Christian Skonberg - Academia.edu (original) (raw)

Papers by Christian Skonberg

Toxicology in vitro : an international journal published in association with BIBRA, 2016

Diclofenac is a widely prescribed NSAID, which by itself and its reactive metabolites (Phase-I an... more Diclofenac is a widely prescribed NSAID, which by itself and its reactive metabolites (Phase-I and Phase-II) may be involved in serious idiosyncratic hepatotoxicity. Mitochondrial injury is one of the mechanisms of drug induced liver injury (DILI). In the present work, an investigation of the inhibitory effects of diclofenac (Dic) and its phase I [4-hydroxy diclofenac (4'-OH-Dic) and 5-hydroxy diclofenac (5-OH-dic)] and Phase-II [diclofenac acyl glucuronide (DicGluA) and diclofenac glutathione thioester (DicSG)] metabolites, on ATP synthesis in rat liver mitochondria was carried out. A mechanism based inhibition of ATP synthesis is exerted by diclofenac and its metabolites. Phase-I metabolite (4'-OH-Dic) and Phase-II metabolites (DicGluA and DicSG) showed potent inhibition (2-5 fold) of ATP synthesis, where as 5-OH-Dic, one of the Phase-I metabolite, was a less potent inhibitor as compared to Dic. The calculated kinetic constants of mechanism based inhibition of ATP synthesi...

Toxicology in vitro : an international journal published in association with BIBRA, Jan 9, 2015

Cyclooxygenase-2 (COX-2) inhibitors (coxibs) are non-steroidal anti-inflammatory drugs (NSAIDs) d... more Cyclooxygenase-2 (COX-2) inhibitors (coxibs) are non-steroidal anti-inflammatory drugs (NSAIDs) designed to selectively inhibit COX-2. However, drugs of this therapeutic class are associated with drug induced liver injury (DILI) and mitochondrial injury is likely to play a role. The effects of selective COX-2 inhibitors on inhibition of oxidative phosphorylation (ATP synthesis) in rat liver mitochondria were investigated. The order of potency of inhibition of ATP synthesis was: lumiracoxib (IC50: 6.48±2.74μM)>celecoxib (IC50: 14.92±6.40μM)>valdecoxib (IC50: 161.4±28.6μM)>rofecoxib (IC50: 238.4±79.2μM)>etoricoxib (IC50: 405.1±116.3μM). Mechanism based inhibition of ATP synthesis (Kinact 0.078min(-1) and KI 21.46μM and Kinact/KI ratio 0.0036min(-1)μM(-1)) was shown by lumiracoxib and data suggest that the opening of the MPT pore may not be the mechanism of toxicity. A positive correlation (with r(2)=0.921) was observed between the potency of inhibition of ATP synthesis and...

Toxicology in vitro : an international journal published in association with BIBRA, Jan 20, 2015

Fenbufen is an arylpropionic acid derivative belonging to the group of non-steroidal anti-inflamm... more Fenbufen is an arylpropionic acid derivative belonging to the group of non-steroidal anti-inflammatory drugs (NSAIDs). Even though fenbufen is considered a safe drug, some adverse reactions including hepatic events have been reported. To investigate whether mitochondrial damage could be involved in the drug induced liver injury (DILI) by fenbufen, the inhibitory effect of fenbufen and its conjugated metabolites on oxidative phosphorylation (ATP synthesis) in rat liver mitochondria was investigated. Fenbufen glucuronide (F-GlcA), fenbufen-N-acetyl cysteine-thioester (F-NAC) and fenbufen-S-glutathione thioester (F-SG) were found to be more potent inhibitors compared to parent fenbufen (F), whereas fenbufen-O-carnitine (F-carn), fenbufen-glycine (F-gly) and fenbufen-N-acetyl lysine amide (F-NAL) were less potent compared to fenbufen. Fenbufen-CoA thioester (F-CoA) was equally potent as fenbufen in inhibiting ATP synthesis. Fenbufen showed time and concentration dependent inhibition of ...

Toxicology in Vitro, 2013

The effect of acetone, acetonitrile, dimethyl sulfoxide (DMSO), ethanol and methanol on oxidative... more The effect of acetone, acetonitrile, dimethyl sulfoxide (DMSO), ethanol and methanol on oxidative phosphorylation (ATP synthesis) in rat liver mitochondria has been studied. All the organic solvents inhibited the oxidative phosphorylation in a concentration dependent manner, but with differences in potencies. Among the tested organic solvents, acetonitrile and acetone were more potent than ethanol, methanol, and DMSO. There was no significant difference in oxidative phosphorylation, compared to controls, when the concentrations of acetone was below 1% (v/v), of acetonitrile below 2% (v/v), of DMSO below 10% (v/v), of ethanol below 5% or of methanol below 2%, respectively. There was complete inhibition of oxidative phosphorylation at 50% (v/v) of acetone, acetonitrile and ethanol. But in the case of DMSO and methanol there were some residual activities observed at the 50% concentration level. DMSO showed least effect on oxidative phosphorylation with an IC50 value of 13.3±1.1% (v/v), followed by methanol (IC50 value 8.3±1.0), ethanol (IC50 value 4.6±1.1), acetone (IC50 value 4.3±1.0) and finally acetonitrile (IC50 value 2.1±1.0). All the organic solvents showed modulatory effects on 2,4-dinitrophenol (DNP) mediated inhibition of oxidative phosphorylation with potentiation of the action of DNP. Acetonitrile showed the highest potentiation effect followed by acetone, ethanol, methanol, and DMSO in presence of DNP. The use of organic solvents for investigation of the effects of compounds on oxidative phosphorylation in mitochondria should therefore include the use of relevant concentrations of the organic solvent in order to validate the contribution.

Chemical Research in Toxicology, 2011

Introduction α-Conotoxins are peptide neurotoxins, which are potent and selective inhibitors of n... more Introduction α-Conotoxins are peptide neurotoxins, which are potent and selective inhibitors of neuronal nicotinic acetylcholine receptors (nAChRs)[1]. They are characterised by the presence of two disulfide bonds and a highly conserved proline residue (Pro6) that contributes to ...

Fundamental Clinical Pharmacology, Apr 1, 2009

J Biol Chem, 2009

Nicotinic acetylcholine receptors (nAChRs) are ligandgated ion channels that belong to the superf... more Nicotinic acetylcholine receptors (nAChRs) are ligandgated ion channels that belong to the superfamily of Cys loop receptors. Valuable insight into the orthosteric ligand binding to nAChRs in recent years has been obtained from the crystal structures of acetylcholine-binding proteins (ACh-BPs) that share significant sequence homology with the amino-terminal domains of the nAChRs. ␣-Conotoxins, which are isolated from the venom of carnivorous marine snails, selectively inhibit the signaling of neuronal nAChR subtypes. Co-crystal structures of ␣-conotoxins in complex with AChBP show that the side chain of a highly conserved proline residue in these toxins is oriented toward the hydrophobic binding pocket in the AChBP but does not have direct interactions with this pocket. In this study, we have designed and synthesized analogues of ␣-conotoxins ImI and PnIA[A10L], by introducing a range of substituents on the Pro 6 residue in these toxins to probe the importance of this residue for their binding to the nAChRs. Pharmacological characterization of the toxin analogues at the ␣ 7 nAChR shows that although polar and charged groups on Pro 6 result in analogues with significantly reduced antagonistic activities, analogues with aromatic and hydrophobic substituents in the Pro 6 position exhibit moderate activity at the receptor. Interestingly, introduction of a 5-(R)-phenyl substituent at Pro 6 in ␣-conotoxin ImI gives rise to a conotoxin analogue with a significantly higher binding affinity and antagonistic activity at the ␣ 7 nAChR than those exhibited by the native conotoxin.

Advances in Experimental Medicine and Biology, 2009

Advances in experimental medicine and biology, 2009

The Analyst, 2012

A temperature controlled (37 °C) metabolic reaction chamber with a volume of 1 mL was coupled dir... more A temperature controlled (37 °C) metabolic reaction chamber with a volume of 1 mL was coupled directly to electrospray ionization mass spectrometry (ESI-MS) by the use of a 50 μm deep counter flow micro-chip electromembrane extraction (EME) system. The EME/ESI-MS system was used to study the in vitro metabolism of amitriptyline in real time. There was no need to stop the metabolisms by protein precipitation as in conventional metabolic studies, since the EME selectively extracted the drug and metabolites from the reaction solution comprised of rat liver microsomes in buffer. Compositional changes in the reaction chamber were continuously detected 9 seconds later in the MS. Most of this time delay was due to transport of the purified extract towards the ESI source. The EME step effectively removed the enzymatic material, buffer and salts from the reaction mixture, and prevented these species from being introduced into the ESI-MS system. The on-chip EME/ESI-MS system provided repeatability for the amitriptyline signal intensity within 3.1% relative standard deviation (RSD) (n = 6), gave a linear response for amitriptyline in the tested concentration range of 0.25 to 15 μM, and was found not to be prone to ion-suppression from major metabolites introduced simultaneously into the EME/ESI-MS system. The setup allowed the study of fast reactions kinetics. The half-life, t(1/2), for the metabolism of 10 μM amitriptyline was 1.4 minutes with a 12.6% RSD (n = 6).

Journal of Pharmaceutical Sciences, 2011

The objective of the present study was to explore the potential of using an in situ suspension fo... more The objective of the present study was to explore the potential of using an in situ suspension forming drug delivery system of celecoxib to provide sustained drug exposure in the joint cavity following intra-articular administration. In vitro, precipitates were formed upon addition of a 400 mg/mL solution of celecoxib in polyethylene glycol 400 (PEG 400) to phosphate buffer, pH 7.4, or synovial fluid. The in vitro release profiles of the in situ formed suspensions were characterized by an initial fast release followed by a slower constant flux. In buffer solutions, these fluxes were comparable to those determined for a preformed suspension containing celecoxib in its most stable crystal form despite the in situ formed precipitates contained a mixture of two crystal forms of celecoxib as determined by X-ray powder diffraction. In situ suspension formation in synovial fluid was subject to considerable variation. A relatively high dose of celecoxib, corresponding to 1.25 mg/kg, in the form of PEG 400 solution (400 mg/mL) was injected into the radiocarpal joint in four horses. Celecoxib was present in serum samples taken over 10 days and in the joint tissue (post mortem), strongly indicating that joint sustained celecoxib exposure can be achieved using in situ suspension formation.

The Journal of Lipid Research, 2010

N-acylethanolamines (NAEs) are a group of lipid mediators synthesized in response to a number of ... more N-acylethanolamines (NAEs) are a group of lipid mediators synthesized in response to a number of physiological and pathological stimuli. Because of the low tissue concentrations of NAEs, analyses often include liquid extraction followed by solid-phase extraction and subsequent quantitation by LC/MS or GC/MS. Reported levels of NAEs vary considerably, however, and often no explanation is given for these discrepancies. Brought on by difficulties encountered during method development, the effects of using four different brands of silica-containing solid phase extraction (SPE) columns and five different brands of chloroform for sample preparation were investigated. Considerable variation in the retention and recoveries of seven NAEs and 2-arachidonoylglycerol existed between the SPE columns. Furthermore, it was found that some chloroforms contained quantifiable amounts of N-palmitoylethanolamine and N-stearoylethanolamine. Finally, it was found that use of one of the chloroforms resulted in a loss of N-oleoylethanolamine from solution due to addition of chlorine to the ω-9 bond. The identity of this reaction product was confirmed by LC-MS/MS and NMR. It is recommended that these aspects of sample preparation and analysis should be thoroughly validated during method development and the relevant information on specific brands used be reported in future communications in order to better estimate the validity of reported quantitative data.

Journal of Biological Chemistry, 2009

Journal of Analytical Atomic Spectrometry, 2009

The metabolism of methylseleninic acid in isolated rat hepatocytes was investigated. Selenium con... more The metabolism of methylseleninic acid in isolated rat hepatocytes was investigated. Selenium containing metabolites excreted from the cells were detected in the supernatant of the incubation sample by LC-ICP-MS. After pre-treatment of the supernatant by preparative ...

Fundamental & Clinical Pharmacology, 2009

European Journal of Pharmaceutical Sciences, 2011

The organic cation transporter 1 (Oct1) has been shown to be one of the most abundant uptake tran... more The organic cation transporter 1 (Oct1) has been shown to be one of the most abundant uptake transporters responsible for the uptake of xenobiotics from the sinusoidal blood across the basolateral membrane of hepatocytes. On the same membrane the multidrug resistance-associated protein 3 (Mrp3) mediates the efflux of xenobiotics or their metabolites from the hepatocytes to the blood allowing their systemic exposure. In the present study we investigated the expression and activity of Oct1 and Mrp3 in suspensions and in monolayer-and sandwich cultures, and activities of CYP2B1/2, 2D1, and 3A1 in monolayerand sandwich cultures of cryopreserved rat hepatocytes. Oct1-mediated active uptake of 10 lM [ 3 H]-1methyl-4-phenylpyridinium (MPP+) into hepatocytes was assessed in the presence of quinidine (1 mM). The results showed the presence of active uptake of MPP+ in suspended hepatocytes ($91 pmol/min/mg protein). In hepatocytes in cultures (monolayer and sandwich) a time-dependent decrease in MPP+ uptake was observed from day 0 to 4, from 80 to 90 pmol/min/mg protein at day 0 to ca. 17 pmol/ min/mg protein at day 4. Mrp3 activity in suspensions and in monolayer-and sandwich cultures were investigated by measuring the efflux of [ 3 H]-taurocholate from hepatocytes in the presence of the Mrp3 inhibitor taurolithocholate-3-sulfate (TLC-S) (500 lM). Cells in suspensions showed efflux of taurocholate by an active transport mechanism indicating Mrp3 activity. Experiments in monolayer-and sandwich cultures also showed Mrp3 activity at day 0 and 1 in culture whereas experiments performed at day 2-4 showed no difference in efflux of taurocholate in the presence or absence of TLC-S, suggesting an absence of Mrp3 activity. The time-dependent decrease in Oct1 activity from day 0 to day 4 in cultures was confirmed by qPCR data also showing a time-dependent decrease in mRNA expression, whereas qPCR data did not support the observed time-dependent decrease in Mrp3 activity in cultures. Timecourse activities of CYP2B1/2, 2D1, and 3A1 were also investigated by using bupropion, bufuralol, and midazolam as respective substrates. Activities of CYP2D1 and 3A1 were reduced by 7575% and 7580%, respectively, from day 0 to day 4 in cultures, whereas activity of CYP2B1/2 was reduced by $50% from day 0 to day 4.

Drug Metabolism Reviews, 2007

N. J. HEWITT ET AL.

Drug Metabolism and Disposition, 2007

Carboxylic acids may be metabolized to acyl glucuronides and acyl-coenzyme A thioesters (acyl-CoA... more Carboxylic acids may be metabolized to acyl glucuronides and acyl-coenzyme A thioesters (acyl-CoAs), which are reactive metabolites capable of reacting with proteins in vivo. In this study, the metabolic activation of tolmetin (Tol) to reactive metabolites and the subsequent formation of Tolprotein adducts in the liver were studied in rats. Two hours after dose administration (100 mg/kg, i.p.), tolmetin acyl-CoA (Tol-CoA) was identified by LC-MS/MS in liver homogenates. Similarly, the acyl-CoA dependent metabolites tolmetin-taurine conjugate (Tol-Tau) and tolmetin-acyl carnitine ester (Tol-Car) were identified in rat livers. In a rat bile study (100 mg/kg, i.p.), the S-acyl glutathione thioester conjugate was identified, providing further evidence of the formation of reactive metabolites such as Tol-CoA or Tol-acyl glucuronide (Tol-O-G), capable of acylating nucleophilic functional groups. Three rats were treated with clofibric acid (150 mg/kg/day, i.p. for 7 days) prior to dose administration of Tol. This resulted in an increase in covalent binding to liver proteins from 0.9 nmol/g liver in control rats to 4.2 nmol/g liver in clofibric acid treated rats.

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2008

Endocannabinoids and N-acylethanolamines are lipid mediators regulating a wide range of biologica... more Endocannabinoids and N-acylethanolamines are lipid mediators regulating a wide range of biological functions including food intake. We investigated short-term effects of feeding rats five different dietary fats (palm oil (PO), olive oil (OA), safflower oil (LA), fish oil (FO) and arachidonic acid (AA)) on tissue levels of 2-arachidonoylglycerol, anandamide, oleoylethanolamide, palmitoylethanolamide, stearoylethanolamide, linoleoylethanolamide, eicosapentaenoylethanolamide, docosahexaenoylethanolamide and tissue fatty acid composition. The LA-diet increased linoleoylethanolamide and linoleic acid in brain, jejunum and liver. The OA-diet increased brain levels of anandamide and oleoylethanolamide (not 2arachidonoylglycerol) without changing tissue fatty acid composition. The same diet increased oleoylethanolamide in liver. All five dietary fats decreased oleoylethanolamide in jejunum without changing levels of anandamide, suggesting that dietary fat may have an orexigenic effect. The AA-diet increased anandamide and 2-arachidonoylglycerol in jejunum without effect on liver. The FO-diet decreased liver levels of all Nacylethanolamines (except eicosapentaenoylethanolamide and docosahexaenoylethanolamide) with similar changes in precursor lipids. The AA-diet and FO-diet had no effect on N-acylethanolamines, endocannabinoids or precursor lipids in brain. All N-acylethanolamines activated PPAR-α. In conclusion, short-term feeding of diets resembling human diets (Mediterranean diet high in monounsaturated fat, diet high in saturated fat, or diet high in polyunsaturated fat) can affect tissue levels of endocannabinoids and N-acylethanolamines.