S9 Fraction

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Markus R Meyer - One of the best experts on this subject based on the ideXlab platform.

  • are pigs a suitable animal model for in vivo metabolism studies of new psychoactive substances a comparison study using different in vitro in vivo tools and u 47700 as model drug
    Toxicology Letters, 2020
    Co-Authors: Frederike Nordmeier, Peter H Schmidt, Nadine Schaefer, Adrian A Doerr, Matthias W Laschke, Michael D Menger, Markus R Meyer
    Abstract:

    Being highly potent, New Synthetic Opioids (NSO) have become a public health concern. Little is known though about the metabolism and toxicokinetics (TK) of many of the non fentanyl NSO such as U-47700. Obtaining such data in humans is challenging and so we investigated if pigs were a suitable model species as TK model for U-47700. The metabolic fate of U-47700 was elucidated after intravenous administration to one pig in vivo and results were compared to metabolic patterns formed by different other in vitro systems (human and pig liver microsomes, human liver S9 Fraction) and compared to rat and human in vivo data. Furthermore, monooxygenase isozymes responsible for the major metabolic steps were elucidated. In total, 12 phase I and 8 phase II metabolites of U-47700 could be identified. The predominant reactions were N-demethylation, hydroxylation, and combination of them followed by glucuronidation or sulfation. The most predominant monooxygenase catalyzed conversions were N-demethylation, and hydroxylation by CYP3A4 and 2B6, and FMO3 catalyzed N-oxidation. Similar main phase I metabolites were found in vitro as compared to in vivo (pig/human). The metabolic pattern elucidated in the pig was comparable to human in vivo data. Thus, pigs seem to be a suitable animal model for metabolism and further TK of U-47700.

  • Toxicokinetics and toxicodynamics of the fentanyl homologs cyclopropanoyl-1-benzyl-4´-fluoro-4-anilinopiperidine and furanoyl-1-benzyl-4-anilinopiperidine
    Archives of Toxicology, 2020
    Co-Authors: Tanja M. Gampfer, Lea Wagmann, Folker Westphal, Yu Mi Park, Annelies Cannaert, Jennifer Herrmann, Svenja Fischmann, Rolf Müller, Christophe P. Stove, Markus R Meyer
    Abstract:

    The two fentanyl homologs cyclopropanoyl-1-benzyl-4´-fluoro-4-anilinopiperidine (4F-Cy-BAP) and furanoyl-1-benzyl-4-anilinopiperidine (Fu-BAP) have recently been seized as new psychoactive substances (NPS) on the drugs of abuse market. As their toxicokinetic and toxicodynamic characteristics are completely unknown, this study focused on elucidating their in vitro metabolic stability in pooled human liver S9 Fraction (pHLS9), their qualitative in vitro (pHLS9), and in vivo (zebrafish larvae) metabolism, and their in vitro isozyme mapping using recombinant expressed isoenzymes. Their maximum-tolerated concentration (MTC) in zebrafish larvae was studied from 0.01 to 100 µM. Their µ-opioid receptor (MOR) activity was analyzed in engineered human embryonic kidney (HEK) 293 T cells. In total, seven phase I and one phase II metabolites of 4F-Cy-BAP and 15 phase I and four phase II metabolites of Fu-BAP were tentatively identified by means of liquid chromatography high-resolution tandem mass spectrometry, with the majority detected in zebrafish larvae. N -Dealkylation, N -deacylation, hydroxylation, and N -oxidation were the most abundant metabolic reactions and the corresponding metabolites are expected to be promising analytical targets for toxicological analysis. Isozyme mapping revealed the main involvement of CYP3A4 in the phase I metabolism of 4F-Cy-BAP and in terms of Fu-BAP additionally CYP2D6. Therefore, drug-drug interactions by CYP3A4 inhibition may cause elevated drug levels and unwanted adverse effects. MTC experiments revealed malformations and changes in the behavior of larvae after exposure to 100 µM Fu-BAP. Both substances were only able to produce a weak activation of MOR and although toxic effects based on MOR activation seem unlikely, activity at other receptors cannot be excluded.

  • the metabolic fate of two new psychoactive substances 2 aminoindane and n methyl 2 aminoindane studied in vitro and in vivo to support drug testing
    Drug Testing and Analysis, 2020
    Co-Authors: Sascha K Manier, Christina Felske, Niels Eckstein, Markus R Meyer
    Abstract:

    The aim of this study was to characterize the in vitro and in vivo metabolism of 2-aminoindane (2,3-dihydro-1H-inden-2-amine, 2-AI), and N-methyl-2-aminoindane (N-methyl-2,3-dihydro-1H-inden-2-amine, NM-2-AI) after incubations using pooled human liver microsomes (pHLMs), pooled human liver S9 Fraction (pS9), and rat urine after oral administration. After analysis using liquid chromatography coupled to high-resolution mass spectrometry, pHLM incubations revealed that 2-AI was left unmetabolized, while NM-2-AI formed a hydroxylamine and diastereomers of a metabolite formed after hydroxylation in beta position. Incubations using pS9 led to the formation of an acetyl conjugation in the case of 2-AI and merely a hydroxylamine for NM-2-AI. Investigations on rat urine showed that 2-AI was hydroxylated also forming diasteromers as described for NM-2-AI or acetylated similar to incubations using pS9. All hydroxylated metabolites of NM-2-AI except the hydroxylamine were found in rat urine as additional sulfates. Assuming similar patterns in humans, urine screening procedures might be focused on the parent compounds but should also include their metabolites. An activity screening using human recombinant N-acetyl transferase (NAT) isoforms 1 and 2 revealed that 2-AI was acetylated exclusively by NAT2, which is polymorphically expressed.

  • phenethylamine derived new psychoactive substances 2c e fly 2c ef fly and 2c t 7 fly investigations on their metabolic fate including isoenzyme activities and their toxicological detectability in urine screenings
    Drug Testing and Analysis, 2019
    Co-Authors: Lea Wagmann, Lilian H J Richter, Nora Hempel, Simon D Brandt, Alexander Stratford, Markus R Meyer
    Abstract:

    Psychoactive substances of the 2C-series are phenethylamine-based designer drugs that can induce psychostimulant and hallucinogenic effects. The so-called 2C-FLY series contains rigidified methoxy groups integrated in a 2,3,6,7-tetrahydrobenzo[1,2-b:4,5-b']difuran core. The aim of the presented work was to investigate the in vivo and in vitro metabolic fate including isoenzyme activities and toxicological detectability of the three new psychoactive substances (NPS) 2C-E-FLY, 2C-EF-FLY, and 2C-T-7-FLY to allow clinical and forensic toxicologists the identification of these novel compounds. Rat urine, after oral administration, and pooled human liver S9 Fraction (pS9) incubations were analyzed by liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS). By performing activity screenings, the human isoenzymes involved were identified and toxicological detectability in rat urine investigated using standard urine screening approaches (SUSAs) based on gas chromatography (GC)-MS, LC-MSn , and LC-HRMS/MS. In total, 32 metabolites were tentatively identified. Main metabolic steps consisted of hydroxylation and N-acetylation. Phase I metabolic reactions were catalyzed by CYP2D6, 3A4, and FMO3 and N-acetylation by NAT1 and NAT2. Methoxyamine was used as a trapping agent for detection of the deaminated metabolite formed by MAO-A and B. Interindividual differences in the metabolism of the 2C-FLY drugs could be caused by polymorphisms of enzymes involved or drug-drug interactions. All three SUSAs were shown to be suitable to detect an intake of these NPS but common metabolites of 2C-E-FLY and 2C-EF-FLY have to be considered during interpretation of analytical findings.

  • studies on the in vitro and in vivo metabolism of the synthetic opioids u 51754 u 47931e and methoxyacetylfentanyl using hyphenated high resolution mass spectrometry
    Scientific Reports, 2019
    Co-Authors: Frederike Nordmeier, Lilian H J Richter, Peter H Schmidt, Nadine Schaefer, Markus R Meyer
    Abstract:

    New Synthetic Opioids (NSOs) are one class of New Psychoactive Substances (NPS) enjoying increasing popularity in Europe. Data on their toxicological or metabolic properties have not yet been published for most of them. In this context, the metabolic fate of three NSOs, namely, trans-3,4-dichloro-N-[2-(dimethylamino)cyclohexyl]-N-methyl-benzenacetamide (U-51754), trans-4-bromo-N-[2-(dimethylamino)cyclohexyl]-N-methyl-benzamide (U-47931E), and 2-methoxy-N-phenyl-N-[1-(2-phenylethyl)piperidin-4-yl] acetamide (methoxyacetylfentanyl), was elucidated by liquid chromatography high-resolution mass spectrometry after pooled human S9 Fraction (phS9) incubations and in rat urine after oral administration. The following major reactions were observed: demethylation of the amine moiety for U-51754 and U-47931E, N-hydroxylation of the hexyl ring, and combinations thereof. N-dealkylation, O-demethylation, and hydroxylation at the alkyl part for methoxyacetylfentanyl. Except for U-47931E, parent compounds could only be found in trace amounts in rat urine. Therefore, urinary markers should preferably be metabolites, namely, the N-demethyl-hydroxy and the hydroxy metabolite for U-51754, the N-demethylated metabolite for U-47931E, and the N-dealkylated metabolite as well as the O-demethylated one for methoxyacetylfentanyl. In general, metabolite formation was comparable in vitro and in vivo, but fewer metabolites, particularly those after multiple reaction steps and phase II conjugates, were found in phS9. These results were consistent with those of comparable compounds obtained from human liver microsomes, human hepatocytes, and/or human case studies.

Muhsin Konuk - One of the best experts on this subject based on the ideXlab platform.

  • detection of genotoxicity and mutagenicity of chlorthiophos using micronucleus chromosome aberration sister chromatid exchange and ames tests
    Environmental Toxicology, 2015
    Co-Authors: Dilek Akyıl, Muhsin Konuk
    Abstract:

    Received 6 December 2013; revised 27 January 2014; accepted 28 January 2014ABSTRACT: Potential mutagenic and genotoxic effects of Chlorthiophos, an organophosphate pesticide,were evaluated using four standard assays. Five different concentrations of the pesticide were tested byan Ames test using Salmonella typhimurium strains TA97, TA98, TA100, and TA102, with and without S9metabolic activation. No concentrations of Chlorthiophos showed mutagenic activity on the TA97, TA100,and TA102 strains, with and without S9 Fraction, but were all mutagenic to the TA98 strain without S9. Sis-ter chromatid exchange (SCE), chromosome aberration (CA), and micronucleus (MN) tests were used toinvestigate the genotoxic effects of Chlorthiophos in human peripheral lymphocytes treated with 25, 50,100, and 200 mg/mL concentrations of Chlorthiophos for 24 and 48 h. The nuclear division index (NDI),replication index (RI), and mitotic index (MI) were also calculated to determine the cytotoxicity of Chlor-thiophos. No increase in SCE frequency was seen for any treatment period or concentration, but Chlor-thiophos at 200 mg/mL increased the frequency of CAs. Increases in MN formation were only observed atChlorthiophos concentrations of 200 mg/mL following 24 and 48 h treatments. Chlorthiophos treatmentreduced the MI and NDI significantly, but had no effect on the RI.

Peter Langguth - One of the best experts on this subject based on the ideXlab platform.

  • presystemic metabolism and intestinal absorption of antipsoriatic fumaric acid esters
    Biopharmaceutics & Drug Disposition, 2003
    Co-Authors: D. Werdenberg, Siegfried Wolffram, Hans P. Merkle, R. Joshi, Peter Langguth
    Abstract:

    Psoriasis is a chronic inflammatory skin disease. Its treatment is based on the inhibition of proliferation of epidermal cells and interference in the inflammatory process. A new systemic antipsoriasis drug, which consists of dimethylfumarate and ethylhydrogenfumarate in the form of their calcium, magnesium and zinc salts has been introduced in Europe with successful results. In the present study, a homologous series of mono- and diesters of fumaric acid has been studied with respect to the sites and kinetics of presystemic ester degradation using pancreas extract, intestinal perfusate, intestinal homogenate and liver S9 Fraction. In addition, intestinal permeability has been determined using isolated intestinal mucosa as well as Caco-2 cell monolayers, in order to obtain estimates of the Fraction of the dose absorbed for these compounds. Relationships between the physicochemical properties of the fumaric acid esters and their biological responses were investigated. The uncharged diester dimethylfumarate displayed a high presystemic metabolic lability in all metabolism models. It also showed the highest permeability in the Caco-2 cell model. However, in permeation experiments with intestinal mucosa in Ussing-type chambers, no undegraded DMF was found on the receiver side, indicating complete metabolism in the intestinal tissue. The intestinal permeability of the monoesters methyl hydrogen fumarate, ethyl hydrogen fumarate, n-propylhydrogen fumarate and n-pentyl hydrogen fumarate increased with an increase in their lipophilicity, however, their presystemic metabolism rates likewise increased with increasing ester chain length. It is concluded that for fumarates, an increase in intestinal permeability of the more lipophilic derivatives is counterbalanced by an increase in first-pass extraction. Copyright © 2003 John Wiley & Sons, Ltd.

  • Presystemic metabolism and intestinal absorption of antipsoriatic fumaric acid esters.
    Biopharmaceutics & drug disposition, 2003
    Co-Authors: D. Werdenberg, Siegfried Wolffram, Hans P. Merkle, R. Joshi, Peter Langguth
    Abstract:

    Psoriasis is a chronic inflammatory skin disease. Its treatment is based on the inhibition of proliferation of epidermal cells and interference in the inflammatory process. A new systemic antipsoriasis drug, which consists of dimethylfumarate and ethylhydrogenfumarate in the form of their calcium, magnesium and zinc salts has been introduced in Europe with successful results. In the present study, a homologous series of mono- and diesters of fumaric acid has been studied with respect to the sites and kinetics of presystemic ester degradation using pancreas extract, intestinal perfusate, intestinal homogenate and liver S9 Fraction. In addition, intestinal permeability has been determined using isolated intestinal mucosa as well as Caco-2 cell monolayers, in order to obtain estimates of the Fraction of the dose absorbed for these compounds. Relationships between the physicochemical properties of the fumaric acid esters and their biological responses were investigated. The uncharged diester dimethylfumarate displayed a high presystemic metabolic lability in all metabolism models. It also showed the highest permeability in the Caco-2 cell model. However, in permeation experiments with intestinal mucosa in Ussing-type chambers, no undegraded DMF was found on the receiver side, indicating complete metabolism in the intestinal tissue. The intestinal permeability of the monoesters methyl hydrogen fumarate, ethyl hydrogen fumarate, n-propylhydrogen fumarate and n-pentyl hydrogen fumarate increased with an increase in their lipophilicity, however, their presystemic metabolism rates likewise increased with increasing ester chain length. It is concluded that for fumarates, an increase in intestinal permeability of the more lipophilic derivatives is counterbalanced by an increase in first-pass extraction.

Michael Mayersohn - One of the best experts on this subject based on the ideXlab platform.

  • IN VITRO TRANSESTERIFICATION OF COCAETHYLENE (ETHYLCOCAINE) IN THE PRESENCE OF ETHANOL Esterase-Mediated Ethyl Ester Exchange
    2020
    Co-Authors: James A Bourland, Michael Mayersohn, And Debra K Martin, Ph.d Michael Mayersohn
    Abstract:

    This paper is available online at http://www.dmd.org ABSTRACT: This study reports that cocaethylene undergoes an esterase-mediated ethyl ester exchange with ethanol, resulting in an increase in the apparent in vitro t 1 ⁄2, compared with control conditions. Homogenized liver from male Sprague Dawley rats in pH 7.4 phosphate buffer was centrifuged at 9000g, and the resulting supernatant (S9) Fraction was collected. Tubes containing the rat S9 Fraction and 50 M cocaethylene plus aqueous buffer (control), 50 mM ethanol, or 51. The coadministration of cocaine (benzoylmethylecgonine) and ethanol produces a unique metabolite, cocaethylene (benzoylethylecgonine, ethylcocaine), which possesses pharmacological activity nearly identical to that of cocaine, a longer elimination half-life, and greater toxicity than cocain

  • in vitro transesterification of cocaethylene ethylcocaine in the presence of ethanol esterase mediated ethyl ester exchange
    Drug Metabolism and Disposition, 1998
    Co-Authors: James A Bourland, Debra K Martin, Michael Mayersohn
    Abstract:

    This study reports that cocaethylene undergoes an esterase-mediated ethyl ester exchange with ethanol, resulting in an increase in the apparent in vitro t1/2, compared with control conditions. Homogenized liver from male Sprague Dawley rats in pH 7.4 phosphate buffer was centrifuged at 9000g, and the resulting supernatant (S9) Fraction was collected. Tubes containing the rat S9 Fraction and 50 microM cocaethylene plus aqueous buffer (control), 50 mM ethanol, or 51. 3 mM 2H6-ethanol were incubated at 37 degrees C for 4 hr. Samples were collected from the incubation tubes at various times, extracted with a solid-phase extraction system, and assayed for cocaethylene and 2H5-cocaethylene by GC/MS. Concentration-time profiles were constructed and kinetic parameters were determined. The experiment was repeated in the presence of specific and nonspecific esterase inhibitors. Enzyme kinetic parameters were also determined. Cocaethylene underwent ethyl ester exchange, being converted to 2H5-cocaethylene in the presence of 2H6-ethanol. The average apparent in vitro t1/2 value for cocaethylene (13.0 +/- 1.4 min) incubated with the S9 Fraction and buffer only was increased approximately 5-fold (67.8 +/- 0.3 min) in the presence of ethanol. Formation of 2H5-cocaethylene was totally blocked with the addition of bis-(p-nitrophenyl)phosphate but was unaffected by physostigmine. The intrinsic metabolite formation clearance of 2H5-cocaethylene from cocaethylene and 2H6-ethanol (1.92 +/- 0.03 microl/min.mg protein) was several times greater than the corresponding value for cocaethylene formation from cocaine and ethanol (0.94 +/- 0.01 microl/min.mg protein) or 2H6-ethanol (0.87 +/- 0.04 microl/min.mg protein).

Dilek Akyıl - One of the best experts on this subject based on the ideXlab platform.

  • detection of genotoxicity and mutagenicity of chlorthiophos using micronucleus chromosome aberration sister chromatid exchange and ames tests
    Environmental Toxicology, 2015
    Co-Authors: Dilek Akyıl, Muhsin Konuk
    Abstract:

    Received 6 December 2013; revised 27 January 2014; accepted 28 January 2014ABSTRACT: Potential mutagenic and genotoxic effects of Chlorthiophos, an organophosphate pesticide,were evaluated using four standard assays. Five different concentrations of the pesticide were tested byan Ames test using Salmonella typhimurium strains TA97, TA98, TA100, and TA102, with and without S9metabolic activation. No concentrations of Chlorthiophos showed mutagenic activity on the TA97, TA100,and TA102 strains, with and without S9 Fraction, but were all mutagenic to the TA98 strain without S9. Sis-ter chromatid exchange (SCE), chromosome aberration (CA), and micronucleus (MN) tests were used toinvestigate the genotoxic effects of Chlorthiophos in human peripheral lymphocytes treated with 25, 50,100, and 200 mg/mL concentrations of Chlorthiophos for 24 and 48 h. The nuclear division index (NDI),replication index (RI), and mitotic index (MI) were also calculated to determine the cytotoxicity of Chlor-thiophos. No increase in SCE frequency was seen for any treatment period or concentration, but Chlor-thiophos at 200 mg/mL increased the frequency of CAs. Increases in MN formation were only observed atChlorthiophos concentrations of 200 mg/mL following 24 and 48 h treatments. Chlorthiophos treatmentreduced the MI and NDI significantly, but had no effect on the RI.