Quantification of plasma carnitine and acylcarnitines by high-performance liquid chromatography-tandem mass spectrometry using online solid-phase extraction

Carnitine is an amino acid derivative that plays a key role in energy metabolism. Endogenous carnitine is found in its free form or esterified with acyl groups of several chain lengths. Quantification of carnitine and acylcarnitines is of particular interest for screening for research and metabolic disorders. We developed a method with online solid-phase extraction coupled to high-performance liquid chromatography and tandem mass spectrometry to quantify carnitine and three acylcarnitines with different polarity (acetylcarnitine, octanoylcarnitine, and palmitoylcarnitine). Plasma samples were deproteinized with methanol, loaded on a cation exchange trapping column and separated on a reversed-phase C8 column using heptafluorobutyric acid as an ion-pairing reagent. Considering the endogenous nature of the analytes, we quantified with the standard addition method and with external deuterated standards. Solid-phase extraction and separation were achieved within 8min. Recoveries of carnitine and acylcarnitines were between 98 and 105%. Both quantification methods were equally accurate (all values within 84 to 116% of target concentrations) and precise (day-to-day variation of less than 18%) for all carnitine species and concentrations analyzed. The method was used successfully for determination of carnitine and acylcarnitines in different human samples. In conclusion, we present a method for simultaneous quantification of carnitine and acylcarnitines with a rapid sample work-up. This approach requires small sample volumes and a short analysis time, and it can be applied for the determination of other acylcarnitines than the acylcarnitines tested. The method is useful for applications in research and clinical routine. Figure A method is presented for the analysis of carnitine and acylcarnitines in urine which includes a precipitation step, on-column extraction and LC-MS/MS. The run time is 8 minutes and the method was validated for carnitine, acetylcarnitine, octanoylcarnitine and palmitoylcarnitine. Analysis of a patient sample with medium-chain acyl-CoA dehydrogenase deficiency is show

Cytochrome P450 Enzymes Involved in Metoprolol Metabolism and Use of Metoprolol as a CYP2D6 Phenotyping Probe Drug

Metoprolol is used for phenotyping of cytochrome P450 (CYP) 2D6, a CYP isoform considered not to be inducible by inducers of the CYP2C, CYP2B, and CYP3A families such as rifampicin. While assessing CYP2D6 activity under basal conditions and after pre-treatment with rifampicin in vivo, we surprisingly observed a drop in the metoprolol/α-OH-metoprolol clearance ratio, suggesting CYP2D6 induction. To study this problem, we performed in vitro investigations using HepaRG cells and primary human hepatocytes (before and after treatment with 20 μM rifampicin), human liver microsomes, and CYP3A4-overexpressing supersomes. While mRNA expression levels of CYP3A4 showed a 15- to 30-fold increase in both cell models, mRNA of CYP2D6 was not affected by rifampicin. 1′-OH-midazolam formation (reflecting CYP3A4 activity) increased by a factor of 5–8 in both cell models, while the formation of α-OH-metoprolol increased by a factor of 6 in HepaRG cells and of 1.4 in primary human hepatocytes. Inhibition studies using human liver microsomes showed that CYP3A4, 2B6, and 2C9 together contributed 19.0 ± 2.6% (mean ± 95%CI) to O-demethylation, 4.0 ± 0.7% to α-hydroxylation, and 7.6 ± 1.7% to N-dealkylation of metoprolol. In supersomes overexpressing CYP3A4, metoprolol was α-hydroxylated in a reaction inhibited by the CYP3A4-specific inhibitor ketoconazole, but not by the CYP2D6-specific inhibitor quinidine. We conclude that metoprolol is not exclusively metabolized by CYP2D6. CYP3A4, 2B6, and 2C9, which are inducible by rifampicin, contribute to α-hydroxylation, O-demethylation, and N-dealkylation of metoprolol. This contribution is larger after CYP induction by rifampicin but is too small to compromise the usability of metoprolol α-hydroxylation for CYP2D6 phenotyping

Clinical Assessment of Potential Drug Interactions of Faldaprevir, a Hepatitis C Virus Protease Inhibitor, With Darunavir/Ritonavir, Efavirenz, and Tenofovir

Faldaprevir is a potent hepatitis C virus NS3/4A protease inhibitor. The findings from 3 phase 1 studies reported here suggest that faldaprevir can be safely coadministered with commonly used antiretroviral

Liver Cirrhosis Affects the Pharmacokinetics of the Six Substrates of the Basel Phenotyping Cocktail Differently.

BACKGROUND Activities of hepatic cytochrome P450 enzymes (CYPs) are relevant for hepatic clearance of drugs and known to be decreased in patients with liver cirrhosis. Several studies have reported the effect of liver cirrhosis on CYP activity, but the results are partially conflicting and for some CYPs lacking. OBJECTIVE In this study, we aimed to investigate the CYP activity in patients with liver cirrhosis with different Child stages (A-C) using the Basel phenotyping cocktail approach. METHODS We assessed the pharmacokinetics of the six compounds and their CYP-specific metabolites of the Basel phenotyping cocktail (CYP1A2: caffeine, CYP2B6: efavirenz, CYP2C9: flurbiprofen, CYP2C19: omeprazole, CYP2D6: metoprolol, CYP3A: midazolam) in patients with liver cirrhosis (n = 16 Child A cirrhosis, n = 15 Child B cirrhosis, n = 5 Child C cirrhosis) and matched control subjects (n = 12). RESULTS While liver cirrhosis only marginally affected the pharmacokinetics of the low to moderate extraction drugs efavirenz and flurbiprofen, the elimination rate of caffeine was reduced by 51% in patients with Child C cirrhosis. For the moderate to high extraction drugs omeprazole, metoprolol, and midazolam, liver cirrhosis decreased the elimination rate by 75%, 37%, and 60%, respectively, increased exposure, and decreased the apparent systemic clearance (clearance/bioavailability). In patients with Child C cirrhosis, the metabolic ratio (ratio of the area under the plasma concentration-time curve from 0 to 24 h of the metabolite to the parent compound), a marker for CYP activity, decreased by 66%, 47%, 92%, 73%, and 43% for paraxanthine/caffeine (CYP1A2), 8-hydroxyefavirenz/efavirenz (CYP2B6), 5-hydroxyomeprazole/omeprazole (CYP2C19), α-hydroxymetoprolol/metoprolol (CYP2D6), and 1'-hydroxymidazolam/midazolam (CYP3A), respectively. In comparison, the metabolic ratio 4-hydroxyflurbiprofen/flurbiprofen (CYP2C9) remained unchanged. CONCLUSIONS Liver cirrhosis affects the activity of CYP isoforms differently. This variability must be considered for dose adjustment of drugs in patients with liver cirrhosis. CLINICAL TRIAL REGISTRATION NCT03337945

Non-immunological toxicological mechanisms of metamizole-associated neutropenia in HL60 cells

Metamizole is an  analgesic and  antipyretic , but can cause  neutropenia and  agranulocytosis . We investigated the toxicity of the metabolites N-methyl-4-aminoantipyrine (MAA),  4-aminoantipyrine (AA), N-formyl-4-aminoantipyrine (FAA) and N-acetyl-4-aminoantipyrine (AAA) on neutrophil granulocytes and on HL60 cells (granulocyte precursor cell line). MAA, FAA, AA, and AAA (up to 100 µM) alone were not toxic for HL60 cells or granulocytes. In the presence of the  myeloperoxidase substrate H2O2, MAA reduced cytotoxicity for HL60 cells at low concentrations (<50 µM), but increased cytotoxicity at 100 µM H2O2. Neutrophil granulocytes were resistant to H2O2 and MAA. Fe2+ and Fe3+ were not toxic to HL60 cells, irrespective of the presence of H2O2 and MAA. Similarly, MAA did not increase the toxicity of  lactoferrin ,  hemoglobin or  methemoglobin for HL60 cells.  Hemin (hemoglobin degradation product containing a  porphyrin ring and Fe3+) was toxic on HL60 cells and cytotoxicity was increased by MAA.  EDTA , N-acetylcystein and  glutathione prevented the toxicity of hemin and hemin/MAA. The absorption spectrum of hemin changed concentration-dependently after addition of MAA, suggesting an interaction between Fe3+and MAA. NMR revealed the formation of a stable MAA reaction product with a reaction pathway involving the formation of an electrophilic intermediate. In conclusion, MAA, the principle metabolite of metamizole, increased cytotoxicity of hemin by a reaction involving the formation of an electrophilic metabolite. Accordingly, cytotoxicity of MAA/hemin could be prevented by the  iron chelator EDTA and by the electron donors NAC and glutathione. Situations with increased production of hemin may represent a risk factor for metamizole-associated  granulocytopenia

Ticagrelor induced systemic inflammatory response syndrome.

BACKGROUND Ticagrelor is a reversible and direct-acting oral antagonist of the adenosine diphosphate receptor P2Y12. Possible adenosine-mediated effects of ticagrelor on inflammation are complex and incompletely understood. To our knowledge, ticagrelor-induced systemic inflammatory response syndrome (SIRS) has not yet been described. CASE PRESENTATION We report the case of an 84 years old patient presenting with SIRS subsequent to initiation of ticagrelor after implantation of two drug eluting stents. A broad diagnostic work-up for alternative causes and therapeutic measures were unrevealing. Discontinuation of the agent was followed by rapid improvement in clinical and laboratory signs of SIRS. CONCLUSIONS After exclusion of other causes, ticagrelor needs to be considered as a possible causative agent for SIRS. Due to the widespread use of ticagrelor, clinicians should be aware of this possible adverse drug reaction

Therapeutic drug monitoring of once daily aminoglycoside dosing: comparison of two methods and investigation of the optimal blood sampling strategy

Purpose: Therapeutic drug monitoring of patients receiving once daily aminoglycoside therapy can be performed using pharmacokinetic (PK) formulas or Bayesian calculations. While these methods produced comparable results, their performance has never been checked against full PK profiles. We performed a PK study in order to compare both methods and to determine the best time-points to estimate AUC0-24 and peak concentrations (C max). Methods: We obtained full PK profiles in 14 patients receiving a once daily aminoglycoside therapy. PK parameters were calculated with PKSolver using non-compartmental methods. The calculated PK parameters were then compared with parameters estimated using an algorithm based on two serum concentrations (two-point method) or the software TCIWorks (Bayesian method). Results: For tobramycin and gentamicin, AUC0-24 and C max could be reliably estimated using a first serum concentration obtained at 1h and a second one between 8 and 10h after start of the infusion. The two-point and the Bayesian method produced similar results. For amikacin, AUC0-24 could reliably be estimated by both methods. C max was underestimated by 10-20% by the two-point method and by up to 30% with a large variation by the Bayesian method. Conclusions: The ideal time-points for therapeutic drug monitoring of once daily administered aminoglycosides are 1h after start of a 30-min infusion for the first time-point and 8-10h after start of the infusion for the second time-point. Duration of the infusion and accurate registration of the time-points of blood drawing are essential for obtaining precise predictions

Influence of Antihypertensive Treatment on RAAS Peptides in Newly Diagnosed Hypertensive Patients.

(1) Background: Recently, influences of antihypertensive treatment on the renin-angiotensin-aldosterone system (RAAS) has gained attention, regarding a possible influence on inflammatory and anti-inflammatory pathways. We aimed to study the effects of newly initiated antihypertensive drugs on angiotensin (Ang) II and Ang (1-7) as representers of two counter-regulatory axes. (2) Methods: In this randomized, open-label trial investigating RAAS peptides after the initiation of perindopril, olmesartan, amlodipine, or hydrochlorothiazide, Ang II and Ang (1-7) equilibrium concentrations were measured at 8 a.m. and 12 a.m. at baseline and after four weeks of treatment. Eighty patients were randomized (1:1:1:1 fashion). (3) Results: Between the four substances, we found significant differences regarding the concentrations of Ang II (p < 0.0005 for 8 a.m., 12 a.m.) and Ang (1-7) (p = 0.019 for 8 a.m., <0.0005 for 12 a.m.) four weeks after treatment start. Ang II was decreased by perindopril (p = 0.002), and increased by olmesartan (p < 0.0005), amlodipine (p = 0.012), and hydrochlorothiazide (p = 0.001). Ang (1-7) was increased by perindopril and olmesartan (p = 0.008/0.002), but not measurably altered by amlodipine and hydrochlorothiazide (p = 0.317/ 0.109). (4) Conclusion: The initiation of all first line antihypertensive treatments causes early and distinct alterations of equilibrium angiotensin levels. Given the additional AT1R blocking action of olmesartan, RAAS peptides shift upon initiation of perindopril and olmesartan appear to work in favor of the anti-inflammatory axis compared to amlodipine and hydrochlorothiazide