The Activity of Members of the UDP-Glucuronosyltransferase Subfamilies UGT1A and UGT2B is Impaired in Patients with Liver Cirrhosis.

BACKGROUND AND OBJECTIVE: The impact of liver cirrhosis on the activity of UDP-glucuronosyltransferases (UGTs) is currently not well characterized. We investigated the glucuronidation capacity and glucuronide accumulation in patients with liver cirrhosis. METHODS: We administered the Basel phenotyping cocktail (caffeine, efavirenz, flurbiprofen, omeprazole, metoprolol, midazolam) to patients with liver cirrhosis (n = 16 Child A, n = 15 Child B, n = 5 Child C) and n = 12 control subjects and obtained pharmacokinetic profiles of substrates and primary metabolites and their glucuronides. RESULTS: Caffeine and its metabolite paraxanthine were only slightly glucuronidated. The metabolic ratio (AUCglucuronide/AUCparent, MR) was not affected for caffeine but decreased by 60% for paraxanthine glucuronide formation in Child C patients. Efavirenz was not glucuronidated whereas 8-hydroxyefavirenz was efficiently glucuronidated. The MR of 8-hydroxyefavirenz-glucuronide formation increased three-fold in Child C patients and was negatively correlated with the glomerular filtration rate. Flurbiprofen and omeprazole were not glucuronidated. 4-Hydroxyflurbiprofen and 5-hydroxyomeprazole were both glucuronidated but the corresponding MRs for glucuronide formation were not affected by liver cirrhosis. Metoprolol, but not α-hydroxymetoprolol, was glucuronidated, and the MR for metoprolol-glucuronide formation dropped by 60% in Child C patients. Both midazolam and its metabolite 1'-hydroxymidazolam underwent glucuronidation, and the corresponding MRs for glucuronide formation dropped by approximately 80% in Child C patients. No relevant glucuronide accumulation occurred in patients with liver cirrhosis. CONCLUSIONS: Detailed analysis revealed that liver cirrhosis may affect the activity of UGTs of the UGT1A and UGT2B subfamilies according to liver function. Clinically significant glucuronide accumulation did not occur in the population investigated. CLINICAL TRIAL REGISTRATION: NCT03337945.

Dose Adjustment in Patients with Liver Cirrhosis - Comparison of Two Different Modeling Approaches.

Failure to perform adequate dose adjustment in patients with liver cirrhosis may be associated with increased toxicity. We compared the prediction of area under the curve (AUC) and clearance for the six compounds of the Basel phenotyping cocktail (caffeine, efavirenz, flurbiprofen, omeprazole, metoprolol, and midazolam) using a well-known physiology-based pharmacokinetic approach (physiologically-based pharmacokinetic [PBPK] approach, Simcyp) and a novel top-down method based on the systemic clearance in healthy volunteers adjusted for markers of liver and renal dysfunction ("top-down approach"). With few exceptions, plasma concentration-time curves were accurately predicted by the PBPK approach. In comparison to the measured AUC and clearance of these drugs in patients with liver cirrhosis and healthy controls, except for efavirenz, the estimates of both approaches were within two standard deviations of the mean for total and free drug concentrations. For both approaches, a correction factor for dose adjustment in patients with liver cirrhosis could be calculated for the drugs administered. AUCs calculated using the adjusted doses were comparable to the AUCs measured in control subjects, with slightly more accurate predictions generated by the PBPK approach. For drugs with a free fraction < 50%, predictions using free drug concentrations were more accurate than with total drug concentrations. In conclusion, both methods provided good qualitative predictions of the changes by liver cirrhosis in the pharmacokinetics of the six compounds investigated. The top-down approach is easier to implement but the PBPK approach predicted changes in drug exposure more accurately than the top-down approach and provided reliable estimates for plasma concentrations.

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.

Cytochrome P450 1A2 is the most important enzyme for hepatic metabolism of the metamizole metabolite 4-methylaminoantipyrine.

AIMS: Metamizole (dipyrone) is a prodrug not detectable in serum or urine after oral ingestion. The primary metabolite, 4-methylaminoantipyrine (4-MAA), can be N-demethylated to 4-aminoantipyrine (4-AA) or oxidized to 4-formylaminoantipyrine (4-FAA) by cytochrome P450 (CYP)-dependent reactions. We aimed to identify the CYPs involved in 4-MAA metabolism and to quantify the effect of CYP inhibition on 4-MAA metabolism. METHODS: We investigated the metabolism of 4-MAA in vitro using CYP expressing supersomes and the pharmacokinetics of metamizole in the presence of CYP inhibitors in male subjects. RESULTS: The experiments in supersomes revealed CYP1A2 as the major CYP for 4-MAA N-demethylation and 4-FAA formation with CYP2C19 and CYP2D6 contributing to N-demethylation. In the clinical study, we investigated the influence of ciprofloxacin (CYP1A2 inhibitor), fluconazole (CYP2C19 inhibitor) and the combination ciprofloxacin/fluconazole on the pharmacokinetics of metamizole in n = 12 male subjects in a randomized, placebo-controlled, double-blind study. The geometric mean ratios for the area under the concentration-time curve of 4-MAA after/before treatment were 1.17 (90% CI 1.09-1.25) for fluconazole, 1.51 (90% CI 1.42-1.60) for ciprofloxacin and 1.92 (90% CI 1.81-2.03) for ciprofloxacin/fluconazole. Fluconazole increased the half-life of 4-MAA from 3.22 hours by 0.47 hours (95% CI 0.13-0.81, P < .05), ciprofloxacin by 0.69 hours (95% CI 0.44-0.94, P < .001) and fluconazole/ciprofloxacin by 2.85 hours (95% CI 2.48-3.22, P < .001). CONCLUSION: CYP1A2 is the major CYP for the conversion of 4-MAA to 4-AA and 4-FAA. The increase in 4-MAA exposure by the inhibition of CYP1A2 and by the combination CYP1A2/CYP2C19 may be relevant for dose-dependent adverse reactions of 4-MAA.

Effect of deglucuronidation on the results of the Basel phenotyping cocktail.

We investigated the effect of deglucuronidation on the plasma concentration of the constituents of the Basel phenotyping cocktail and on the interpretation of the phenotyping results under basal conditions and after cytochrome P450 (CYP) induction with metamizole. The cocktail containing caffeine (CYP1A2), efavirenz (CYP2B6), flurbiprofen (CYP2C9), omeprazole (CYP2C19), metoprolol (CYP2D6) and midazolam (CYP3A4) was administered to 12 healthy subjects before (basal) and after treatment with metamizole for 1 week. In the basal state, deglucuronidation caused an increase in the plasma concentrations and area under the curve (AUC) of metoprolol, 8'-hydroxyefavirenz, 4'-hydroxyflurbiprofen and 1'-hydroxymidazolam. This effect could be visualized in Bland-Altman plots, where the values for 8'-hydroxyefavirenz, 4'-hydroxyflurbiprofen and 1'-hydroxymidazolam were mostly above the +20% threshold. As a result, the metabolic ratio (MR), calculated as AUCparent drug /AUCmetabolite , decreased with deglucuronidation for CYP2B6, CYP2C9 and CYP3A4 and increased for CYP2D6. Treatment with metamizole, a constitutive androstane receptor-dependent inducer of CYP2B6, CYP2C9, CYP2C19 and CYP3A4, accentuated the effect of deglucuronidation on AUC and MR. The correlation of MRs calculated as the plasma concentration ratio parent drug/metabolite with the MR calculated as the AUC ratio showed that 1 sample obtained between 2 and 6 hours after cocktail ingestion and analysed with and without deglucuronidation is sufficient to obtain reliable phenotyping results. Importantly, CYP2C9 and 3A4 induction would have been missed without deglucuronidation of the plasma samples. In conclusion, deglucuronidation of the plasma samples improves the stability of the phenotyping results of the Basel phenotyping cocktail and is necessary to reliably detect CYP induction.

Metamizole is a Moderate Cytochrome P450 Inducer Via the Constitutive Androstane Receptor and a Weak Inhibitor of CYP1A2.

Metamizole is an analgesic and antipyretic drug used intensively in certain countries. Previous studies have shown that metamizole induces cytochrome (CYP) 2B6 and possibly CYP3A4. So far, it is unknown whether metamizole induces additional CYPs and by which mechanism. Therefore, we assessed the activity of 6 different CYPs in 12 healthy male subjects before and after treatment with 3 g of metamizole per day for 1 week using a phenotyping cocktail approach. In addition, we investigated whether metamizole induces CYPs by an interaction with the constitutive androstane receptor (CAR) or the pregnane X receptor (PXR) in HepaRG cells. In the clinical study, we confirmed a moderate induction of CYP2B6 (decrease in the efavirenz area under the plasma concentration time curve (AUC) by 79%) and 3A4 (decrease in the midazolam AUC by 68%) by metamizole. In addition, metamizole weakly induced CYP2C9 (decrease in the flurbiprofen AUC by 22%) and moderately CYP2C19 (decrease in the omeprazole AUC by 66%) but did not alter CYP2D6 activity. In addition, metamizole weakly inhibited CYP1A2 activity (1.79-fold increase in the caffeine AUC). We confirmed these results in HepaRG cells, where 4-MAA, the principal metabolite of metamizole, induced the mRNA expression of CYP2B6, 2C9, 2C19, and 3A4. In HepaRG cells with a stable knockout of PXR or CAR, we could demonstrate that CYP induction by 4-MAA depends on CAR and not on PXR. In conclusion, metamizole is a broad CYP inducer by an interaction with CAR and an inhibitor of CYP1A2. Regarding the widespread use of metamizole, these findings are of substantial clinical relevance.

Development and validation of an LC-MS/MS method for the bioanalysis of the major metamizole metabolites in human plasma.

Aim: Metamizole is a frequently used antipyretic and analgesic prodrug, yet its pharmacokinetics has not been thoroughly studied in infants and with coadministered medications. Thus, an LC-MS/MS method was developed to quantify the four major metamizole metabolites in human plasma. Methodology: Pre- and postcolumn infusion was installed to enable robust analyte retention and electrospray ionization following deproteinization of plasma samples. Results: The method was linear (R > 0.996), accurate (93.1-106.0%) and precise (≤12.7%). Mean recovery was more than 91.8% and ion suppression less than 13.1% for all analytes. Pharmacokinetic profiles were reproducible after 4 years at -80°C except for the formylated metabolite (-22.2%). Conclusion: The method fulfilled pertinent criteria of validation guidelines and required only little sample volume. The method therefore qualifies for metamizole analyses in children.

Dose evaluation of intravenous metamizole (dipyrone) in infants and children: a prospective population pharmacokinetic study.

PURPOSE: The prodrug metamizole is prescribed intravenously for postoperative pain in children, including off-label use in infants < 1 year. We aimed to assess the pharmacokinetics of the main metabolites of metamizole in children aged 3-72 months. METHODS: A single dose of 10 mg/kg metamizole was administered intravenously for postoperative analgesia. Pharmacokinetic samples were drawn at predefined time points. Pharmacokinetics of the main active metabolite 4-methylaminoantipyrine and three other metabolites was characterized by both non-compartmental and population pharmacokinetic analysis. AUC0-inf of 4-methylaminoantipyrine was calculated by non-compartmental analysis for two age cohorts (3-23 months, 2-6 years) and compared with the 80-125% range of adult dose-adjusted reference exposure (AUCref). Population pharmacokinetic analysis investigated age and weight dependency of the pharmacokinetics and optimal dosing strategies to achieve equivalent adult exposure. RESULTS: A total of 25 children aged 5 months-5.8 years (7.8-24.8 kg) with at least one concentration sample were included; 19 children had ≥ 5 predefined samples up to 10 h after metamizole dose administration. AUC0-inf of 4-methylaminoantipyrine in children 2-6 years was 29.9 mg/L/h (95% CI 23.4-38.2), significantly lower than AUCref (80-125% range 39.2-61.2 mg/L/h). AUC0-inf of 4-methylaminoantipyrine in infants < 2 years was 43.6 mg/L/h (95% CI 15.8-119.0), comparable with AUCref, while infants < 12 months showed increased exposure. Observed variability could be partially explained by covariates weight and age. CONCLUSIONS: Age-related changes in pharmacokinetics of 4-methylaminoantipyrine requires reduced weight-based IV dosing in infants < 1 year compared with infants and children up to 6 years (5 versus 10-20 mg/kg) to achieve equivalent adult exposure. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT02660177 .

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.

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.

N-demethylation of N-methyl-4-aminoantipyrine, the main metabolite of metamizole.

Metamizole is an old analgesic used frequently in some countries. Active metabolites of metamizole are the non-enzymatically generated N-methyl-4-aminoantipyrine (4-MAA) and its demethylation product 4-aminoantipyrine (4-AA). Previous studies suggested that 4-MAA demethylation can be performed by hepatic cytochrome P450 (CYP) 3A4, but the possible contribution of other CYPs remains unclear. Using human liver microsomes (HLM), liver homogenate and HepaRG cells, we could confirm 4-MAA demethylation by CYPs. Based on CYP induction (HepaRG cells) and CYP inhibition (HLM) we could identify CYP2B6, 2C8, 2C9 and 3A4 as major contributors to 4-MAA demethylation. The 4-MAA demethylation rate by HLM was 280 pmol/mg protein/h, too low to account for in vivo 4-MAA demethylation in humans. Since peroxidases can perform N-demethylation, we investigated horseradish peroxidase and human myeloperoxidase (MPO). Horse radish peroxidase efficiently demethylated 4-MAA, depending on the hydrogen peroxide concentration. This was also true for MPO; this reaction was saturable with a Km of 22.5 µM and a maximal velocity of 14 nmol/min/mg protein. Calculation of the entire body MPO capacity revealed that the demethylation capacity by granulocyte/granulocyte precursors was approximately 600 times higher than the liver capacity and could account for 4-MAA demethylation in humans. 4-MAA demethylation could also be demonstrated in MPO-expressing granulocyte precursor cells (HL-60). In conclusion, 4-MAA can be demethylated in the liver by several CYPs, but hepatic metabolism cannot fully explain 4-MAA demethylation in humans. The current study suggests that the major part of 4-MAA is demethylated by circulating granulocytes and granulocyte precursors in bone marrow.

Potential Antiosteoporotic Natural Product Lead Compounds That Inhibit 17ß-Hydroxysteroid Dehydrogenase Type 2.

17ß-Hydroxysteroid dehydrogenase type 2 (17ß-HSD2) converts the active steroid hormones estradiol, testosterone, and 5α-dihydrotestosterone into their weakly active forms estrone, Δ4-androstene-3,17-dione, and 5α-androstane-3,17-dione, respectively, thereby regulating cell- and tissue-specific steroid action. As reduced levels of active steroids are associated with compromised bone health and onset of osteoporosis, 17ß-HSD2 is considered a target for antiosteoporotic treatment. In this study, a pharmacophore model based on 17ß-HSD2 inhibitors was applied to a virtual screening of various databases containing natural products in order to discover new lead structures from nature. In total, 36 hit molecules were selected for biological evaluation. Of these compounds, 12 inhibited 17ß-HSD2 with nanomolar to low micromolar IC50 values. The most potent compounds, nordihydroguaiaretic acid (1), IC50 0.38 ± 0.04 µM, (-)-dihydroguaiaretic acid (4), IC50 0.94 ± 0.02 µM, isoliquiritigenin (6), IC50 0.36 ± 0.08 µM, and ethyl vanillate (12), IC50 1.28 ± 0.26 µM, showed 8-fold or higher selectivity over 17ß-HSD1. As some of the identified compounds belong to the same structural class, structure-activity relationships were derived for these molecules. Thus, this study describes new 17ß-HSD2 inhibitors from nature and provides insights into the binding pocket of 17ß-HSD2, offering a promising starting point for further research in this area.

Ligand-based pharmacophore modeling and virtual screening for the discovery of novel 17ß-hydroxysteroid dehydrogenase 2 inhibitors.

17ß-Hydroxysteroid dehydrogenase 2 (17ß-HSD2) catalyzes the inactivation of estradiol into estrone. This enzyme is expressed only in a few tissues, and therefore its inhibition is considered as a treatment option for osteoporosis to ameliorate estrogen deficiency. In this study, ligand-based pharmacophore models for 17ß-HSD2 inhibitors were constructed and employed for virtual screening. From the virtual screening hits, 29 substances were evaluated in vitro for 17ß-HSD2 inhibition. Seven compounds inhibited 17ß-HSD2 with low micromolar IC50 values. To investigate structure-activity relationships (SAR), 30 more derivatives of the original hits were tested. The three most potent hits, 12, 22, and 15, had IC50 values of 240 nM, 1 µM, and 1.5 µM, respectively. All but 1 of the 13 identified inhibitors were selective over 17ß-HSD1, the enzyme catalyzing conversion of estrone into estradiol. Three of the new, small, synthetic 17ß-HSD2 inhibitors showed acceptable selectivity over other related HSDs, and six of them did not affect other HSDs.