Physiologically Based Pharmacokinetic Modeling to Predict the Impact of Liver Cirrhosis on Glucuronidation via UGT1A4 and UGT2B7/2B4-A Case Study with Midazolam.

Hepatic impairment, due to liver cirrhosis, decreases the activity of cytochrome P450 enzymes (CYPs). The use of physiologically based pharmacokinetic (PBPK) modeling to predict this effect for CYP substrates has been well-established, but the effect of cirrhosis on uridine-glucuronosyltransferase (UGT) activities is less studied and few PBPK models have been reported. UGT enzymes are involved in primary N-glucuronidation of midazolam and glucuronidation of 1'-OH-midazolam following CYP3A hydroxylation. In this study, Simcyp was used to establish PBPK models for midazolam, its primary metabolites midazolam-N-glucuronide (UGT1A4) and 1'-OH midazolam (CYP3A4/3A5), and the secondary metabolite 1'-OH-midazolam-O-glucuronide (UGT2B7/2B4), allowing to simulate the impact of liver cirrhosis on the primary and secondary glucuronidation of midazolam. The model was verified in noncirrhotic subjects before extrapolation to cirrhotic patients of Child-Pugh (CP) classes A, B, and C. Our model successfully predicted the exposures of midazolam and its metabolites in noncirrhotic and cirrhotic patients, with 86% of observed plasma concentrations within 5th-95th percentiles of predictions and observed geometrical mean of area under the plasma concentration curve between 0 hours to infinity and maximal plasma concentration within 0.7- to 1.43-fold of predictions. The simulated metabolic ratio defined as the ratio of the glucuronide metabolite AUC over the parent compound AUC (AUCglucuronide/AUCparent, metabolic ratio [MR]), was calculated for midazolam-N-glucuronide to midazolam (indicative of UGT1A4 activity) and decreased by 40% (CP A), 48% (CP B), and 75% (CP C). For 1'-OH-midazolam-O-glucuronide to 1'-OH-midazolam, the MR (indicative of UGT2B7/2B4 activity) dropped by 35% (CP A), 51% (CP B), and 64% (CP C). These predicted MRs were corroborated by the observed data. This work thus increases confidence in Simcyp predictions of the effect of liver cirrhosis on the pharmacokinetics of UGT1A4 and UGT2B7/UGT2B4 substrates. SIGNIFICANCE STATEMENT: This article presents a physiologically based pharmacokinetic model for midazolam and its metabolites and verifies the accurate simulation of pharmacokinetic profiles when using the Simcyp hepatic impairment population models. Exposure changes of midazolam-N-glucuronide and 1'-OH-midazolam-O-glucuronide reflect the impact of decreases in UGT1A4 and UGT2B7/2B4 glucuronidation activity in cirrhotic patients. The approach used in this study may be extended to verify the modeling of other uridine glucuronosyltransferase enzymes affected by liver cirrhosis.

The pharmacokinetics and drug-drug interactions of ivermectin in Aedes aegypti mosquitoes.

Mosquitoes are vectors of major diseases such as dengue fever and malaria. Mass drug administration of endectocides to humans and livestock is a promising complementary approach to current insecticide-based vector control measures. The aim of this study was to establish an insect model for pharmacokinetic and drug-drug interaction studies to develop sustainable endectocides for vector control. Female Aedes aegypti mosquitoes were fed with human blood containing either ivermectin alone or ivermectin in combination with ketoconazole, rifampicin, ritonavir, or piperonyl butoxide. Drug concentrations were quantified by LC-MS/MS at selected time points post-feeding. Primary pharmacokinetic parameters and extent of drug-drug interactions were calculated by pharmacometric modelling. Lastly, the drug effect of the treatments was examined. The mosquitoes could be dosed with a high precision (%CV: ≤13.4%) over a range of 0.01-1 µg/ml ivermectin without showing saturation (R2: 0.99). The kinetics of ivermectin were characterised by an initial lag phase of 18.5 h (CI90%: 17.0-19.8 h) followed by a slow zero-order elimination rate of 5.5 pg/h (CI90%: 5.1-5.9 pg/h). By contrast, ketoconazole, ritonavir, and piperonyl butoxide were immediately excreted following first order elimination, whereas rifampicin accumulated over days in the mosquitoes. Ritonavir increased the lag phase of ivermectin by 11.4 h (CI90%: 8.7-14.2 h) resulting in an increased exposure (+29%) and an enhanced mosquitocidal effect. In summary, this study shows that the pharmacokinetics of drugs can be investigated and modulated in an Ae. aegypti animal model. This may help in the development of novel vector-control interventions and further our understanding of toxicology in arthropods.

Rats with Long-Term Cholestasis Have a Decreased Cytosolic but Maintained Mitochondrial Hepatic CoA Pool.

Previous studies showed that rats with long-term bile duct ligation have reduced coenzyme A stores per g of liver but maintained mitochondrial CoA stores. Based on these observations, we determined the CoA pool in the liver homogenate, liver mitochondria, and liver cytosol of rats with bile duct ligation for 4 weeks (BDL rats, n = 9) and sham-operated control rats (CON rats, n = 5). In addition, we tested the cytosolic and mitochondrial CoA pools by assessing the metabolism of sulfamethoxazole and benzoate in vivo and of palmitate in vitro. The hepatic total CoA content was lower in BDL than CON rats (mean ± SEM; 128 ± 5 vs. 210 ± 9 nmol/g), affecting all subfractions equally (free CoA (CoASH), short- and long-chain acyl-CoA). In BDL rats, the hepatic mitochondrial CoA pool was maintained, and the cytosolic pool was reduced (23.0 ± 0.9 vs. 84.6 ± 3.7 nmol/g liver; CoA subfractions were affected equally). The urinary excretion of hippurate after i.p. benzoate administration (measuring mitochondrial benzoate activation) was reduced in BDL rats (23.0 ± 0.9 vs. 48.6 ± 3.7% of dose/24 h), whereas the urinary elimination of N-acetylsulfamethoxazole after i.p. sulfamethoxazole administration (measuring the cytosolic acetyl-CoA pool) was maintained (36.6 ± 3.0 vs. 35.1 ± 2.5% of dose/24 h BDL vs. CON rats). Palmitate activation was impaired in the liver homogenate of BDL rats but the cytosolic CoASH concentration was not limiting. In conclusion, BDL rats have reduced hepatocellular cytosolic CoA stores, but this reduction does not limit sulfamethoxazole N-acetylation or palmitate activation. The hepatocellular mitochondrial CoA pool is maintained in BDL rats. Impaired hippurate formation in BDL rats is explained best by mitochondrial dysfunction.

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.

Mitochondrial Toxicity Associated with Imatinib and Sorafenib in Isolated Rat Heart Fibers and the Cardiomyoblast H9c2 Cell Line.

Tyrosine kinase inhibitors (TKIs) are associated with cardiac toxicity, which may be caused by mitochondrial toxicity. The underlying mechanisms are currently unclear and require further investigation. In the present study, we aimed to investigate in more detail the role of the enzyme complexes of the electron transfer system (ETS), mitochondrial oxidative stress, and mechanisms of cell death in cardiac toxicity associated with imatinib and sorafenib. Cardiac myoblast H9c2 cells were exposed to imatinib and sorafenib (1 to 100 µM) for 24 h. Permeabilized rat cardiac fibers were treated with both drugs for 15 min. H9c2 cells exposed to sorafenib for 24 h showed a higher membrane toxicity and ATP depletion in the presence of galactose (favoring mitochondrial metabolism) compared to glucose (favoring glycolysis) but not when exposed to imatinib. Both TKIs resulted in a higher dissipation of the mitochondrial membrane potential in galactose compared to glucose media. Imatinib inhibited Complex I (CI)- and CIII- linked respiration under both conditions. Sorafenib impaired CI-, CII-, and CIII-linked respiration in H9c2 cells cultured with glucose, whereas it inhibited all ETS complexes with galactose. In permeabilized rat cardiac myofibers, acute exposure to imatinib and sorafenib decreased CI- and CIV-linked respiration in the presence of the drugs. Electron microscopy showed enlarged mitochondria with disorganized cristae. In addition, both TKIs caused mitochondrial superoxide accumulation and decreased the cellular GSH pool. Both TKIs induced caspase 3/7 activation, suggesting apoptosis as a mechanism of cell death. Imatinib and sorafenib impaired the function of cardiac mitochondria in isolated rat cardiac fibers and in H9c2 cells at plasma concentrations reached in humans. Both imatinib and sorafenib impaired the function of enzyme complexes of the ETS, which was associated with mitochondrial ROS accumulation and cell death by apoptosis.

PGC-1α and MEF2 Regulate the Transcription of the Carnitine Transporter OCTN2 Gene in C2C12 Cells and in Mouse Skeletal Muscle.

OCTN2 (SLC22A5) is a carnitine transporter whose main function is the active transport of carnitine into cells. In skeletal muscle and other organs, the regulation of the SLC22A5 gene transcription has been shown to depend on the nuclear transcription factor PPAR-α. Due to the observation that the muscle OCTN2 mRNA level is maintained in PPAR-α knock-out mice and that PGC-1α overexpression in C2C12 myoblasts increases OCTN2 mRNA expression, we suspected additional regulatory pathways for SLC22A5 gene transcription. Indeed, we detected several binding sites of the myocyte-enhancing factor MEF2 in the upstream region of the SLC22A5 gene, and MEF2C/MEF2D stimulated the activity of the OCTN2 promoter in gene reporter assays. This stimulation was increased by PGC-1α and was blunted for a SLC22A5 promoter fragment with a mutated MEF2 binding site. Further, we demonstrated the specific binding of MEF2 to the SLC22A5 gene promoter, and a supershift of the MEF2/DNA complex in electrophoretic mobility shift assays. In immunoprecipitation experiments, we could demonstrate the interaction between PGC-1α and MEF2. In addition, SB203580, a specific inhibitor of p38 MAPK, blocked and interferon-γ stimulated the transcriptional activity of the SLC22A5 gene promoter. Finally, mice with muscle-specific overexpression of OCTN2 showed an increase in OCTN2 mRNA and protein expression in skeletal muscle. In conclusion, we detected and characterized a second stimulatory pathway of SLC22A5 gene transcription in skeletal muscle, which involves the nuclear transcription factor MEF2 and co-stimulation by PGC-1α and which is controlled by the p38 MAPK signaling cascade.

The Risk of Muscular Events Among New Users of Hydrophilic and Lipophilic Statins: an Observational Cohort Study.

BACKGROUND: Statins are effective lipid-lowering drugs for the prevention of cardiovascular disease, but muscular adverse events can limit their use. Hydrophilic statins (pravastatin, rosuvastatin) may cause less muscular events than lipophilic statins (e.g. simvastatin, atorvastatin) due to lower passive diffusion into muscle cells. OBJECTIVE: To compare the risk of muscular events between statins at comparable lipid-lowering doses and to evaluate if hydrophilic statins are associated with a lower muscular risk than lipophilic statins. DESIGN/SETTING: Propensity score-matched cohort study using data from the United Kingdom-based Clinical Practice Research Datalink (CPRD) GOLD. PATIENTS: New statin users. Cohort 1: pravastatin 20-40 mg (hydrophilic) vs simvastatin 10-20 mg (lipophilic), cohort 2: rosuvastatin 5-40 mg (hydrophilic) vs atorvastatin 10-80 mg (lipophilic), and cohort 3: simvastatin 40-80 mg vs atorvastatin 10-20 mg. MAIN MEASURES: The outcome was a first record of a muscular event (myopathy, myalgia, myositis, rhabdomyolysis) during a maximum follow-up of 1 year. KEY RESULTS: The propensity score-matched cohorts consisted of 1) 9,703, 2) 7,032, and 3) 37,743 pairs of statin users. Comparing the risk of muscular events between low-intensity pravastatin vs low-intensity simvastatin yielded a HR of 0.86 (95% CI 0.64-1.16). In the comparison of moderate- to high-intensity rosuvastatin vs equivalent doses of atorvastatin, we observed a HR of 1.17 (95% CI 0.88-1.56). Moderate- to high-intensity simvastatin was associated with a HR of 1.33 (95% CI 1.16-1.53), when compared with atorvastatin at equivalent doses. LIMITATIONS: We could not conduct other pairwise comparisons of statins due to small sample size. In the absence of a uniform definition on the comparability of statin doses, the applied dose ratios may not fully match with all literature sources. CONCLUSIONS: Our results do not suggest a systematically lower risk of muscular events for hydrophilic statins when compared to lipophilic statins at comparable lipid-lowering doses.

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.

Antiseizure drugs and risk of developing smoking-related chronic obstructive pulmonary disease or lung cancer: A population-based case-control study.

AIMS: To determine whether enzyme-inducing antiseizure drugs (ASDs) affect the risk of developing chronic obstructive pulmonary disease (COPD) or lung cancer in smokers. METHODS: Cases of COPD and lung cancer and matched controls without these conditions were identified from a population of smokers with ≥1 prescription for any type of ASD in the Clinical Practice Research Datalink UK database of patients managed in primary care (1995-2016). A matched case-control study was performed utilising multivariate logistic regression analyses of exposure to enzyme-inducing ASDs compared to non-enzyme-inducing ASDs. The duration of ASD exposure and level of tobacco exposure were also assessed. RESULTS: We identified 5952 incident COPD and 1373 incident lung cancer cases, and 59 328 and 13 681 matched controls, respectively. Compared with never use, ever use of enzyme-inducing ASDs was associated with slightly decreased risk estimates of COPD (adjusted odds ratio: 0.85, 95% confidence interval: 0.81-0.89) and lung cancer (adjusted odds ratio: 0.82, 95% confidence interval: 0.73-0.92). These risk estimates were attenuated in heavy smokers. CONCLUSION: We found slightly decreased risk estimates of COPD and lung cancer among smokers taking enzyme-inducing ASDs and hypothesise that this may be related to induction of detoxification of tobacco-specific lung toxins.

Effects of Hypericum perforatum (St John's wort) on the pharmacokinetics and pharmacodynamics of rivaroxaban in humans.

AIMS: To investigate the influence of a cytochrome P450 CYP3A4 and efflux transporter P-glycoprotein (P-gp) inducing Hypericum perforatum extract on the pharmacokinetics and pharmacodynamics of rivaroxaban. METHODS: Open-label, nonrandomized, sequential treatment interaction study. Following CYP3A4 and P-gp phenotyping using low-dose midazolam and fexofenadine, 12 healthy volunteers received a single oral dose of 20 mg rivaroxaban and rivaroxaban plasma concentrations and inhibition of the activated coagulation factor X (factor Xa) activity were measured prior to and up to 48 h postdosing. The procedures were repeated after 2 weeks' treatment with the H. perforatum extract. RESULTS: The geometric mean ratios for the area under the concentration-time curve and Cmax of rivaroxaban after/before induction with the H. perforatum extract were 0.76 (90% confidence interval [CI] 0.70, 0.82) and 0.86 (90% CI 0.76, 0.97), respectively. Inhibition of factor Xa activity was reduced with a geometric mean area under the effect-time curve ratio after/before induction of 0.80 (90% CI 0.71, 0.89). No clinically significant differences were found regarding Tmax (median 1.5 vs 1 h, P = .26) and terminal elimination half-life (mean 10.6 vs 10.8 h, P = .93) of rivaroxaban. The H. perforatum extract significantly induced CYP3A4 and P-gp activity, as evidenced by phenotyping. CONCLUSION: The CYP3A4/P-gp inducing H. perforatum extract caused a decrease of rivaroxaban exposure with a proportional decrease of the pharmacodynamic effect. Although the data do not justify a contraindication for the combination or a systematic adjustment of rivaroxaban dosage, avoidance of the combination or laboratory monitoring should be considered in patients taking hyperforin-containing H. perforatum extracts with rivaroxaban.

Effects of Simvastatin on Lipid Metabolism in Wild-Type Mice and Mice with Muscle PGC-1α Overexpression.

Previous studies suggest that statins may disturb skeletal muscle lipid metabolism potentially causing lipotoxicity with insulin resistance. We investigated this possibility in wild-type mice (WT) and mice with skeletal muscle PGC-1α overexpression (PGC-1α OE mice). In WT mice, simvastatin had only minor effects on skeletal muscle lipid metabolism but reduced glucose uptake, indicating impaired insulin sensitivity. Muscle PGC-1α overexpression caused lipid droplet accumulation in skeletal muscle with increased expression of the fatty acid transporter CD36, fatty acid binding protein 4, perilipin 5 and CPT1b but without significant impairment of muscle glucose uptake. Simvastatin further increased the lipid droplet accumulation in PGC-1α OE mice and stimulated muscle glucose uptake. In conclusion, the impaired muscle glucose uptake in WT mice treated with simvastatin cannot be explained by lipotoxicity. PGC-1α OE mice are protected from lipotoxicity of fatty acids and triglycerides by increased the expression of FABP4, formation of lipid droplets and increased expression of CPT1b.

Identification of miR-199a-5p, miR-214-3p and miR-99b-5p as Fibrosis-Specific Extracellular Biomarkers and Promoters of HSC Activation.

Liver fibrosis is characterized by the accumulation of extracellular matrix (ECM) resulting in the formation of fibrous scars. In the clinic, liver biopsies are the standard diagnostic method despite the potential for clinical complications. miRNAs are single-stranded, non-coding RNAs that can be detected in tissues, body fluids and cultured cells. The regulation of many miRNAs has been linked to tissue damage, including liver fibrosis in patients, resulting in aberrant miRNA expression/release. Experimental evidence also suggests that miRNAs are regulated in a similar manner in vitro and could thus serve as translational in vitro-in vivo biomarkers. In this work, we set out to identify and characterize biomarkers for liver fibrosis that could be used in vitro and clinically for research and diagnostic purposes. We focused on miRNAs released from hepatic 3D cultures exposed to methotrexate (MTX), which causes fibrosis, and acetaminophen (APAP), an acute hepatotoxicant with no clinically relevant association to liver fibrosis. Using a 3D in vitro model, we corroborated compound-specific responses as we show MTX induced a fibrotic response, and APAP did not. Performing miRNA-seq of cell culture supernatants, we identified potential miRNA biomarkers (miR-199a-5p, miR-214-3p, niRNA-125a-5p and miR-99b-5p) that were associated with a fibrotic phenotype and not with hepatocellular damage alone. Moreover, transfection of HSC with miR-199a-5p led to decreased expression of caveolin-1 and increased α-SMA expression, suggesting its role in HSC activation. In conclusion, we propose that extracellular miR-214-3p, miR-99b-5p, miR-125a-5p and specifically miR-199a-5p could contribute towards a panel of miRNAs for identifying liver fibrosis and that miR-199a-5p, miR-214-3p and miR-99b-5p are promoters of HSC activation.

The effect of food on the pharmacokinetics of oral ivermectin.

BACKGROUND: Ivermectin is an older anthelminthic agent that is being studied more intensely given its potential for mass drug administration against scabies, malaria and other neglected tropical diseases. Its pharmacokinetics (PK) remain poorly characterized. Furthermore, the majority of PK trials are performed under fasted-state dosing conditions, and the effect of food is therefore not well known. To better plan and design field trials with ivermectin, a model that can account for both conditions would be valuable. OBJECTIVES: To develop a PK model and characterize the food effect with single oral doses of ivermectin. PATIENTS AND METHODS: We performed a population-based PK analysis of data pooled from two previous trials of a single dose of 12 mg ivermectin, one with dosing after a high-fat breakfast (n=12) and one with fasted-state dosing (n=3). RESULTS: The final model described concentration-time profiles after fed and fasted dosing accurately, and estimated the food effect associated with relative bioavailability to 1.18 (95% CI 1.10-1.67). CONCLUSIONS: In this analysis, the effect of a high-fat breakfast compared with a fasted-state administration of a single oral dose of 12 mg ivermectin was minimal.

Mechanisms of statin-associated skeletal muscle-associated symptoms.

Statins lower the serum low-density lipoprotein cholesterol and prevent cardiovascular events by inhibiting 3-hydroxy-3-methyl-glutaryl-CoA reductase. Although the safety of statins is documented, many patients ingesting statins may suffer from skeletal muscle-associated symptoms (SAMS). Importantly, SAMS are a common reason for stopping the treatment with statins. Statin-associated muscular symptoms include fatigue, weakness and pain, possibly accompanied by elevated serum creatine kinase activity. The most severe muscular adverse reaction is the potentially fatal rhabdomyolysis. The frequency of SAMS is variable but in up to 30% of the patients ingesting statins, depending on the population treated and the statin used. The mechanisms leading to SAMS are currently not completely clarified. Over the last 15 years, several research articles focused on statin-induced mitochondrial dysfunction as a reason for SAMS. Statins can impair the function of the mitochondrial respiratory chain, thereby reducing ATP and increasing ROS production. This can induce mitochondrial membrane permeability transition, release of cytochrome c into the cytosol and induce apoptosis. In parallel, statins inhibit activation of Akt, mainly due to reduced function of mTORC2, which may be related to mitochondrial dysfunction. Mitochondrial dysfunction by statins is also responsible for activation of AMPK, which is associated with impaired activation of mTORC1. Reduced activation of mTORC1 leads to increased skeletal muscle protein degradation, impaired protein synthesis and stimulation of apoptosis. In this paper, we discuss some of the different hypotheses how statins affect skeletal muscle in more detail, focusing particularly on those related to mitochondrial dysfunction and the impairment of the Akt/mTOR pathway.

Pharmacokinetics and phenotyping properties of the Basel phenotyping cocktail combination capsule in healthy male adults.

AIMS: We compared the phenotyping metrics of a combination capsule formulation to its individual components of the newly composed Basel phenotyping cocktail. Moreover, we investigated a reduced sampling regimen for clinical applications. METHODS: We performed in vitro experiments and a crossover pharmacokinetic study in twelve healthy male subjects to compare the Basel phenotyping cocktail capsule containing 6 cytochrome P450 (CYP) probe drugs with individual administration of the same drugs. Parent compounds and selected metabolites were determined by liquid chromatography-tandem mass spectrometry. Metabolic ratios (MR) for are under the curve (AUC) and single time point measurements and their correlation were determined. RESULTS: Experiments with human liver microsomes and primary human hepatocytes in 3D co-culture confirmed that flurbiprofen is a suitable CYP2C9 substrate. Both cocktail formulations (capsule and individual probe drug administration) were well-tolerated and yielded reproducible MRs, which were almost identical. Correlations between single time point ratios and the corresponding AUC ratios depended on the sampling time point and the concentration time curve of the probe drugs. The MR of the capsule (Spearman rank correlation coefficient, Rs : 0.77-0.97) as well as the individual components (Rs : 0.69-0.99) correlated best at 6 h post-treatment considering all 6 CYPs. Moreover, the 2-h time points of the capsule agreed suitably with the AUC; however, the MR of omeprazole could not be determined for 10 out of 12 subjects. CONCLUSION: The capsule is easy to swallow, well tolerated and provides reliable estimates for CYP activity. The optimal sampling point for the capsule formulation is 6 h after intake.

Para-Halogenation of Amphetamine and Methcathinone Increases the Mitochondrial Toxicity in Undifferentiated and Differentiated SH-SY5Y Cells.

Halogenation of amphetamines and methcathinones has become a common method to obtain novel psychoactive substances (NPS) also called "legal highs". The para-halogenated derivatives of amphetamine and methcathinone are available over the internet and have entered the illicit drug market but studies on their potential neurotoxic effects are rare. The primary aim of this study was to explore the neurotoxicity of amphetamine, methcathinone and their para-halogenated derivatives 4-fluoroamphetamine (4-FA), 4-chloroamphetamine (PCA), 4-fluoromethcathinone (4-FMC), and 4-chloromethcathinone (4-CMC) in undifferentiated and differentiated SH-SY5Y cells. We found that 4-FA, PCA, and 4-CMC were cytotoxic (decrease in cellular ATP and plasma membrane damage) for both cell types, whereby differentiated cells were less sensitive. IC50 values for cellular ATP depletion were in the range of 1.4 mM for 4-FA, 0.4 mM for PCA and 1.4 mM for 4-CMC. The rank of cytotoxicity observed for the para-substituents was chloride > fluoride > hydrogen for both amphetamines and cathinones. Each of 4-FA, PCA and 4-CMC decreased the mitochondrial membrane potential in both cell types, and PCA and 4-CMC impaired the function of the electron transport chain of mitochondria in SH-SY5Y cells. 4-FA, PCA, and 4-CMC increased the ROS level and PCA and 4-CMC induced apoptosis by the endogenous pathway. In conclusion, para-halogenation of amphetamine and methcathinone increases their neurotoxic properties due to the impairment of mitochondrial function and induction of apoptosis. Although the cytotoxic concentrations were higher than those needed for pharmacological activity, the current findings may be important regarding the uncontrolled recreational use of these compounds.

[Opioid therapy in patients with liver cirrhosis]. / Therapie mit Opioiden bei Patienten mit Leberzirrhose.

Opioid therapy in patients with liver cirrhosis Abstract. There are currently no endogenous markers representing the metabolic activity and the extent of portacaval shunts in patients with liver cirrhosis. The pharmacokinetic properties of the applied drugs must therefore be taken into account when adjusting the dose in patients with liver cirrhosis. For drugs with a high degradation during the first passage across the liver (first-pass metabolism) the bioavailability is going to increase and the clearance to decrease after oral application. After topical, buccal or parenteral administration, only the impaired clearance will play a role. For drugs with a high bioavailability (> 70 %) only the impaired hepatic clearance has to be considered. In the following article, the relevant pharmacokinetic properties of the most common opioids in Switzerland and the resulting consequences regarding dose adjustment in liver cirrhosis are going to be discussed. Buprenorphine, fentanyl, hydromorphone, morphine, naloxone und tapentadol are drugs with a high first-pass effect, while the bioavailability of methadone, oxycodone and tramadol is > 70 %.

OATP1B3-1B7 (LST-3TM12) Is a Drug Transporter That Affects Endoplasmic Reticulum Access and the Metabolism of Ezetimibe.

Drug transporters play a crucial role in pharmacokinetics. One subfamily of transporters with proven clinical relevance are the OATP1B transporters. Recently we identified a new member of the OATP1B family named OATP1B3-1B7 (LST-3TM12). This functional transporter is encoded by SLCO1B3 and SLCO1B7 OATP1B3-1B7 is expressed in hepatocytes and is located in the membrane of the smooth endoplasmic reticulum (SER). One aim of this study was to test whether OATP1B3-1B7 interacts with commercial drugs. First, we screened a selection of OATP1B substrates for inhibition of OATP1B3-1B7-mediated transport of dehydroepiandrosterone sulfate and identified several inhibitors. One such inhibitor was ezetimibe, which not only inhibited OATP1B3-1B7 but is also a substrate, as its cellular content was significantly increased in cells heterologously expressing the transporter. In humans, ezetimibe is extensively metabolized by hepatic and intestinal uridine-5'-diphospho-glucuronosyltransferases (UGTs), the catalytic site of which is located within the SER lumen. After verification of OATP1B3-1B7 expression in the small intestine, we determined in microsomes whether SER access can be modulated by inhibitors of OATP1B3-1B7. We were able to show that these compounds significantly reduced accumulation in small intestinal and hepatic microsomes, which influenced the rate of ezetimibe ß-D-glucuronide formation as determined in microsomes treated with bromsulphthalein. Notably, this molecule not only inhibits the herein reported transporter but also other transport systems. In conclusion, we report that multiple drugs interact with OATP1B3-1B7; for ezetimibe, we were able to show that SER access and metabolism is significantly reduced by bromsulphthalein, which is an inhibitor of OATP1B3-1B7. SIGNIFICANCE STATEMENT: OATP1B3-1B3 (LST-3TM12) is a transporter that has yet to be fully characterized. We provide valuable insight into the interaction potential of this transporter with several marketed drugs. Ezetimibe, which interacted with OATP1B3-1B7, is highly metabolized by uridine-5'-diphospho-glucuronosyltransferases (UGTs), whose catalytic site is located within the smooth endoplasmic reticulum (SER) lumen. Through microsomal assays with ezetimibe and the transport inhibitor bromsulphthalein we investigated the interdependence of SER access and the glucuronidation rate of ezetimibe. These findings led us to the hypothesis that access or exit of drugs to the SER is orchestrated by SER transporters such as OATP1B3-1B7.

Pharmacokinetics of oxycodone/naloxone and its metabolites in patients with end-stage renal disease during and between haemodialysis sessions.

BACKGROUND: The pharmacokinetics of oxycodone in patients with end-stage renal disease (ESRD) requiring haemodialysis are largely unknown. Therefore, we investigated the pharmacokinetics of oxycodone/naloxone prolonged release and their metabolites in patients with ESRD during and between haemodialysis sessions. METHODS: Single doses of oxycodone/naloxone (5/2.5 or 10/5 mg) were administered in nine patients with ESRD using a cross-over design on the day of dialysis and on a day between dialysis sessions. Plasma, dialysate and urine concentrations of oxycodone, naloxone and their metabolites were determined up to 48 h post-dosing using a liquid chromatography-tandem mass spectrometry system. RESULTS: Haemodialysis performed 6-10 h after dosing removed ∼10% of the administered dose of oxycodone predominantly as unconjugated oxycodone and noroxycodone or conjugated oxymorphone and noroxymorphone. The haemodialysis clearance of oxycodone based on its recovery in dialysate was (mean ± SD) 8.4 ± 2.1 L/h. The geometric mean (coefficient of variation) plasma elimination half-life of oxycodone during the 4-h haemodialysis period was 3.9 h (39%) which was significantly shorter than the 5.7 h (22%) without haemodialysis. Plasma levels of the active metabolite oxymorphone in its unconjugated form were very low. CONCLUSIONS: Oxycodone is removed during haemodialysis. The pharmacokinetics including the relatively short half-life of oxycodone in patients with ESRD with or without haemodialysis and the absence of unconjugated active metabolites indicate that oxycodone can be used at usual doses in patients requiring dialysis.

Population pharmacokinetics of oral ivermectin in venous plasma and dried blood spots in healthy volunteers.

AIMS: The anthelminthic ivermectin is receiving new attention as it is being repurposed for new indications such as mass drug administrations for the treatment of scabies or in malaria vector control. As its pharmacokinetics are still poorly understood, we aimed to characterize the population pharmacokinetics of ivermectin in plasma and dried blood spots (DBS), a sampling method better suited to field trials, with special focus on the influence of body composition and enterohepatic circulation. METHODS: We performed a clinical trial in 12 healthy volunteers who each received a single oral dose of 12 mg ivermectin, and collected peripheral venous and capillary DBS samples. We determined ivermectin concentrations in plasma and DBS by liquid chromatography tandem mass spectrometry using a fully automated and scalable extraction system for DBS sample processing. Pharmacokinetic data were analysed using non-linear mixed effects modelling. RESULTS: A two-compartment model with a transit absorption model, first-order elimination, and weight as an influential covariate on central volume of distribution and clearance best described the data. The model estimates (inter-individual variability) for a 70 kg subject were: apparent population clearance 7.7 (25%) l h-1 , and central and peripheral volumes of distribution 89 (10%) l and 234 (20%) l, respectively. Concentrations obtained from DBS samples were strongly linearly correlated (R2  = 0.97) with plasma concentrations, and on average 30% lower. CONCLUSION: The model accurately depicts population pharmacokinetics of plasma and DBS concentrations over time for oral ivermectin. The proposed analytical workflow is scalable and applicable to the requirements of mass drug administrations.