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Paediatric clinical pharmacology
Published in Evelyne Jacqz-Aigrain, Imti Choonara, Paediatric Clinical Pharmacology, 2021
Evelyne Jacqz-Aigrain, Imti Choonara
Computer simulation is being employed increasingly within the pharmaceutical industry. The prediction of absorption, distribution, metabolism and excretion parameters for a specific drug are becoming possible from its chemical structure alone [142,143]. An area of rapid development is the prediction of in vivo pharmacokinetics parameters from in vitro data. Such in vitro-in vivo extrapolation (IVIVE) depends on the availability of good quality in vitro enzyme kinetic data, such as maximum enzyme velocity (Vmax), Michaelis-Menten constant (Km) for the drug substrate and inhibitor constant (Ki) and inhibitor concentration (I) for the inhibitor.
Detection of Lysosomal Membrane Permeabilization
Published in Bruno Gasnier, Michael X. Zhu, Ion and Molecule Transport in Lysosomes, 2020
Anne-Marie Ellegaard, Line Groth-Pedersen, Marja Jäättelä
Plot the time vs. fluorescence and calculate the Vmax from the linear part of the curve. Use these data to calculate the relative cysteine cathepsin activity later (see calculations and Figure 8.2e).
Placental Biosynthesis, Metabolism, and Transport of Eicosanoids
Published in Murray D. Mitchell, Eicosanoids in Reproduction, 2020
Purification of the PGDH enzyme from human placenta by a factor of 3110 was achieved by Thaler Dao et al.,67 with a specific activity of 1866 nmoles/min/mg protein. The km and Vmax values reported were similar to those found by Jarabak.66 The enzyme was found to have a molecular weight of 50 to 55,000 by size exclusion chromatography and 42,000 on SDS-PAGE. The PGDH enzyme was concurrently purified by Schlegel et al.,68 but slightly different km and Vmax values were reported, perhaps due to differences in the purity of the enzyme preparations. However, a similar enzyme activity (2000 nmoles/min/mg protein) was reported, together with reaction rates that agree with those reported for lung tissue. Schlegel et al.68 reported the pH optimum of the enzyme activity to be between 7.5 and 8.8; however, they described the presence of several bands on polyacrylamide gel electrophoresis, suggesting that there may be isoenzymes of PGDH present. Further, they reported that there was less enzyme activity in crude homogenates from immature placenta than in term placenta, but that no difference existed in ammonium sulfate precipitates of homogenates from the same placentae. This finding was interpreted as implying the presence of an inhibitor of enzyme activity in the immature placenta. However, alternative explanations could be the presence of the δ13-reductase enzyme or of NADH oxidase in homogenates from immature placenta.
Elucidating the pathogenesis of adenosine deaminase 2 deficiency: current status and unmet needs
Published in Expert Opinion on Orphan Drugs, 2021
Teresa K Tarrant, Susan J. Kelly, Michael S Hershfield
From the perspective of this review, the Ado/NETs hypothesis rests on the premise that ADA2, as the major ADA isozyme in plasma, regulates levels of extracellular Ado, whereas ADA1 is primarily involved with intracellular Ado. This premise also underlies some published notions of how ADA2 might function as an extracellular growth factor. It should be appreciated that ADA2 is found to be the major ADA isozyme in plasma when reaction rates are measured at a substrate (Ado) concentration saturating for both isozymes, i.e. far higher than Ado levels in vivo. Use of this condition yields the maximal velocity (Vmax) for each enzyme, and if other critical assay variables are also comparable, it allows results from different laboratories to be compared. But even under this non-physiologic assay condition, substantial ADA1 activity is found in plasma. For example, in studies of healthy adults in Japan, Iran, and the US conducted over a 30-year span using assays with 6–12 mM Ado, mean ADA1 (EHNA-inhibitable) and ADA2 (EHNA-resistant) activities in plasma ranged from 3 to 5 U/L and 9 to 13 U/mL, respectively [48–50] (and unpublished).
Linear and branched β-Glucans degrading enzymes from versatile Bacteroides uniformis JCM 13288T and their roles in cooperation with gut bacteria
Published in Gut Microbes, 2020
Ravindra Pal Singh, Sivasubramanian Rajarammohan, Raksha Thakur, Mohsin Hassan
Michaelis Menten parameters of each enzyme in terms of Km, Vmax and Kcat were determined using various concentrations of different substrates, such as laminarin, curdlan, lichenan, and lentinan. Different concentrations of these substrates, ranging from 0.001 to 20 mg/ml were used for kinetics parameters with suitable concentration of enzyme in 50 mM sodium – phosphate buffer (pH 7.0) at 37°C for 2 h of incubation. Afterward, reaction was stopped by incubating at 100°C for 5 minutes. Enzymatic assays with these substrates were determined with DNS assays, as above mentioned,44 and thin-layer chromatography (TLC). TLC analysis was carried out on Silica Gel 60 F254 (Merck) and generated mono- and oligo-saccharides were visualized by spraying TLC with 5% H2SO4 in ethanol, followed by charring. Kinetics parameters such as Km, Vmax, and Kcat were analyzed on GraphPad Prism software. Three independent tests were performed for each experiment.
Osthole inhibited the activity of CYP2C9 in human liver microsomes and influenced indomethacin pharmacokinetics in rats
Published in Xenobiotica, 2020
Hui He, Yuandong Zhang, Dezhang Zhao, Junhao Jiang, Baogang Xie, Limei Ma, Xueqing Liu, Chao Yu
Diclofenac (4.2, 8.4, 16.8 and 24.0 μM) was incubated with RLMs and pooled HLMs or recombinant CYP2C9 in the presence of osthole at different concentrations (1–10 μg/ml), and the concentration of 4′-hydroxydiclofenac was measured. The Ki values of osthole were calculated via nonlinear regression of the data to the equations for competitive inhibition (Equation (1)), noncompetitive inhibition (Equation (2)) or mixed inhibition (Equation (3)), using GraphPad Prism 5 software (GraphPad Software, La Jolla, CA): v represents the velocity of the reaction; S and I are the concentrations of substrate and inhibitor, respectively; and Km is the substrate concentration that yields a half-maximal velocity (Vmax). The inhibition type was determined from Lineweaver–Burk’s plot, the Dixon plot, and fits to the enzyme inhibition kinetic models. Kinetic constants were reported as the mean ± S.E. of the parameter estimate.