequestering them in plasma, thereby reducing the free inhibitor concentration, reversing fXa inhibition, and normalizing thrombin generation. The decreased unbound concentrations of the direct fXa inhibitors (apixaban, rivaroxaban and edoxaban) cause a portion of the extravascular fXa inhibitors to move into the vasculature. Reversal of fXa inhibitor-induced anticoagulation requires a molar excess of AnXa relative to the total amount of fXa inhibitor in the blood - i.e., the initial fXa inhibitor plasma concentration plus the redistributed amount. We report a PKPD model that accounts for this extravascular-intravascular redistribution and allows determination of the appropriate AnXa dose to reverse the anti-fXa activity for each approved direct fXa inhibitor.MethodsA model for rivaroxaban was constructed first by jointly analyzing AnXa concentrations, total and unbound rivaroxaban concentrations, and anti-fXa activity for 5 different doses of AnXa (each administered at the peak plasma level of 20 mg QD rivaroxaban at steady state). Model parameters were estimated by maximum likelihood using nonlinear regression (NONMEM, v. 7.2.0). The model was used to simulate the potential level of reversal of anti-fXa activity after 2.5-40 mg/day rivaroxaban (QD and BID) for different bolus doses of AnXa and to predict maintenance of reversal by various follow-on infusion rates. A similar model was developed to characterize the interaction between AnXa and apixaban. Learnings from the rivaroxaban and apixaban models were used to develop a universal model to predict the level of reversal of anticoagulation for 60 mg edoxaban after a particular dose of AnXa. The model accurately predicted the degree of reversal of edoxaban anticoagulation. The final model was used to construct a nomogram of AnXa doses that could be used for each fXa inhibitor at each approved dose based on timing of the last dose of the inhibitor.ResultsThe final PKPD model was a three-compartment PK model for the fXa inhibitors, including one central and two tissue compartments. The two tissue compartments included one that rapidly equilibrated with the central compartment and a second that equilibrated slowly. The combined volume of the central and rapidly equilibrating compartments was similar to the volume of central compartment of the fXa inhibitor PK models in the absence of AnXa.The AnXa PK was best described by a two-compartment PK model with an additional saturable binding component. The AnXa-fXa inhibitor complex moved to a "sequestration" compartment. Upon release from AnXa, the fXa inhibitor was able to return to the central compartment. The fXa inhibitor did not clear with AnXa. The binding between AnXa and free fXa inhibitor was characterized by a reversible binding equilibrium. There was a direct PKPD linear relationship between free fXa inhibitor concentrations and anti-fXa activity.Simulations were used to estimate the best dosing regimen to reverse anticoagulation for each direct fXa inhibitor. A bolus dose of 800 mg of AnXa followed by an 8 mg/min infusion was sufficient to fully reverse the peak level of anti-fXa activity after the 20 mg QD dose of rivaroxaban. This represents a >90% decrease in anti-fXa activity compared to pre-AnXa administration. Sustained reversal of peak anti-fXa levels for the 5 mg BID dose of apixaban required a 400 mg bolus AnXa dose with a follow-on infusion of 4 mg/min. The peak anticoagulant activity of the 60 mg dose of edoxaban could be reversed by an 800 mg bolus dose of AnXa followed by an 8 mg/min infusion.Conclusions•A PKPD model was constructed that accurately predicted the AnXa dose necessary to reverse coagulation inhibition of each direct fXa inhibitor. The model incorporated the PK of AnXa, the anti-fXa activity, unbound and total levels of the fXa inhibitor, and the redistribution of each fXa inhibitor between the extravascular and intravascular compartments.•Simulations were used to predict the AnXa dose necessary to reverse each approved dose of each direct fXa inhibitor and for different times after the last dose taken of the fXa inhibitor. DisclosuresJanet:Portola Pharmaceuticals, Inc.: Employment. Lu:Portola Pharmaceuticals, Inc.: Employment. Curnutte:3-V Biosciences: Equity Ownership; Sea Lane Biotechnologies: Consultancy; Portola Pharmaceuticals, Inc.: Employment. Conley:Portola Pharmaceuticals, Inc.: Employment. Mandema:Portola Pharmaceuticals, Inc.: Consultancy.

Chitosan coating with trans-cinnamaldehyde improves structural integrity and antioxidant metabolism of fresh-cut melon

This study evaluated a 2% chitosan-based coating with 500mgL−1trans-cinnamaldehyde on quality attributes, pro- and anti-oxidative metabolism, and structural integrity of biological membranes and cell wall of fresh-cut melon (Cucumis melo var. Cantalupensis Naud.) stored for 20 days at 4°C. Coating improved (P<0.05) samples’ firmness, colour, soluble solids, total vitamin C and carotenoid content for up to 15 days of storage, thereafter samples showed signs of decay with softening of processed melon resulting from structural changes in both biological membranes and cell walls. Coating also reduced hydrogen peroxide radical levels leading to lower antioxidant enzymatic activity and lipid peroxidation degree and electrolyte leakage levels compared with control. Coating improved produce visual quality due to reduction in the activity of browning-associated enzymes, G-POD and PPO. Results indicate that coating improved quality of fresh cut Cantaloupe melons by acting as a physical barrier to gas exchange decreasing respiration rate and consequently, O2-dependent processes, while trans-cinnamaldehyde acted as free radical scavenger and enzyme inhibitor. The lower respiration rate inhibited the establishment of an oxidative stress induced by fruit processing and senescence with less damage to biological membranes, lower cell wall hydrolase activities and loss of bioactive compounds.