Cytochromes P450 2C8 and 3A Catalyze the Metabolic Activation of the Tyrosine Kinase Inhibitor Masitinib.

Masitinib is a small molecule tyrosine kinase inhibitor under investigation for the treatment of amyotrophic lateral sclerosis, mastocytosis, and COVID-19. Hepatotoxicity has been reported in some patients while taking masitinib. The liver injury is thought to involve hepatic metabolism of masitinib by cytochrome P450 (P450) enzymes to form chemically reactive, potentially toxic metabolites. The goal of the current investigation was to determine the P450 enzymes involved in the metabolic activation of masitinib in vitro. In initial studies, masitinib (30 µM) was incubated with pooled human liver microsomes in the presence of NADPH and potassium cyanide to trap reactive iminium ion metabolites as cyano adducts. Masitinib metabolites and cyano adducts were analyzed using reversed-phase liquid chromatography-tandem mass spectrometry. The primary active metabolite, N-desmethyl masitinib (M485), and several oxygenated metabolites were detected along with four reactive metabolite cyano adducts (MCN510, MCN524, MCN526, and MCN538). To determine which P450 enzymes were involved in metabolite formation, reaction phenotyping experiments were conducted by incubation of masitinib (2 µM) with a panel of recombinant human P450 enzymes and by incubation of masitinib with human liver microsomes in the presence of P450-selective chemical inhibitors. In addition, enzyme kinetic assays were conducted to determine the relative kinetic parameters (apparent Km and Vmax) of masitinib metabolism and cyano adduct formation. Integrated analysis of the results from these experiments indicates that masitinib metabolic activation is catalyzed primarily by P450 3A4 and 2C8, with minor contributions from P450 3A5 and 2D6. These findings provide further insight into the pathways involved in the generation of reactive, potentially toxic metabolites of masitinib. Future studies are needed to evaluate the impact of masitinib metabolism on the toxicity of the drug in vivo.

Resistance Exercise Increases Gastrointestinal Symptoms, Markers of Gut Permeability, and Damage in Resistance-Trained Adults.

PURPOSE: This study aimed to determine the influence of acute resistance exercise (RE) and biological sex on subjective gastrointestinal (GI) symptoms, GI epithelial damage, and GI permeability in resistance-trained males and females. METHODS: Thirty resistance-trained men ( n = 15) and women ( n = 15) completed an RE bout and a nonexercise control (CON) session in a randomized counterbalanced design. The RE protocol used a load of 70% one-repetition maximum for 4 sets of 10 repetitions with a 90-s rest period length between sets and a 120-s rest period between exercises (squat, seated shoulder press, deadlift, bent-over row, and leg press). Blood samples were collected before exercise (PRE), immediately postexercise (IP), and 15-, 30-, and 60-min postexercise. Participants completed GI symptom questionnaires to assess subjective GI symptoms PRE, IP, and 60-min postexercise. Blood samples were assayed to quantify small intestine damage (I-FABP) and GI permeability (lactulose-rhamnose [L/R] ratio). Data were analyzed via separate repeated-measures ANOVA, and area under the curve (AUC) analyses were completed via one-way ANOVA. RESULTS: Participants reported greater GI symptoms in RE at IP compared with CON ( P < 0.001) with 70% of participants reporting at least one GI symptom with no differences between sexes. Nausea was the most reported GI symptom (63.3%), followed by vomiting (33.3%). I-FABP and L/R ratio did not exhibit differential responses between conditions. However, L/R ratio AUC was greater in males after RE than male CON ( P = 0.002) and both conditions for females ( P < 0.05). Furthermore, I-FABP AUC in the male RE condition was greater than both female conditions ( P < 0.05). CONCLUSIONS: Resistance-trained individuals experience GI distress after RE, with males incurring the greatest increases in markers of GI damage and permeability.

Interindividual Variability in Cytochrome P450 3A and 1A Activity Influences Sunitinib Metabolism and Bioactivation.

Sunitinib is an orally administered tyrosine kinase inhibitor associated with idiosyncratic hepatotoxicity; however, the mechanisms of this toxicity remain unclear. We have previously shown that cytochromes P450 1A2 and 3A4 catalyze sunitinib metabolic activation via oxidative defluorination leading to a chemically reactive, potentially toxic quinoneimine, trapped as a glutathione (GSH) conjugate (M5). The goals of this study were to determine the impact of interindividual variability in P450 1A and 3A activity on sunitinib bioactivation to the reactive quinoneimine and sunitinib N-dealkylation to the primary active metabolite N-desethylsunitinib (M1). Experiments were conducted in vitro using single-donor human liver microsomes and human hepatocytes. Relative sunitinib metabolite levels were measured by liquid chromatography-tandem mass spectrometry. In human liver microsomes, the P450 3A inhibitor ketoconazole significantly reduced M1 formation compared to the control. The P450 1A2 inhibitor furafylline significantly reduced defluorosunitinib (M3) and M5 formation compared to the control but had minimal effect on M1. In CYP3A5-genotyped human liver microsomes from 12 individual donors, M1 formation was highly correlated with P450 3A activity measured by midazolam 1'-hydroxylation, and M3 and M5 formation was correlated with P450 1A2 activity estimated by phenacetin O-deethylation. M3 and M5 formation was also associated with P450 3A5-selective activity. In sandwich-cultured human hepatocytes, the P450 3A inducer rifampicin significantly increased M1 levels. P450 1A induction by omeprazole markedly increased M3 formation and the generation of a quinoneimine-cysteine conjugate (M6) identified as a downstream metabolite of M5. The nonselective P450 inhibitor 1-aminobenzotriazole reduced each of these metabolites (M1, M3, and M6). Collectively, these findings indicate that P450 3A activity is a key determinant of sunitinib N-dealkylation to the active metabolite M1, and P450 1A (and potentially 3A5) activity influences sunitinib bioactivation to the reactive quinoneimine metabolite. Accordingly, modulation of P450 activity due to genetic and/or nongenetic factors may impact the risk of sunitinib-associated toxicities.

Performance of a UV-A LED system for degradation of aflatoxins B1 and M1 in pure water: kinetics and cytotoxicity study.

The efficacy of a UV-A light emitting diode system (LED) to reduce the concentrations of aflatoxin B1, aflatoxin M1 (AFB1, AFM1) in pure water was studied. This work investigates and reveals the kinetics and main mechanism(s) responsible for the destruction of aflatoxins in pure water and assesses the cytotoxicity in liver hepatocellular cells. Irradiation experiments were conducted using an LED system operating at 365 nm (monochromatic wave-length). Known concentrations of aflatoxins were spiked in water and irradiated at UV-A doses ranging from 0 to 1,200 mJ/cm2. The concentration of AFB1 and AFM1 was determined by HPLC with fluorescence detection. LC-MS/MS product ion scans were used to identify and semi-quantify degraded products of AFB1 and AFM1. It was observed that UV-A irradiation significantly reduced aflatoxins in pure water. In comparison to control, at dose of 1,200 mJ/cm2 UV-A irradiation reduced AFB1 and AFM1 concentrations by 70 ± 0.27 and 84 ± 1.95%, respectively. We hypothesize that the formation of reactive species initiated by UV-A light may have caused photolysis of AFB1 and AFM1 molecules in water. In cell culture studies, our results demonstrated that the increase of UV-A dosage decreased the aflatoxins-induced cytotoxicity in HepG2 cells, and no significant aflatoxin-induced cytotoxicity was observed at UV-A dose of 1,200 mJ/cm2. Further results from this study will be used to compare aflatoxins detoxification kinetics and mechanisms involved in liquid foods such as milk and vegetable oils.

Role of Cytochrome P450 Enzymes in the Metabolic Activation of Tyrosine Kinase Inhibitors.

Tyrosine kinase inhibitors are a rapidly expanding class of molecular targeted therapies for the treatment of various types of cancer and other diseases. An increasing number of clinically important small molecule tyrosine kinase inhibitors have been shown to undergo cytochrome P450-mediated bioactivation to form chemically reactive, potentially toxic products. Metabolic activation of tyrosine kinase inhibitors is proposed to contribute to the development of serious adverse reactions, including idiosyncratic hepatotoxicity. This article will review recent findings and ongoing studies to elucidate the link between drug metabolism and tyrosine kinase inhibitor-associated hepatotoxicity.

UV-C Irradiation on the Quality of Green Tea: Effect on Catechins, Antioxidant Activity, and Cytotoxicity.

Short-wavelength ultraviolet (UV-C) irradiation is a nonthermal processing technique that is a possible alternative to the heat-pasteurization of tea beverages. This study investigated the effect of UV-C irradiation on the polyphenolic and total phenolic contents of a green tea beverage and analyzed cytotoxicity of irradiated green tea using a novel continuous flow-through UV system. UV-C fluence levels ranging from 0 to 240 mJ/cm2 were delivered to green tea, and polyphenols were chemically profiled. Continuous-flow UV-C irradiation of the green tea beverage at a fluence of 68 mJ/cm2  induced a minor reduction in the concentration of the most abundant catechin in green tea, (-)-epigallocatechin gallate (EGCG), from 145 to 131.1 µg/mL. The total phenolic content of the green tea beverage was 0.19 µg GAE/uL and remained constant at all UV fluence levels. The UV-treated green tea beverage showed no cytotoxic effects on normal intestinal cells with healthy colonic cells (CCD-18Co) maintained at 90% viability for the UV-treated green tea beverages and the control. The treated and nontreated green tea have comparable inhibitory effects on the survival of human colon cancer cells. Overall, these results demonstrate that the UV-C irradiation did not significantly deplete catechins or produce cytotoxic byproducts. PRACTICAL APPLICATION: Short wavelength ultraviolet (UV-C) irradiation is a nonthermal processing technique that is a possible alternative to the heat pasteurization of tea beverages. This study investigated the effect of UV-C irradiation on the antioxidant concentration of green tea and analyzed cytotoxicity of irradiated a green tea beverage using a novel continuous flow-through UV system. The results demonstrated that the UV-C irradiation did not significantly deplete catechins or produce cytotoxic byproducts.

Effect of UV irradiation on aflatoxin reduction: a cytotoxicity evaluation study using human hepatoma cell line.

In this proof-of-concept study, the efficacy of a medium-pressure UV (MPUV) lamp source to reduce the concentrations of aflatoxin B1, aflatoxin B2, and aflatoxin G1 (AFB1, AFB2, and AFG1) in pure water is investigated. Irradiation experiments were conducted using a collimated beam system operating between 200 to 360 nm. The optical absorbance of the solution and the irradiance of the lamp are considered in calculating the average fluence rate. Based on these factors, the UV dose was quantified as a product of average fluence rate and treatment time. Known concentrations of aflatoxins were spiked in water and irradiated at UV doses ranging from 0, 1.22, 2.44, 3.66, and 4.88 J cm-2. The concentration of aflatoxins was determined by HPLC with fluorescence detection. LC-MS/MS product ion scans were used to identify and semi-quantify degraded products of AFB1, AFB2, and AFG1. It was observed that UV irradiation significantly reduced aflatoxins in pure water (p < 0.05). Irradiation doses of 4.88 J cm-2 reduced concentrations 67.22% for AFG1, 29.77% for AFB2, and 98.25% for AFB1 (p < 0.05). Using this technique, an overall reduction of total aflatoxin content of ≈95% (p < 0.05) was achieved. We hypothesize that the formation of ˙OH radicals initiated by UV light may have caused photolysis of AFB1, AFB2, and AFG1 molecules. In cell culture studies, our results demonstrated that the increase of UV dosage decreased the aflatoxin-induced cytotoxicity in HepG2 cells. Therefore, our research finding suggests that UV irradiation can be used as an effective technique for the reduction of aflatoxins.

MALDI-TOF/TOF CID study of poly(alpha-methylstyrene) fragmentation reactions.

MALDI-TOF/TOF CID experiments are reported for hydroxylated poly(alpha-methylstyrene) precursor ions (PAMS: m/z 1,445.9 (n = 10), 2,036.3 (n = 15), 2,626.7 (n = 20), 3,217.1 (n = 25), and 3,807.5 (n = 30), where the number of repeat units n corresponds to the oligomer mass numbers). The influences of structure, molecular weight, and kinetic energy on degradation mechanisms were examined to test the generality of our multi-chain fragmentation model developed for polystyrene. Our results indicate that poly(alpha-methylstyrene) free radicals are formed initially through multiple chain breaks and subsequently undergo a variety of depolymerization reactions to yield predominantly monomer and dimer species; the intensity of each species depends on the effective kinetic energy selected for the CID process. Each depolymerization mechanism is presented in detail with experimental and computational data to justify/rationalize the process and its kinetic energy dependence. These processes show the complex interrelationships between the various pathways along with preferred production of tertiary radicals, which suppresses the appearance of primary radicals. Additionally, Py-GC/MS experimental data are presented to allow a comparison of the multimolecular free radical reactions in pyrolysis with the unimolecular fragmentation reactions of MS/MS.

MALDI-TOF/TOF CID study of 4-alkyl-substituted polystyrene fragmentation reactions.

MALDI-TOF/TOF CID experiments were conducted on a variety of hydrogen-terminated poly(4-methylstyrene), hydroxylated poly(t-butylstyrene), and polystyrene precursor ions: n = 10, 15, 20, 25, and 30, where the number of repeat units n corresponds to the oligomer mass number. The influences of structure, molecular weight, and effective collision kinetic energy on degradation mechanisms were examined to test the generality of our multi-chain fragmentation model developed for polystyrene. Each depolymerization mechanism is presented in detail with experimental and computational data to justify/rationalize its occurrence and effective kinetic energy dependence. These processes show the complex interrelationship between the various pathways along with preferred production of secondary radicals, which suppresses the appearance of primary radicals. Additionally, Py-GC/MS experimental data are presented, for comparison of the multimolecular free radical reactions in pyrolysis with the unimolecular fragmentation reactions of MS/MS.

Synthesis and characterization of a pegylated derivative of 3-(1,2,3,6-tetrahydro-pyridin-4yl)-1H-indole (IDT199): a high affinity SERT ligand for conjugation to quantum dots.

Quantum dots consisting of a cadmium selenide core encapsulated in a shell of cadmium doped zinc sulfide have the potential to revolutionize fluorescent imaging of live cell cultures. In order to utilize these fluorescent probes it is necessary to functionalize them with biologically active ligands. In this paper we report the design and synthesis of a ligand that has a high affinity for the serotonin transporter (SERT) that may be conjugated to quantum dots.

Cleavage of peptides and proteins using light-generated radicals from titanium dioxide.

Protein identification and characterization often requires cleavage into distinct fragments. Current methods require proteolytic enzymes or chemical agents and typically a second reagent to discontinue cleavage. We have developed a selective cleavage process for peptides and proteins using light-generated radicals from titanium dioxide. The hydroxyl radicals, produced at the TiO(2) surface using UV light, are present for only hundreds of microseconds and are confined to a defined reagent zone. Peptides and proteins can be moved past the "reagent zone", and cleavage is tunable through residence time, illumination time, and intensity. Using this method, products are observed consistent with cleavage at proline residues. These initial experiments indicate the method is rapid, specific, and reproducible. In certain configurations, cleavage products are produced in less than 10 s. Reproducible product patterns consistent with cleavage of the peptide bond at proline for angiotensin I, Lys-bradykinin, and myoglobin are demonstrated using capillary electrophoresis. Mass characterization of fragments produced in the cleavage of angiotensin I was obtained using liquid chromatography-mass spectrometry. In addition to the evidence supporting cleavage at proline, enkephalin and peptide A-779, two peptides that do not contain proline, showed no evidence of cleavage under the same conditions.

Preparation of Pt-Re/Vulcan carbon nanocomposites using a single-source molecular precursor and relative performance as a direct methanol fuel cell electrooxidation catalyst.

Pt-Re/Vulcan carbon powder nanocomposites have been prepared with total metal loadings of 18 wt.% and 40 wt.% using a new non-cluster (1:1)-PtRe bimetallic precursor as the source of metal. Pt-Re nanoparticles having an average diameter of ca. 6 nm and atomic stoichiometry near 1:1 are formed. TEM, on-particle HR-EDS, and powder XRD data are consistent with the formation of Pt-Re alloy nanoparticles having a hexagonal unit cell with cell constants of a = 2.77 A and c = 4.47 A. A nanocomposite prepared at higher total metal loading under more rigorous thermal treatment also contains Pt-Re alloy nanoparticles having a fcc unit cell structure (a = 3.95 A). The precise dependence of Pt-Re nanocrystal structure on the thermal history of the nanocomposite specimen has not been investigated in detail. While these Pt-Re/carbon nanocomposites are active as anode catalysts in operating direct methanol fuel cells, the measured performance is less than that of commercial Pt-Ru/carbon catalysts and has marginal practical importance.

Combinations of microphase separation and terminal multiple hydrogen bonding in novel macromolecules.

The synthesis and characterization of terminal multiple hydrogen-bonded (MHB) polymers, such as poly(styrene) (PS), poly(isoprene) (PI), and microphase separated PS-b-PI block copolymers, possessing controlled molecular weights and narrow molecular distributions are described. Hydroxyl-terminated polymeric precursors were prepared using living anionic polymerization and subsequent quantitative termination with ethylene oxide. MHB polymers were synthesized in a controlled fashion via end-group modification of these well-defined macromolecular alcohols with excess isophorone diisocyanate and subsequent derivatization of the isocyanate-terminated polymeric intermediate with methyl isocytosine. The glass transition temperatures of the terminal MHB polymers were reproducibly higher than both nonfunctionalized and hydroxyl-terminated polymers at nearly equivalent number average molecular weights. Thin-layer chromatography analysis indicated that the interaction of terminal MHB polymers with silica was stronger as compared to both nonfunctionalized and hydroxyl-terminated polymers. Rheological characterization indicated that the melt viscosity at constant shear rate for various MHB polymers was more than 100 times higher than those for nonfunctionalized and hydroxyl-terminated polymers. Interestingly, the melt viscosity of MHB polymers was higher than those of nonfunctionalized polymers with twice the number average molecular weight. In addition, DSC and rheological characterization also suggested that terminal MHB polymers formed aggregates and not simple dimers in the melt state, and the aggregates were observed to completely dissociate at 80 degrees C.