Establishment of reference values for the lysine acetylation marker Nɛ-acetyllysine in small volume human plasma samples by a multi-target LC-MS/MS method.

Cardiovascular disease (CVD) and chronic kidney disease (CKD) constitute substantial burdens for public health. The identification and validation of risk markers for CVD and CKD in epidemiological studies requires frequent adaption of existing analytical methods as well as development of new methods. In this study, an analytical procedure to simultaneously quantify ten endogenous biomarkers for CVD and CKD is described. An easy-to-handle sample preparation requiring only 20 µL of human plasma is followed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The method was successfully validated according to established guidelines meeting required criteria for accuracy, precision, recovery, linearity, selectivity, and limits of quantification. The scalability of the method for application in larger cohorts was assessed using a set of plasma samples from healthy volunteers (n = 391) providing first reference values for the recently established biomarker Nɛ-acetyllysine (Nɛ-AcLys). Other biomarkers analyzed were creatinine, ß-aminoisobutyric acid (ß-AIB), carnitine, 1-methylnicotinamide (1-MNA), citrulline, symmetric dimethylarginine (SDMA), asymmetric dimethylarginine (ADMA), homoarginine (hArg), and ornithine. All obtained results are within reference values specified elsewhere. Overall, these results demonstrate the suitability of the method for simultaneous quantification of ten endogenous biomarkers for CVD and CKD in plasma samples from larger cohorts and allow validation of Nɛ-AcLys as a biomarker in large cohorts.

N(1)-methylnicotinamide as an endogenous probe for drug interactions by renal cation transporters: studies on the metformin-trimethoprim interaction.

PURPOSE: N(1)-methylnicotinamide (NMN) was proposed as an in vivo probe for drug interactions involving renal cation transporters, which, for example, transport the oral antidiabetic drug metformin, based on a study with the inhibitor pyrimethamine. The role of NMN for predicting other interactions with involvement of renal cation transporters (organic cation transporter 2, OCT2; multidrug and toxin extrusion proteins 1 and 2-K, MATE1 and MATE2-K) is unclear. METHODS: We determined inhibition of metformin or NMN transport by trimethoprim using cell lines expressing OCT2, MATE1, or MATE2-K. Moreover, a randomized, open-label, two-phase crossover study was performed in 12 healthy volunteers. In each phase, 850 mg metformin hydrochloride was administered p.o. in the evening of day 4 and in the morning of day 5. In phase B, 200 mg trimethoprim was administered additionally p.o. twice daily for 5 days. Metformin pharmacokinetics and effects (measured by OGTT) and NMN pharmacokinetics were determined. RESULTS: Trimethoprim inhibited metformin transport with K i values of 27.2, 6.3, and 28.9 µM and NMN transport with IC50 values of 133.9, 29.1, and 0.61 µM for OCT2, MATE1, and MATE2-K, respectively. In the clinical study, trimethoprim increased metformin area under the plasma concentration-time curve (AUC) by 29.5 % and decreased metformin and NMN renal clearances by 26.4 and 19.9 %, respectively (p ≤ 0.01). Moreover, decreases of NMN and metformin renal clearances due to trimethoprim correlated significantly (r S=0.727, p=0.010). CONCLUSIONS: These data on the metformin-trimethoprim interaction support the potential utility of N(1)-methylnicotinamide as an endogenous probe for renal drug-drug interactions with involvement of renal cation transporters.

Interaction of the cardiovascular risk marker asymmetric dimethylarginine (ADMA) with the human cationic amino acid transporter 1 (CAT1).

Elevated plasma concentrations of endogenously formed asymmetric (ADMA) and symmetric dimethyl-l-arginine (SDMA) are associated with adverse clinical outcomes. Our aim was to investigate the cellular uptake properties of ADMA by the human cationic amino acid transporter 1 (CAT1; SLC7A1). Human embryonic kidney cells (HEK293) stably overexpressing CAT1 (HEK-CAT1) and vector-transfected control cells (HEK-VC) were established to determine cellular uptake of labeled [(3)H]ADMA and [(3)H]l-arginine. Uptake of ADMA and l-arginine were significantly (p<0.001) higher in HEK-CAT1 than in HEK-VC at all investigated concentrations. Apparent V(max) values of cellular ADMA and l-arginine uptake by CAT1 were 26.9 ± 0.8 and 11.0 ± 0.2 nmol mg protein(-1) min(-1), respectively. K(m) values were 183 ± 21 µmoll(-1) (ADMA) and 519 ± 36 µmoll(-1) (l-arginine). Uptake of ADMA was inhibited by l-arginine and SDMA with IC(50) values (95% CI) of 227 (69-742) µmoll(-1) and 273 (191-390) µmoll(-1), respectively. ADMA and SDMA inhibited CAT1-mediated uptake of l-arginine with IC(50) values of 758 (460-1251) µmoll(-1) and 789 (481-1295) µmoll(-1), respectively. Efflux of ADMA was significantly increased in HEK-CAT1 cells as compared to HEK-VC (p<0.05). CAT1 mediates the cellular uptake of ADMA. In its physiological concentration range ADMA is unlikely to impair CAT1-mediated transport of l-arginine. Conversely, high (but still physiological) concentrations of l-arginine can inhibit CAT1-mediated cellular uptake of ADMA.

Automotive Radar Processing With Spiking Neural Networks: Concepts and Challenges.

Frequency-modulated continuous wave radar sensors play an essential role for assisted and autonomous driving as they are robust under all weather and light conditions. However, the rising number of transmitters and receivers for obtaining a higher angular resolution increases the cost for digital signal processing. One promising approach for energy-efficient signal processing is the usage of brain-inspired spiking neural networks (SNNs) implemented on neuromorphic hardware. In this article we perform a step-by-step analysis of automotive radar processing and argue how spiking neural networks could replace or complement the conventional processing. We provide SNN examples for two processing steps and evaluate their accuracy and computational efficiency. For radar target detection, an SNN with temporal coding is competitive to the conventional approach at a low compute overhead. Instead, our SNN for target classification achieves an accuracy close to a reference artificial neural network while requiring 200 times less operations. Finally, we discuss the specific requirements and challenges for SNN-based radar processing on neuromorphic hardware. This study proves the general applicability of SNNs for automotive radar processing and sustains the prospect of energy-efficient realizations in automated vehicles.

Characterization of beta-adrenoceptor antagonists as substrates and inhibitors of the drug transporter P-glycoprotein.

Transporter proteins such as P-glycoprotein are major determinants of intracellular drug concentrations. Moreover, inhibition or induction of transporters is an important mechanism underlying drug interactions in humans. However, very little is known whether beta-adrenoceptor antagonists are substrates and/or inhibitors of P-glycoprotein. Therefore, we investigated the P-glycoprotein-mediated transport of propranolol, metoprolol, bisoprolol, carvedilol and sotalol in P-glycoprotein-expressing Caco-2 monolayers and inhibition of P-glycoprotein-mediated digoxin transport by the beta-adrenoceptor antagonists. A significant inhibition of polarized, basal to apical drug transport by the P-glycoprotein inhibitor PSC-833 was observed for bisoprolol (0.5 and 5 microm) and carvedilol (0.5 microm). Moreover, propranolol and carvedilol inhibited P-glycoprotein-mediated digoxin transport with IC(50) values of 24.8 and 0.16 microm, respectively, whereas metoprolol and sotalol had no effect. Bisoprolol significantly inhibited directional digoxin transport at 50 and 250 microm by 31% and 44%, respectively. Taken together, P-glycoprotein is likely to be one determinant of bisoprolol and carvedilol disposition in humans. In addition, the beta-adrenoceptor antagonists propranolol and carvedilol significantly inhibit P-glycoprotein function thereby possibly contributing to drug interactions in humans (e.g. digoxin-carvedilol and cyclosporine-carvedilol).

Aceclofenac spares cyclooxygenase 1 as a result of limited but sustained biotransformation to diclofenac.

OBJECTIVE: The mechanism of action of aceclofenac is currently unclear. This study investigated whether biotransformation to metabolites (4'-hydroxy-aceclofenac, diclofenac, 4'-hydroxy-diclofenac) contributes to inhibitory effects on the cyclooxygenase (COX) isozymes in vitro and ex vivo. METHODS: In vitro investigations were performed with human whole blood and human blood monocytes. A randomized crossover study was performed in volunteers receiving 100 mg aceclofenac or a sustained-release resinate formulation of 75 mg diclofenac to assess the pharmacokinetics and the ex vivo inhibition of COX-1. RESULTS: In short-term in vitro assays, neither aceclofenac nor 4'-hydroxy-aceclofenac affected COX-1 or COX-2, whereas diclofenac and 4'-hydroxy-diclofenac inhibited both isoforms. In long-term in vitro assays, aceclofenac and 4'-hydroxy-aceclofenac suppressed both COX isoforms. However, this inhibition was paralleled by a conversion to diclofenac and 4'-hydroxy-diclofenac, respectively. Maximal plasma concentrations of diclofenac after oral administration of aceclofenac (0.39 micromol/L) or diclofenac (1.28 micromol/L) were sufficient for a greater than 97% inhibition of COX-2 (50% inhibitory concentration, 0.024 micromol/L) and a 46% (aceclofenac treatment) or 82% inhibition (diclofenac treatment) of COX-1 (50% inhibitory concentration, 0.43 micromol/L). Moreover, ex vivo COX-1-dependent thromboxane B(2) synthesis was inhibited significantly less by aceclofenac than by diclofenac. CONCLUSIONS: Inhibition of COX isozymes by aceclofenac requires conversion into diclofenac. Although 100 mg aceclofenac yielded diclofenac concentrations substantially lower than 75 mg diclofenac, these were sufficient for a sustained block of COX-2 but caused a minor and shorter inhibition of COX-1 than 75 mg diclofenac. In conclusion, both COX-1-sparing and COX-2-inhibitory actions of aceclofenac may rest in its limited but sustained biotransformation to diclofenac.

The prostaglandin transporter OATP2A1 is expressed in human ocular tissues and transports the antiglaucoma prostanoid latanoprost.

PURPOSE: Latanoprost, a prostaglandin F(2alpha) analogue, has become one of the most widely used medications for the treatment of glaucoma. The authors hypothesized that organic anion transporting polypeptides (OATPs) are responsible for the uptake of latanoprost into ocular tissues and, hence, that they contribute to the interindividual differences in drug concentrations and effects. METHODS: Expression of prostaglandin (PG) transporters (OATP2A1, OATP2B1) in human ocular tissues was determined using real-time RT-PCR and immunofluorescence. The inhibitory interactions between latanoprost and its active metabolite (the free acid) and the uptake of prototypical substrates (PGE(2) and bromosulfophthalein) were tested in stably transfected human embryonic kidney cells overexpressing either OATP2A1 or OATP2B1. These cells were also used to investigate whether latanoprost and latanoprost acid are substrates of OATP2A1 or OATP2B1. RESULTS: OATP2A1 and OATP2B1 mRNA expression was highest in the choroid/retinal pigment epithelium (RPE) complex and ciliary body. OATP2A1 protein expression was most prominent in the RPE and in epithelial and endothelial cell layers of anterior segment tissues, such as cornea, conjunctiva, iris, and ciliary body, whereas OATP2B1 protein was additionally expressed in trabecular meshwork, Schlemm canal, and choroidal vasculature. Latanoprost and latanoprost acid significantly inhibited both OATP2A1 and OATP2B1. Uptake experiments demonstrated that latanoprost acid is effectively transported by OATP2A1 (affinity constant [K(m)], 5.4 microM; maximum uptake rate [V(max)], 21.5 pmol/mg protein/min) and less effectively by OATP2B1. CONCLUSIONS: The results presented herein suggest that at least OATP2A1 plays a role in the intraocular disposition of the therapeutically used prostanoid latanoprost.

The influence of macrolide antibiotics on the uptake of organic anions and drugs mediated by OATP1B1 and OATP1B3.

Macrolides may cause severe drug interactions due to the inhibition of metabolizing enzymes. Transporter-mediated uptake of drugs into cells [e.g., by members of the human organic anion transporting polypeptide (OATP) family] is a determinant of drug disposition and a prerequisite for subsequent metabolism. However whether macrolides are also inhibitors of uptake transporters, thereby providing an additional mechanism of drug interactions, has not been systematically studied. The human OATP family members OATP1B1 and OATP1B3 mediate the uptake of endogenous substances and drugs such as antibiotics and HMG-CoA reductase inhibitors (statins) into hepatocytes. In this study we investigated the potential role of these uptake transporters on macrolide-induced drug interactions. By using sulfobromophthalein (BSP) and the HMG-CoA reductase inhibitor pravastatin as substrates, the effects of the macrolides azithromycin, clarithromycin, erythromycin, and roxithromycin and of the ketolide telithromycin on the OATP1B1- and OATP1B3-mediated uptake were analyzed. These experiments demonstrated that the OATP1B1- and OATP1B3-mediated uptake of BSP and pravastatin can be inhibited by increasing concentrations of all macrolides except azithromycin. The IC50 values for the inhibition of OATP1B3-mediated BSP uptake were 11 microM for telithromycin, 32 microM for clarithromycin, 34 microM for erythromycin, and 37 microM for roxithromycin. These IC50 values were lower than the IC50 values for inhibition of OATP1B1-mediated BSP uptake (96-217 microM). These macrolides also inhibited in a concentration-dependent manner the OATP1B1- and OATP1B3-mediated uptake of pravastatin. In summary, these results indicate that alterations of uptake transporter function by certain macrolides/ketolides have to be considered as a potential additional mechanism underlying drug-drug interactions.

Simultaneous determination of aceclofenac and three of its metabolites in human plasma by high-performance liquid chromatography.

Aceclofenac [[2-(2',6'-dichlorophenyl)amino]phenylacetoxyacetic acid] is a phenylacetic acid derivative with potent analgesic and anti-inflammatory properties and an improved gastro-intestinal tolerance. In the present study, a liquid-liquid extraction-based reversed-phase HPLC method with UV detection was validated and applied for the analysis of aceclofenac and three of its metabolites (4'-hydroxy-aceclofenac, diclofenac, 4'-hydroxy-diclofenac) in human plasma. The analytes were separated using an acetonitrile-phosphate buffer gradient at a flow rate of 1 mL/min, and UV detection at 282 nm. The retention times for aceclofenac, diclofenac, 4'-hydroxy-aceclofenac, 4'-hydroxy-diclofenac and ketoprofen (internal standard) were 69.1, 60.9, 46.9, 28.4 and 21.2 min, respectively. The validated quantitation range of the method was 10-10000 ng/mL for aceclofenac, 4'-hydroxy-aceclofenac and diclofenac, and 25-10000 ng/mL for 4'-hydroxy-diclofenac. The developed procedure was applied to assess the pharmacokinetics of aceclofenac and its metabolites following administration of a single 100 mg oral dose of aceclofenac to three healthy male volunteers.