Finding a cell-permeable compound to inhibit inflammatory cytokines: Uptake, biotransformation, and anti-cytokine activity of javamide-I/-II esters.

AIM: Currently, there is limited information available about cell-permeability and anti-cytokine activity of javamide-I/-II esters in monocyte/macrophage-like cells. Therefore, the aim of this study was to investigate their cell-permeability and anti-cytokine activity in the cells. MATERIALS AND METHODS: The uptake of javamide-I/-II and esters was studied in THP-1 cells and PBMCs. Also, kinetic and inhibition studies were conducted using THP-1 cells. Western Blot was performed to determine the level of ATF-2 phosphorylation in THP-1 cells, and ELISA assays were carried out to measure TNF-alpha, MCP-1, IL-1beta and IL-8 levels in PBMCs. KEY FINDINGS: In THP-1 cells, the uptake of javamide-I/-II esters was significantly higher than javamide-I/-II (P < 0.001), and the Km for javamide-I ester was 27 µM. Also, the uptake of the esters was inhibited by PepT2 substrate/blocker. In THP-1 cells, javamide-I/-II esters were also biotransformed into javamide-I/-II. Furthermore, javamide-I ester could inhibit ATF-2 phosphorylation better than javamide-I in the cells, suggesting that the ester could be transported inside the cells better than javamide-I. Similarly, javamide-I/-II esters were found to be transported and biotransformed in PBMCs involved in inflammation processes. As anticipated, the esters were found to inhibit TNF-alpha and MCP-1 significantly in PBMCs (P < 0.005). Especially, javamide-I ester inhibited TNF-alpha, MCP-1, IL-1beta and IL-8 with IC50 values of 1.79, 0.88, 0.91 and 2.57 µM in PBMCs. SIGNIFICANCE: Javamide-I/-II esters can be transported, biotransformed and inhibit inflammatory cytokines significantly in monocyte/macrophage-like cells, suggesting that they may be utilized as a potent cell-permeable carrier to inhibit inflammatory cytokines in the cells. CHEMICAL COMPOUNDS: Javamide-I, javamide-I-O-methyl ester, javamide-II, javamide-II-O-methyl ester, tryptophan, coumaric acid, caffeic acid, GlySar, enalapril.

Enhanced Efficiency of the Removal of Cytostatic Anthracycline Drugs Using Immobilized Mycelium of Bjerkandera adusta CCBAS 930.

The aim of this study was to evaluate the bioremoval of anthracycline antibiotics (daunomycin-DNR, doxorubicin-DOX, and mitoxantrone-MTX) by immobilized mycelium of B. adusta CCBAS 930. The activity of oxidoreductases: versatile peroxidases (VP), superoxide dismutase (SOD), catalase (CAT), and glucose oxidase (GOX), and the levels of phenolic compounds (PhC) and free radicals (SOR) were determined during the biotransformation of anthracyclines by B. adusta strain CCBAS 930. Moreover, the phytotoxicity (Lepidium sativum L.), biotoxicity (MARA assay), and genotoxicity of anthracyclines were evaluated after biological treatment. After 120 h, more than 90% of anthracyclines were removed by the immobilized mycelium of B. adusta CCBAS 930. The effective biotransformation of anthracyclines was correlated with detoxification and reduced genotoxicity.

Xenobiotic transport and metabolism in the human brain.

Organisms have metabolic pathways responsible for eliminating endogenous and exogenous toxicants. Generally, we associate the liver par excellence as the organ in charge of detoxifying the body; however, this process occurs in all tissues, including the brain. Due to the presence of the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB), the Central Nervous System (CNS) is considered a partially isolated organ, but similar to other organs, the CNS possess xenobiotic transporters and metabolic pathways associated with the elimination of xenobiotic agents. In this review, we describe the different systems related to the detoxification of xenobiotics in the CNS, providing examples in which their association with neurodegenerative processes is suspected. The CNS detoxifying systems include carrier-mediated, active efflux and receptor-mediated transport, and detoxifying systems that include phase I and phase II enzymes, as well as those enzymes in charge of neutralizing compounds such as electrophilic agents, reactive oxygen species (ROS), and free radicals, which are products of the bioactivation of xenobiotics. Moreover, we discuss the differential expression of these systems in different regions of the CNS, showing the different detoxifying needs and the composition of each region in terms of the cell type, neurotransmitter content, and the accumulation of xenobiotics and/or reactive compounds.

Quantitation of Plasma Membrane Drug Transporters in Kidney Tissue and Cell Lines Using a Novel Proteomic Approach Enabled a Prospective Prediction of Metformin Disposition.

The successful prospective incorporation of in vitro transporter kinetics in physiologically based pharmacokinetic (PBPK) models to describe drug disposition remains challenging. Although determination of scaling factors to extrapolate in vitro to in vivo transporter kinetics has been facilitated by quantitative proteomics, no robust assessment comparing membrane recoveries between different cells/tissues has been made. HEK293 cells overexpressing OCT2, MATE1, and MATE2K or human kidney cortex were homogenized and centrifuged to obtain the total membrane fractions, which were subsequently subjected to liquid-liquid extraction followed by centrifugation and precipitation to isolate plasma membrane fractions. Plasma membrane recoveries determined by quantitation of the marker Na+/K+-ATPase in lysate and plasma membrane fractions were ≤20% but within 3-fold across different cells and tissues. A separate study demonstrated that recoveries are comparable between basolateral and apical membranes of renal proximal tubules, as measured by Na+/K+-ATPase and γ-glutamyl transpeptidase 1, respectively. The plasma membrane expression of OCT2, MATE1, and MATE2K was quantified and relative expression factors (REFs) were determined as the ratio between the tissue and cell concentrations. Corrections using plasma membrane recovery had minimal impact on REF values (<2-fold). In vitro transporter kinetics of metformin were extrapolated to in vivo using the corresponding REFs in a PBPK model. The simulated metformin exposures were within 2-fold of clinical exposure. These results demonstrate that transporter REFs based on plasma membrane expression enable a prediction of transporter-mediated drug disposition. Such REFs may be estimated without the correction of plasma membrane recovery when the same procedure is applied between different matrices. SIGNIFICANCE STATEMENT: Transporter REFs based on plasma membrane expression enable in vitro-in vivo extrapolation of transporter kinetics. Plasma membrane recoveries as determined by the quantification of sodium-potassium adenosine triphosphatase were comparable between the in vitro and in vivo systems used in the present study, and therefore had minimal impact on the transporter REF values.

Prediction of Pregnancy-Induced Changes in Secretory and Total Renal Clearance of Drugs Transported by Organic Anion Transporters.

Pregnancy can significantly change the pharmacokinetics of drugs, including those renally secreted by organic anion transporters (OATs). Quantifying these changes in pregnant women is logistically and ethically challenging. Hence, predicting the in vivo plasma renal secretory clearance (CLsec) and renal CL (CLrenal) of OAT drugs in pregnancy is important to design correct dosing regimens of OAT drugs. Here, we first quantified the fold-change in renal OAT activity in pregnant versus nonpregnant individual using available selective OAT probe drug CLrenal data (training dataset; OAT1: tenofovir, OAT2: acyclovir, OAT3: oseltamivir carboxylate). The fold-change in OAT1 activity during the 2nd and 3rd trimester was 2.9 and 1.0 compared with nonpregnant individual, respectively. OAT2 activity increased 3.1-fold during the 3rd trimester. OAT3 activity increased 2.2, 1.7 and 1.3-fold during the 1st, 2nd, and 3rd trimester, respectively. Based on these data, we predicted the CLsec, CLrenal and total clearance ((CLtotal) of drugs in pregnancy, which are secreted by multiple OATs (verification dataset; amoxicillin, pravastatin, cefazolin and ketorolac, R-ketorolac, S-ketorolac). Then, the predicted clearances (CLs) were compared with the observed values. The predicted/observed CLsec, CLrenal, and CLtotal of drugs in pregnancy of all verification drugs were within 0.80-1.25 fold except for CLsec of amoxicillin in the 3rd trimester (0.76-fold) and cefazolin in the 2nd trimester (1.27-fold). Overall, we successfully predicted the CLsec, CLrenal, and CLtotal of drugs in pregnancy that are renally secreted by multiple OATs. This approach could be used in the future to adjust dosing regimens of renally secreted OAT drugs which are administered to pregnant women. SIGNIFICANCE STATEMENT: To the authors' knowledge, this is the first report to successfully predict renal secretory clearance and renal clearance of multiple OAT substrate drugs during pregnancy. The data presented here could be used in the future to adjust dosing regimens of renally secreted OAT drugs in pregnancy. In addition, the mechanistic approach used here could be extended to drugs transported by other renal transporters.

Static and Dynamic Projections of Drug-Drug Interactions Caused by Cytochrome P450 3A Time-Dependent Inhibitors Measured in Human Liver Microsomes and Hepatocytes.

Cytochrome P450 3A (CYP3A) is a frequent target for time-dependent inhibition (TDI) that can give rise to drug-drug interactions (DDI). Yet many drugs that exhibit in vitro TDI for CYP3A do not result in DDI. There were 23 drugs with published clinical DDI evaluated for CYP3A TDI in human liver microsomes (HLM) and hepatocytes (HHEP), and these data were used in static and dynamic models for projecting DDI caused by inactivation of CYP3A in both liver and intestine. TDI parameters measured in HHEP, particularly the maximal rate of enzyme inactivation, were generally lower than those measured in HLM. In static models, the use of estimated average unbound organ exit concentrations offered the most accurate projections of DDI with geometric mean fold errors of 2.0 and 1.7 for HLM and HHEP, respectively. Use of maximum organ entry concentrations yielded marked overestimates of DDI. When evaluated in a binary fashion (i.e., projection of DDI of 1.25-fold or greater), data from HLM offered the greatest sensitivity (100%) and specificity (67%) and yielded no missed DDI when average unbound organ exit concentrations were used. In dynamic physiologically based pharmacokinetic modeling, accurate projections of DDI were obtained with geometric mean fold errors of 1.7 and 1.6 for HLM and HHEP, respectively. Sensitivity and specificity were 100% and 67% when using TDI data generated in HLM and Simcyp modeling. Overall, DDI caused by CYP3A-mediated TDI can be reliably projected using dynamic or static models. For static models, average organ unbound exit concentrations should be used as input values otherwise DDI will be markedly overestimated. SIGNIFICANCE STATEMENT: CYP3A time-dependent inhibitors (TDI) are important in the design and development of new drugs. The prevalence of CYP3A TDI is high among newly synthesized drug candidates, and understanding the potential need for running clinical drug-drug interaction (DDI) studies is essential during drug development. Ability to reliably predict DDI caused by CYP3A TDI has been difficult to achieve. We report a thorough evaluation of CYP3A TDI and demonstrate that DDI can be predicted when using appropriate models and input parameters generated in human liver microsomes or hepatocytes.

Identification and Toxicity Prediction of Biotransformation Molecules of Organophosphate Flame Retardants by Microbial Reactions in a Wastewater Treatment Plant.

Organophosphate flame retardants (OPFRs) are substances added to plastics, textiles, and furniture, and are used as alternatives to brominated flame retardants. As the use of OPFRs increases in the manufacturing industry, the concentration in the aquatic environment is also increasing. In this study, OPFRs introduced into a wastewater treatment plant (WWTP) were identified, and the toxicity of biotransformation molecules generated by the biological reaction was predicted. Tris(2-butoxyethyl) phosphate, tris(2-butoxyethyl) phosphate, and triphenyl phosphate were selected as research analytes. Chemicals were analyzed using high-resolution mass spectrometry, and toxicity was predicted according to the structure. As a result, tris(1-chloro-2-propyl) phosphate showed the highest concentration, and the removal rate of OPFRs in the WWTP was 0-57%. A total of 15 biotransformation products were produced by microorganisms in the WWTP. Most of the biotransformation products were predicted to be less toxic than the parent compound, but some were highly toxic. These biotransformation products, as well as OPFRs, could flow into the water from the WWTP and affect the aquatic ecosystem.

Effects of anthropogenic activities on microplastics in deposit-feeders (Diptera: Chironomidae) in an urban river of Taiwan.

The presence of microplastics (MPs) in the environment has generated global concerns. However, the explicit assessment of the effect of multiple anthropogenic activities on the existence of MPs in the freshwater system is scarcely reported. This study quantified anthropogenic activities and analyzed their relationship with MPs on a freshwater organism: the midge larvae (Diptera: Chironomidae). The study took place in an urban river and consisted of comparing the abundance and types of MPs. Our results highlight that, while industrial area was the most important variable contributing to the total MP concentration in midge larvae, residential area also influenced the concentration of microfibers in midge larvae. The impact of a residential area on the relative abundance of microfibers in each sample site was diluted by the proximity to an industrial area. In conclusion, we suggest that industrial areas are a potential source of MP pollution in river sediment, and midge larvae can be a good indicator of the MP concentrations in urban river systems. Quantifying anthropogenic activities can help discern their effects on MP concentration in a river system and promote management strategies.

Evaluation of the carcinogenicity of dichloromethane in rats, mice, hamsters and humans.

Dichloromethane (DCM) is a high production volume chemical (>1000 t/a) mainly used as an industrial solvent. Carcinogenicity studies in rats, mice and hamsters have demonstrated a malignant tumor inducing potential of DCM only in the mouse (lung and liver) at 1000-4000 ppm whereas human data do not support a conclusion of cancer risk. Based on this, DCM has been classified as a cat. 2 carcinogen. Dose-dependent toxicokinetics of DCM suggest that DCM is a threshold carcinogen in mice, initiating carcinogenicity via the low affinity/high capacity GSTT1 pathway; a biotransformation pathway that becomes relevant only at high exposure concentrations. Rats and hamsters have very low activities of this DCM-metabolizing GST and humans have even lower activities of this enzyme. Based on the induction of specific tumors selectively in the mouse, the dose- and species-specific toxicokinetics in this species, and the absence of a malignant tumor response by DCM in rats and hamsters having a closer relationship to DCM toxicokinetics in humans and thus being a more relevant animal model, the current classification of DCM as human carcinogen cat. 2 remains appropriate.

Transformation of pig manure by passage through the gut of black soldier fly larvae (Hermetia illucens): Metal speciation, potential pathogens and metal-related functional profiling.

Black soldier fly larvae (BSFL) have great potential in livestock manure disposal. However, the changes in metal speciation, microbial communities, potential pathogens during the manure transformation process by BSFL is still largely uncharacterized, as well as the underlying metal tolerance mechanism of larval gut microbiome. Here we used BSFL to convert pig manure (PM) into larval feces (BF), and investigated the metal and microbial changes in the conversion process. Physicochemical parameters (e.g. pH, electrical conductivity, total nitrogen, total phosphorus and total potassium) in PM were significantly altered compared to BF. After conversion, less than 10% of Cu and Zn were accumulated in larval bodies. The bioavailable fraction of Cu (88.3%-86.2%) and Zn (80.6%-82.3%) occupied as the primary form in PM and BF. Genera Enterococcus, Clostridium_sensu_stricto_1, Terrisporobacter and Romboutsia were substantially enriched in the final BSFL gut (GF) compared with initial gut (GI). BSFL transformation substantially reduced pathogen abundances (decreased by 89%) derived from pig manure. Functional genes involved in metal homeostasis and resistance (e.g. CutC, pcoC, cusR, zurR and zntB) were obviously strengthened (by 2.3-7.7 folds) in GF than in GI, which might partly explain the metal tolerance ability of BSFL during the livestock manure transformation process.

Detoxication versus Bioactivation Pathways of Lapatinib In Vitro: UGT1A1 Catalyzes the Hepatic Glucuronidation of Debenzylated Lapatinib.

O-Dealkylation of the tyrosine kinase inhibitor lapatinib by cytochrome P450 3A enzymes is implicated in the development of lapatinib-induced hepatotoxicity. Conjugative metabolism of debenzylated lapatinib (M1) via glucuronidation and sulfation is thought to be a major detoxication pathway for lapatinib in preclinical species (rat and dog), limiting formation of the quinoneimine reactive metabolite. Glucuronidation of M1 by human recombinant UDP-glucuronosyltransferases (UGTs) has been reported in vitro; however, the relative UGT enzyme contributions are unknown, and the interspecies differences in the conjugation versus bioactivation pathways of M1 have not been fully elucidated. In the present study, reaction phenotyping experiments using human recombinant UGT enzymes and enzyme-selective chemical inhibitors demonstrated that UGT1A1 was the major hepatic UGT enzyme involved in lapatinib M1 glucuronidation. Formation of the M1-glucuronide by human liver microsomes from UGT1A1-genotyped donors was significantly correlated with UGT1A1 activity as measured by 17ß-estradiol 3-glucuronidation (R 2 = 0.90). Interspecies differences were found in the biotransformation of M1 in human, rat, and dog liver microsomal and 9000g supernatant (S9) fractions via glucuronidation, sulfation, aldehyde oxidase-mediated oxidation, and bioactivation to the quinoneimine trapped as a glutathione (GSH) conjugate. Moreover, we demonstrated the sequential metabolism of lapatinib in primary human hepatocytes to the M1-glucuronide, M1-sulfate, and quinoneimine-GSH conjugate. M1 glucuronidation was highly correlated with the rates of M1 formation, suggesting that O-dealkylation may be the rate-limiting step in lapatinib biotransformation. Interindividual variability in the formation and clearance pathways of lapatinib M1 likely influences the hepatic exposure to reactive metabolites and may affect the risk for hepatotoxicity. SIGNIFICANCE STATEMENT: We used an integrated approach to examine the interindividual and interspecies differences in detoxication versus bioactivation pathways of lapatinib, which is associated with idiosyncratic hepatotoxicity. In addition to cytochrome P450 (P450)-mediated bioactivation, we report that multiple non-P450 pathways are involved in the biotransformation of the primary phenolic metabolite of lapatinib in vitro, including glucuronidation, sulfation, and aldehyde oxidase mediated oxidation. UGT1A1 was identified as the major hepatic enzyme involved in debenzylated lapatinib glucuronidation, which may limit hepatic exposure to the potentially toxic quinoneimine.

Fermentation Enhanced Biotransformation of Compounds in the Kernel of Chrysophyllum albidum.

Chrysophyllum albidum Linn (African star apple) is a fruit with extensive nutritional and medicinal benefits. The fruit and kernel in the seed are both edible. Strains of lactic acid bacteria (LAB) were isolated from fermented seeds and assessed for probiotic characteristics. The extracts in both the unfermented and the fermented aqueous extracts from the kernels obtained from the seeds of C. albidum were subjected to analysis using the gas chromatography/mass spectrometry (GC-MS) method. This analysis identified the bioactive compounds present as possible substrate(s) for the associated organisms inducing the fermentation and the resultant biotransformed products formed. Three potential probiotic LAB strains identified as Lactococcus raffinolactis (ProbtA1), Lactococcus lactis (ProbtA2a), and Pediococcus pentosaceus (ProbtA2b) were isolated from the fermented C. albidum seeds. All strains were non hemolytic, which indicated their safety, Probt (A1, A2a, and A2b) grew in an acidic environment (pH 3.5) during the 48-h incubation time, and all three strains grew in 1% bile, and exhibited good hydrophobicity and auto-aggregation properties. Mucin binding proteins was not detected in any strain, and bile salt hydrolase was detected in all the strains. l-lactic acid (28.57%), norharman (5.07%), formyl 7E-hexadecenoate (1.73%), and indole (1.51%) were the four major constituents of the fermented kernel of the C. albidum, while 2,5-dimethylpyrazine (C1, 1.27%), 3,5-dihydroxy-6-methyl-2,3-dihydropyran-4-one (C2, 2.90%), indole (C3, 1.31%), norharman (C4, 3.01%), and methyl petroselinate (C5, 4.33%) were the five major constituents of the unfermented kernels. The isolated LAB are safe for consumption. The fermenting process metabolized C1, C2, and C5, which are possible starter cultures for the growth of probiotics. Fermentation is an essential tool for bioengineering molecules in foods into safe and health beneficial products.

Assembly of Plant Enzymes in E. coli for the Production of the Valuable (-)-Podophyllotoxin Precursor (-)-Pluviatolide.

Lignans are plant secondary metabolites with a wide range of reported health-promoting bioactivities. Traditional routes toward these natural products involve, among others, the extraction from plant sources and chemical synthesis. However, the availability of the sources and the complex chemical structures of lignans often limit the feasibility of these approaches. In this work, we introduce a newly assembled biosynthetic route in E. coli for the efficient conversion of the common higher-lignan precursor (+)-pinoresinol to the noncommercially available (-)-pluviatolide via three intermediates. (-)-Pluviatolide is considered a crossroad compound in lignan biosynthesis, because the methylenedioxy bridge in its structure, resulting from the oxidation of (-)-matairesinol, channels the biosynthetic pathway toward the microtubule depolymerizer (-)-podophyllotoxin. This oxidation reaction is catalyzed with high regio- and enantioselectivity by a cytochrome P450 monooxygenase from Sinopodophyllum hexandrum (CYP719A23), which was expressed and optimized regarding redox partners in E. coli. Pinoresinol-lariciresinol reductase from Forsythia intermedia (FiPLR), secoisolariciresinol dehydrogenase from Podophyllum pleianthum (PpSDH), and CYP719A23 were coexpressed together with a suitable NADPH-dependent reductase to ensure P450 activity, allowing for four sequential biotransformations without intermediate isolation. By using an E. coli strain coexpressing the enzymes originating from four plants, (+)-pinoresinol was efficiently converted, allowing the isolation of enantiopure (-)-pluviatolide at a concentration of 137 mg/L (ee ≥99% with 76% isolated yield).

Influence of breast cancer risk factors and intramammary biotransformation on estrogen homeostasis in the human breast.

Understanding intramammary estrogen homeostasis constitutes the basis of understanding the role of lifestyle factors in breast cancer etiology. Thus, the aim of the present study was to identify variables influencing levels of the estrogens present in normal breast glandular and adipose tissues (GLT and ADT, i.e., 17ß-estradiol, estrone, estrone-3-sulfate, and 2-methoxy-estrone) by multiple linear regression models. Explanatory variables (exVARs) considered were (a) levels of metabolic precursors as well as levels of transcripts encoding proteins involved in estrogen (biotrans)formation, (b) data on breast cancer risk factors (i.e., body mass index, BMI, intake of estrogen-active drugs, and smoking) collected by questionnaire, and (c) tissue characteristics (i.e., mass percentage of oil, oil%, and lobule type of the GLT). Levels of estrogens in GLT and ADT were influenced by both extramammary production (menopausal status, intake of estrogen-active drugs, and BMI) thus showing that variables known to affect levels of circulating estrogens influence estrogen levels in breast tissues as well for the first time. Moreover, intratissue (biotrans)formation (by aromatase, hydroxysteroid-17beta-dehydrogenase 2, and beta-glucuronidase) influenced intratissue estrogen levels, as well. Distinct differences were observed between the exVARs exhibiting significant influence on (a) levels of specific estrogens and (b) the same dependent variables in GLT and ADT. Since oil% and lobule type of GLT influenced levels of some estrogens, these variables may be included in tissue characterization to prevent sample bias. In conclusion, evidence for the intracrine activity of the human breast supports biotransformation-based strategies for breast cancer prevention. The susceptibility of estrogen homeostasis to systemic and tissue-specific modulation renders both beneficial and adverse effects of further variables associated with lifestyle and the environment possible.

Metabolism of a Bioorthogonal PET Tracer Candidate [19F/18F]SiFA-Tetrazine in Mouse Liver Microsomes: Biotransformation Pathways and Defluorination Investigated by UHPLC-HRMS.

Organofluorosilicon based 18F-radiolabeling is an efficient method for incorporating fluorine-18 into 18F-radiopharmaceuticals for positron emission tomography (PET) by 19F/18F isotopic exchange (IE). The first PET radiopharmaceutical, 18F-SiFAlin-TATE, radiolabeled with a silicon-based [18F]fluoride acceptor (SiFA), namely, a para-substituted di-tert-butyl[18F]fluorosilylbenzene, has entered clinical trials, and is paving the way for other potential [18F]SiFA-labeled radiopharmaceuticals for diagnostic use. In this study, we report the in vitro metabolism of an oxime-linked SiFA tetrazine (SiFA-Tz), a new PET-radiotracer candidate, recently evaluated for pretargeted PET imaging and macromolecule labeling. Metabolism of SiFA-Tz was studied in mouse liver microsomes (MLM) for elucidating its major biotransformation pathways. Nontargeted screening by ultrahigh performance liquid chromatography high-resolution mass spectrometry (UHPLC-HRMS) was utilized for detection of unknown metabolites. The oxime bond between the SiFA and Tz groups forms two geometric (E/Z) isomers, which underwent the same biotransformations, but unexpectedly with different kinetics. In total, nine proposed metabolites of SiFA-Tz from phase I and II reactions were detected, five of which were defluorinated in MLMs, elucidating the metabolic pathway leading to previously reported defluorination of [18F]SiFA-Tz in vivo. Based on the HRMS studies a biotransformation pathway is proposed: hydroxylation (+O) to tert-butyl group adjacent to the silicon, followed by oxidative defluorination (+OH/-F) cleaving the fluorine off the silicon. Interestingly, eight proposed metabolites of a reduced dihydrotetrazine analogue, SiFA-H2Tz, from phase I and II reactions were additionally detected. To the best of our knowledge, this is the first reported comprehensive investigation of enzyme mediated metabolic pathway of tetrazines and para-substituted di-tert-butylfluorosilylbenzene fluoride acceptors, providing novel structural information on the biotransformation and fragmentation patterns of radiotracers bearing these structural motifs. By investigating the metabolism preceding defluorination, structurally optimized new SiFA compounds can be designed for expanding the portfolio of efficient 19F/18F isotopic exchange labeling probes for PET imaging.

Overexpression of the constitutive androstane receptor and shaken 3D-culturing increase biotransformation and oxidative phosphorylation and sensitivity to mitochondrial amiodarone toxicity of HepaRG cells.

The liver cell line HepaRG is one of the preferred sources of human hepatocytes for in vitro applications. However, mitochondrial energy metabolism is relatively low, which affects hepatic functionality and sensitivity to hepatotoxins. Culturing in a bioartificial liver (BAL) system with high oxygen, medium perfusion, low substrate stiffness, and 3D conformation increases HepaRG functionality and mitochondrial activity compared to conventional monolayer culturing. In addition, drug metabolism has been improved by overexpression of the constitutive androstane receptor (CAR), a regulator of drug and energy metabolism in the new HepaRG-CAR line. Here, we investigated the effect of BAL culturing on the HepaRG-CAR line by applying a simple and downscaled BAL culture procedure based on shaking 3D cultures, named Bal-in-a-dish (BALIAD). We compared monolayer and BALIAD cultures of HepaRG and HepaRG-CAR cells. CAR overexpression and BALIAD culturing synergistically or additively increased transcript levels of CAR and three of the seven tested CAR target genes in biotransformation. Additionally, Cytochrome P450 3A4 activity was 35-fold increased. The mitochondrial energy metabolism was enhanced; lactate production and glucose consumption switched into lactate elimination and glucose production. BALIAD culturing alone reduced glycogen content and increased oxygen consumption and mitochondrial content. Both CAR overexpression and BALIAD culturing decreased mitochondrial superoxide levels. HepaRG-CAR BALIADs were most sensitive to mitochondrial toxicity induced by the hepatotoxin amiodarone, as indicated by oxygen consumption and mitochondrial superoxide accumulation. These data show that BALIAD culturing of HepaRG-CAR cells induces high mitochondrial energy metabolism and xenobiotic metabolism, increasing its potential for drug toxicity studies.

Molecular mechanisms of ethanol biotransformation: enzymes of oxidative and nonoxidative metabolic pathways in human.

Ethanol, as a small-molecule organic compound exhibiting both hydrophilic and lipophilic properties, quickly pass through the biological barriers. Over 95% of absorbed ethanol undergoes biotransformation, the remaining amount is excreted unchanged, mainly with urine and exhaled air.The main route of ethyl alcohol metabolism is its oxidation to acetaldehyde, which is converted into acetic acid with the participation of cytosolic NAD+ - dependent alcohol (ADH) and aldehyde (ALDH) dehydrogenases. Oxidative biotransformation pathways of ethanol also include reactions catalyzed by the microsomal ethanol oxidizing system (MEOS), peroxisomal catalase and aldehyde (AOX) and xanthine (XOR) oxidases. The resulting acetic acid can be activated to acetyl-CoA by the acetyl-CoA synthetase (ACS).It is also possible, to a much smaller extent, non-oxidative routes of ethanol biotransformation including its esterification with fatty acids by ethyl fatty acid synthase (FAEES), re-esterification of phospholipids, especially phosphatidylcholines, with phospholipase D (PLD), coupling with sulfuric acid by alcohol sulfotransferase (SULT) and with glucuronic acid using UDP-glucuronyl transferase (UGT, syn. UDPGT).The intestinal microbiome plays a significant role in the ethanol biotransformation and in the initiation and progression of liver diseases stimulated by ethanol and its metabolite - acetaldehyde, or by lipopolysaccharide and ROS.

Biotransformation Pathways and Metabolite Profiles of Oral [14C]Alisertib (MLN8237), an Investigational Aurora A Kinase Inhibitor, in Patients with Advanced Solid Tumors.

Alisertib (MLN8237) is an investigational, orally available, selective aurora A kinase inhibitor in clinical development for the treatment of solid tumors and hematologic malignancies. This metabolic profiling analysis was conducted as part of a broader phase 1 study evaluating mass balance, pharmacokinetics, metabolism, and routes of excretion of alisertib following a single 35-mg dose of [14C]alisertib oral solution (∼80 µCi) in three patients with advanced malignancies. On average, 87.8% and 2.7% of the administered dose was recovered in feces and urine, respectively, for a total recovery of 90.5% by 14 days postdose. Unchanged [14C]alisertib was the predominant drug-related component in plasma, followed by O-desmethyl alisertib (M2), and alisertib acyl glucuronide (M1), which were present at 47.8%, 34.6%, and 12.0% of total plasma radioactivity. In urine, of the 2.7% of the dose excreted, unchanged [14C]alisertib was a negligible component (trace), with M1 (0.84% of dose) and glucuronide conjugate of hydroxy alisertib (M9; 0.66% of dose) representing the primary drug-related components in urine. Hydroxy alisertib (M3; 20.8% of the dose administered) and unchanged [14C]alisertib (26.3% of the dose administered) were the major drug-related components in feces. In vitro, oxidative metabolism of alisertib was primarily mediated by CYP3A. The acyl glucuronidation of alisertib was primarily mediated by uridine 5'-diphospho-glucuronosyltransferase 1A1, 1A3, and 1A8 and was stable in 0.1 M phosphate buffer and in plasma and urine. Further in vitro evaluation of alisertib and its metabolites M1 and M2 for cytochrome P450-based drug-drug interaction (DDI) showed minimal potential for perpetrating DDI with coadministered drugs. Overall, renal elimination played an insignificant role in the disposition of alisertib, and metabolites resulting from phase 1 oxidative pathways contributed to >58% of the alisertib dose recovered in urine and feces over 192 hours postdose. SIGNIFICANCE STATEMENT: This study describes the primary clearance pathways of alisertib and illustrates the value of timely conduct of human absorption, distribution, metabolism, and excretion studies in providing guidance to the clinical pharmacology development program for oncology drugs, for which a careful understanding of sources of exposure variability is crucial to inform risk management for drug-drug interactions given the generally limited therapeutic window for anticancer drugs and polypharmacy that is common in cancer patients.

Unexpected intracellular biodegradation and recrystallization of gold nanoparticles.

Gold nanoparticles are used in an expanding spectrum of biomedical applications. However, little is known about their long-term fate in the organism as it is generally admitted that the inertness of gold nanoparticles prevents their biodegradation. In this work, the biotransformations of gold nanoparticles captured by primary fibroblasts were monitored during up to 6 mo. The combination of electron microscopy imaging and transcriptomics study reveals an unexpected 2-step process of biotransformation. First, there is the degradation of gold nanoparticles, with faster disappearance of the smallest size. This degradation is mediated by NADPH oxidase that produces highly oxidizing reactive oxygen species in the lysosome combined with a cell-protective expression of the nuclear factor, erythroid 2. Second, a gold recrystallization process generates biomineralized nanostructures consisting of 2.5-nm crystalline particles self-assembled into nanoleaves. Metallothioneins are strongly suspected to participate in buildings blocks biomineralization that self-assembles in a process that could be affected by a chelating agent. These degradation products are similar to aurosomes structures revealed 50 y ago in vivo after gold salt therapy. Overall, we bring to light steps in the lifecycle of gold nanoparticles in which cellular pathways are partially shared with ionic gold, revealing a common gold metabolism.

Biotransformation of the New Synthetic Cannabinoid with an Alkene, MDMB-4en-PINACA, by Human Hepatocytes, Human Liver Microsomes, and Human Urine and Blood.

Although at a slower rate, new psychoactive substances continue to appear on the illicit drug market, challenging their detection in biological specimens by forensic and clinical toxicologists. Here, we report in vitro and in vivo metabolism of a new synthetic cannabinoid, methyl 3,3-dimethyl-2-[1-(pent-4-en-1-yl)-1H-indazole-3-carboxamido] butanoate (MDMB-4en-PINACA). This is the first report on metabolism of a synthetic cannabinoid with an alkene functional group at the alkyl side chain. MDMB-4en-PINACA was incubated with both human hepatocytes and human liver microsomes (HLM) for up to 5 h and 1 h, respectively. The samples were analyzed by liquid chromatography-quadrupole time-of-flight mass spectrometry. An authentic human urine and a corresponding blood sample were analyzed to confirm the in vitro metabolites. A total of 32 metabolites were detected, of which 11 metabolites were detected in hepatocyte samples, 31 in HLM, 2 in urine, and 1 in blood. Analysis of the metabolites revealed that the main metabolic pathway of the terminal alkene group of the pentenyl side chain is dihydrodiol formation, most likely via epoxidation. The majority of the metabolites were generated from ester hydrolysis and/or dihydrodiol formation with further hydroxylation and/or dehydrogenation. Two most abundant metabolites in hepatocyte incubation samples, M8 (ester hydrolysis and dihydrodiol) and M30 (ester hydrolysis), coincided the two detected urinary metabolites. Based on the results, M8 and M30 are proposed to be appropriate urinary markers for MDMB-4en-PINACA intake for screening, while the inclusion of the parent drug itself and M29 (hydroxylation) may be useful for confirmation purposes.