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.

Drug disposition in neonatal Göttingen Minipigs: Exploring Effects of Perinatal Asphyxia and Therapeutic Hypothermia.

Asphyxiated neonates often undergo therapeutic hypothermia (TH) to reduce morbidity and mortality. Since both perinatal asphyxia (PA) and TH influence physiology, altered pharmacokinetics (PK) and pharmacodynamics (PD) are expected. Given that TH is the standard of care for PA with moderate to severe hypoxic-ischemic encephalopathy (HIE), disentangling the effect of PA versus TH on PK/PD is not possible in clinical settings. However, animal models can provide insights into this matter. The (neonatal) Göttingen Minipig, the recommended strain for nonclinical drug development, was selected as translational model. Four drugs - midazolam (MDZ), fentanyl (FNT), phenobarbital (PHB), and topiramate (TPM), were intravenously administered under four conditions: control (C), therapeutic hypothermia (TH), hypoxia (H), hypoxia + TH (H+TH). Each group included six healthy male neonatal Göttingen Minipigs anesthetized for 24 hours. Blood samples were drawn at 0 (pre-dose), and 0.5, 2, 2.5, 3, 4, 4.5, 6, 8, 12, 24 hours post-drug administration. Drug plasma concentrations were determined using validated bioanalytical assays. The PK parameters were estimated through compartmental and non-compartmental PK analysis (NCA). The study showed a statistically significant decrease in FNT clearance (CL, 66% decrease) with approximately 3-fold longer half-life (t1/2) in the TH group. The H+TH group showed a 17% reduction in FNT CL with a 62% longer t1/2 compared to the C group, however non-statistically significant. Trends towards lower CL and longer t1/2 were observed in the TH and H+TH groups for MDZ and PHB. Additionally, TPM demonstrated a 28% decrease in CL in the H group compared to controls. Significance Statement The overarching goal of this study using the neonatal Göttingen Minipig model was to disentangle the effects of systemic hypoxia and TH on PK, using four model drugs. Such insights can subsequently be used to inform and develop a physiologically-based pharmacokinetic (PBPK) model, which is useful for drug exposure prediction in human neonates.

Automated Generation of hiPSC-Derived Hepatic Progeny by Cost-Efficient Compounds.

Human pluripotent stem cell (hPSC)-derived hepatocyte-like cells (HLCs) hold great promise for liver disease modeling, drug discovery, and drug toxicity screens. Yet, several hurdles still need to be overcome, including among others decrease in the cost of goods to generate HLCs and automation of the differentiation process. We here describe that the use of an automated liquid handling system results in highly reproducible HLC differentiation from hPSCs. This enabled us to screen 92 chemicals to replace expensive growth factors at each step of the differentiation protocol to reduce the cost of goods of the differentiation protocol by approximately 79%. In addition, we also evaluated several recombinant extracellular matrices to replace Matrigel. We demonstrated that differentiation of hPSCs on Laminin-521 using an optimized small molecule combination resulted in HLCs that were transcriptionally identical to HLCs generated using the growth factor combinations. In addition, the HLCs created using the optimized small molecule combination secreted similar amounts of albumin and urea, and relatively low concentrations of alfa-fetoprotein, displayed similar CYP3A4 functionality, and a similar drug toxicity susceptibility as HLCs generated with growth factor cocktails. The broad applicability of the new differentiation protocol was demonstrated for 4 different hPSC lines. This allowed the creation of a scalable, xeno-free, and cost-efficient hPSC-derived HLC culture, suitable for high throughput disease modeling and drug screenings, or even for the creation of HLCs for regenerative therapies.

PBPK Modelling for Drugs Cleared by Non-CYP Enzymes: State-of-the-Art and Future Perspectives.

Physiologically-based pharmacokinetic (PBPK) modeling has become the established method for predicting human pharmacokinetics (PK) and drug-drug interactions (DDI). The number of drugs cleared by non-CYP enzyme metabolism has increased steadily and to date, there is no consolidated overview of PBPK modeling for drugs cleared by non-CYP enzymes. This review aims to describe the state-of-the-art for PBPK modeling for drugs cleared via non-CYP enzymes, to identify successful strategies, to describe gaps and to provide suggestion to overcome them. To this end, we conducted a detailed literature search and found 58 articles published before the 1st of January 2023 containing 95 examples of clinical PBPK models for 62 non-CYP enzyme substrates. Reviewed articles covered the drug clearance by uridine 5'-diphospho-glucuronosyltransferases (UGTs), aldehyde oxidase (AO), flavin-containing monooxygenases (FMOs), sulfotransferases (SULTs) and carboxylesterases (CES), with UGT2B7, UGT1A9, CES1, FMO3 and AO being the enzymes most frequently involved. In vitro-in vivo extrapolation (IVIVE) of intrinsic clearance and the bottom-up PBPK modeling involving non-CYP enzymes remains challenging. We observed that the middle-out modeling approach was applied in 80% of the cases, with metabolism parameters optimized in 73% of the models. Our review could not identify a standardized approach used for model optimization based on clinical data, with manual optimization employed most frequently. Successful development of models for UGT2B7, UGT1A9, CES1, and FMO3 substrates provides a foundation for other drugs metabolized by these enzymes and guides the way forward in creating PBPK models for other enzymes in these families. Significance Statement Our review charts the rise of PBPK modeling for drugs cleared by non-CYP enzymes. Analyzing 58 articles and 62 non-CYP enzyme substrates, we found that UGTs, AO, FMOs, SULTs, and CES were the main enzyme families involved and that UGT2B7, UGT1A9, CES1, FMO3 and AO are the individual enzymes with the strongest PBPK modeling precedents. Approaches established for these enzymes can now be extended to additional substrates and to drugs metabolized by enzymes that are similarly well characterized.

Physiologically based pharmacokinetic modelling to inform combination dosing regimens of ceftaroline and daptomycin in special populations.

AIMS: The combination of daptomycin and ceftaroline used as salvage therapy is associated with higher survival and decreased clinical failure in complicated methicillin-resistant Staphylococcus aureus (MRSA) infections that are resistant to standard MRSA treatment. This study aimed to evaluate dosing regimens for coadministration of daptomycin and ceftaroline in special populations including paediatrics, renally impaired (RI), obese and geriatrics that generate sufficient coverage against daptomycin-resistant MRSA. METHODS: Physiologically based pharmacokinetic models were developed from pharmacokinetic studies of healthy adults, geriatric, paediatric, obese and RI patients. The predicted profiles were used to evaluate joint probability of target attainment (PTA), as well as tissue-to-plasma ratios. RESULTS: The adult dosing regimens of 6 mg/kg every (q)24h or q48h daptomycin and 300-600 mg q12h ceftaroline fosamil by RI categories achieved ≥90% joint PTA when the minimum inhibitory concentrations in the combination are at or below 1 and 4 µg/mL against MRSA. In paediatrics, wherein there is no recommended daptomycin dosing regimen for S. aureus bacteraemia, ≥90% joint PTA is achieved when the minimum inhibitory concentrations in the combination are up to 0.5 and 2 µg/mL for standard paediatric dosing regimens of 7 mg/kg q24h daptomycin and 12 mg/kg q8h ceftaroline fosamil. Model predicted tissue-to-plasma ratios of 0.3 and 0.7 in the skin and lung, respectively, for ceftaroline and 0.8 in the skin for daptomycin. CONCLUSION: Our work illustrates how physiologically based pharmacokinetic modelling can inform appropriate dosing of adult and paediatric patients and thereby enable prediction of target attainment in the patients during multitherapies.

Unravelling the Hepatic Elimination Mechanisms of Colistin.

PURPOSE: Colistin is an antibiotic which is increasingly used as a last-resort therapy in critically-ill patients with multidrug resistant Gram-negative infections. The purpose of this study was to evaluate the mechanisms underlying colistin's pharmacokinetic (PK) behavior and to characterize its hepatic metabolism. METHODS: In vitro incubations were performed using colistin sulfate with rat liver microsomes (RLM) and with rat and human hepatocytes (RH and HH) in suspension. The uptake of colistin in RH/HH and thefraction of unbound colistin in HH (fu,hep) was determined. In vitro to in vivo extrapolation (IVIVE) was employed to predict the hepatic clearance (CLh) of colistin. RESULTS: Slow metabolism was detected in RH/HH, with intrinsic clearance (CLint) values of 9.34± 0.50 and 3.25 ± 0.27 mL/min/kg, respectively. Assuming the well-stirred model for hepatic drug elimination, the predicted rat CLh was 3.64± 0.22 mL/min/kg which could explain almost 70% of the reported non-renal in vivo clearance. The predicted human CLh was 91.5 ± 8.83 mL/min, which was within two-fold of the reported plasma clearance in healthy volunteers. When colistin was incubated together with the multidrug resistance-associated protein (MRP/Mrp) inhibitor benzbromarone, the intracellular accumulation of colistin in RH/HH increased significantly. CONCLUSION: These findings indicate the major role of hepatic metabolism in the non-renal clearance of colistin, while MRP/Mrp-mediated efflux is involved in the hepatic disposition of colistin. Our data provide detailed quantitative insights into the hereto unknown mechanisms responsible for non-renal elimination of colistin.

Favipiravir at high doses has potent antiviral activity in SARS-CoV-2-infected hamsters, whereas hydroxychloroquine lacks activity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapidly spread around the globe after its emergence in Wuhan in December 2019. With no specific therapeutic and prophylactic options available, the virus has infected millions of people of which more than half a million succumbed to the viral disease, COVID-19. The urgent need for an effective treatment together with a lack of small animal infection models has led to clinical trials using repurposed drugs without preclinical evidence of their in vivo efficacy. We established an infection model in Syrian hamsters to evaluate the efficacy of small molecules on both infection and transmission. Treatment of SARS-CoV-2-infected hamsters with a low dose of favipiravir or hydroxychloroquine with(out) azithromycin resulted in, respectively, a mild or no reduction in virus levels. However, high doses of favipiravir significantly reduced infectious virus titers in the lungs and markedly improved lung histopathology. Moreover, a high dose of favipiravir decreased virus transmission by direct contact, whereas hydroxychloroquine failed as prophylaxis. Pharmacokinetic modeling of hydroxychloroquine suggested that the total lung exposure to the drug did not cause the failure. Our data on hydroxychloroquine (together with previous reports in macaques and ferrets) thus provide no scientific basis for the use of this drug in COVID-19 patients. In contrast, the results with favipiravir demonstrate that an antiviral drug at nontoxic doses exhibits a marked protective effect against SARS-CoV-2 in a small animal model. Clinical studies are required to assess whether a similar antiviral effect is achievable in humans without toxic effects.

Ceftriaxone dosing based on the predicted probability of augmented renal clearance in critically ill patients with pneumonia.

OBJECTIVES: PTA of protein-unbound ceftriaxone may be compromised in critically ill patients with community-acquired pneumonia (CAP) with augmented renal clearance (ARC). We aimed to determine an optimized ceftriaxone dosage regimen based on the probability of developing ARC on the next day (PARC,d+1; www.arcpredictor.com). PATIENTS AND METHODS: Thirty-three patients enrolled in a prospective cohort study were admitted to the ICU with severe CAP and treated with ceftriaxone 2 g once daily. Patients contributed 259 total ceftriaxone concentrations, collected during 1 or 2 days (±7 samples/day). Unbound fractions of ceftriaxone were determined in all peak and trough samples (n = 76). Population pharmacokinetic modelling and simulation were performed using NONMEM7.4. Target attainment was defined as an unbound ceftriaxone concentration >4 mg/L throughout the dosing interval. RESULTS: A two-compartment population pharmacokinetic model described the data well. The maximal protein-bound ceftriaxone concentration decreased with lower serum albumin. Ceftriaxone clearance increased with body weight and PARC,d+1 determined on the previous day. A high PARC,d+1 was identified as a clinically relevant predictor for underexposure on the next day (area under the receiver operating characteristics curve 0.77). Body weight had a weak predictive value and was therefore considered clinically irrelevant. Serum albumin had no predictive value. An optimal PARC,d+1 threshold of 5.7% was identified (sensitivity 73%, specificity 69%). Stratified once- or twice-daily 2 g dosing when below or above the 5.7% PARC,d+1 cut-off, respectively, was predicted to result in 81% PTA compared with 47% PTA under population-level once-daily 2 g dosing. CONCLUSIONS: Critically ill patients with CAP with a high PARC,d+1 may benefit from twice-daily 2 g ceftriaxone dosing for achieving adequate exposure on the next day.

Pharmacokinetics of 2 oral paracetamol formulations in hospitalized octogenarians.

AIMS: It is currently unclear how paracetamol should be dosed in order to increase its efficacy while warranting safety in very old adults. The objective was to evaluate the pharmacokinetics of 2 oral paracetamol formulations and its metabolites in hospitalized octogenarians. METHODS: Geriatric inpatients aged 80 years and older received a 1000-mg paracetamol tablet or granulate at 08.00, 14.00 and 20.00. After at least 4 consecutive gifts, plasma samples were collected around the 08.00 dose (trough, +0.5, +1, +2, +4, +5 and +6 h). Plasma concentrations of paracetamol and its metabolites were determined and individual pharmacokinetic parameters were derived. The Edmonton Frail Scale was used to assess frailty. An analgesic plasma target was defined as an average plasma concentration (Cavg ) of 10 mg/L. RESULTS: The mean (±standard deviation) age was 86.78 (±4.20) years. The majority (n = 26/36, 72%) received the tablet, 10 (28%) the granulate. Thirty patients (85%) were classified with moderate to severe frailty. Seven (21%) patients had a Cavg above 10 mg/L. The median [interquartile range] time to reach the peak concentration was 50.5 [31.50-92.50] and 42.50 [33.75-106.75] min for the tablet and granulate, respectively. The coefficient of variation was 95% for time to reach the peak concentration and 30% for Cavg of paracetamol. A correlation of Cavg of paracetamol was observed with female sex and total serum bilirubin. CONCLUSION: Large interindividual differences were found for pharmacokinetic parameters of oral paracetamol in frail inpatients after multiple dosing. Female sex and higher total serum bilirubin concentrations were associated with paracetamol exposure. No significant differences were observed between the tablet and granulate.

Current knowledge, challenges and innovations in developmental pharmacology: A combined conect4children Expert Group and European Society for Developmental, Perinatal and Paediatric Pharmacology White Paper.

Developmental pharmacology describes the impact of maturation on drug disposition (pharmacokinetics, PK) and drug effects (pharmacodynamics, PD) throughout the paediatric age range. This paper, written by a multidisciplinary group of experts, summarizes current knowledge, and provides suggestions to pharmaceutical companies, regulatory agencies and academicians on how to incorporate the latest knowledge regarding developmental pharmacology and innovative techniques into neonatal and paediatric drug development. Biological aspects of drug absorption, distribution, metabolism and excretion throughout development are summarized. Although this area made enormous progress during the last two decades, remaining knowledge gaps were identified. Minimal risk and burden designs allow for optimally informative but minimally invasive PK sampling, while concomitant profiling of drug metabolites may provide additional insight in the unique PK behaviour in children. Furthermore, developmental PD needs to be considered during drug development, which is illustrated by disease- and/or target organ-specific examples. Identifying and testing PD targets and effects in special populations, and application of age- and/or population-specific assessment tools are discussed. Drug development plans also need to incorporate innovative techniques such as preclinical models to study therapeutic strategies, and shift from sequential enrolment of subgroups, to more rational designs. To stimulate appropriate research plans, illustrations of specific PK/PD-related as well as drug safety-related challenges during drug development are provided. The suggestions made in this joint paper of the Innovative Medicines Initiative conect4children Expert group on Developmental Pharmacology and the European Society for Developmental, Perinatal and Paediatric Pharmacology, should facilitate all those involved in drug development.

Effects of Drugs Formerly Proposed for COVID-19 Treatment on Connexin43 Hemichannels.

Connexin43 (Cx43) hemichannels form a pathway for cellular communication between the cell and its extracellular environment. Under pathological conditions, Cx43 hemichannels release adenosine triphosphate (ATP), which triggers inflammation. Over the past two years, azithromycin, chloroquine, dexamethasone, favipiravir, hydroxychloroquine, lopinavir, remdesivir, ribavirin, and ritonavir have been proposed as drugs for the treatment of the coronavirus disease 2019 (COVID-19), which is associated with prominent systemic inflammation. The current study aimed to investigate if Cx43 hemichannels, being key players in inflammation, could be affected by these drugs which were formerly designated as COVID-19 drugs. For this purpose, Cx43-transduced cells were exposed to these drugs. The effects on Cx43 hemichannel activity were assessed by measuring extracellular ATP release, while the effects at the transcriptional and translational levels were monitored by means of real-time quantitative reverse transcriptase polymerase chain reaction analysis and immunoblot analysis, respectively. Exposure to lopinavir and ritonavir combined (4:1 ratio), as well as to remdesivir, reduced Cx43 mRNA levels. None of the tested drugs affected Cx43 protein expression.

Bosentan Alters Endo- and Exogenous Bile Salt Disposition in Sandwich-Cultured Human Hepatocytes.

Bosentan, a well-known cholestatic agent, was not identified as cholestatic at concentrations up to 200 µM based on the drug-induced cholestasis (DIC) index value, determined in a sandwich-cultured human hepatocyte (SCHH)-based DIC assay. To obtain further quantitative insights into the effects of bosentan on cellular bile salt handling by human hepatocytes, the present study determined the effect of 2.5-25 µM bosentan on endogenous bile salt levels and on the disposition of 10 µM chenodeoxycholic acid (CDCA) added to the medium in SCHHs. Bosentan reduced intracellular as well as extracellular concentrations of both endogenous glycochenodeoxycholic acid (GCDCA) and glycocholic acid in a concentration-dependent manner. When exposed to 10 µM CDCA, bosentan caused a shift from canalicular efflux to sinusoidal efflux of GCDCA. CDCA levels were not affected. Our mechanistic model confirmed the inhibitory effect of bosentan on canalicular GCDCA clearance. Moreover, our results in SCHHs also indicated reduced GCDCA formation. We confirmed the direct inhibitory effect of bosentan on CDCA conjugation with glycine in incubations with liver S9 fraction. SIGNIFICANCE STATEMENT: Bosentan was evaluated at therapeutically relevant concentrations (2.5-25 µM) in sandwich-cultured human hepatocytes. It altered bile salt disposition and inhibited canalicular secretion of glycochenodeoxycholic acid (GCDCA). Within 24 hours, bosentan caused a shift from canalicular to sinusoidal efflux of GCDCA. These results also indicated reduced GCDCA formation. This study confirmed a direct effect of bosentan on chenodeoxycholic acid conjugation with glycine in liver S9 fraction.

Population pharmacokinetics of propofol in neonates and infants: Gestational and postnatal age to determine clearance maturation.

AIMS: Develop a population pharmacokinetic model describing propofol pharmacokinetics in (pre)term neonates and infants, that can be used for precision dosing (e.g. during target-controlled infusion) of propofol in this population. METHODS: A nonlinear mixed effects pharmacokinetic analysis (Monolix 2018R2) was performed, based on a pooled study population in 107 (pre)term neonates and infants. RESULTS: In total, 836 blood samples were collected from 66 (pre)term neonates and 41 infants originating from 3 studies. Body weight (BW) of the pooled study population was 3.050 (0.580-11.440) kg, postmenstrual age (PMA) was 36.56 (27.00-43.00) weeks and postnatal age (PNA) was 1.14 (0-104.00) weeks (median and min-max range). A 3-compartment structural model was identified and the effect of BW was modelled using fixed allometric exponents. Elimination clearance maturation was modelled accounting for the maturational effect on elimination clearance until birth (by gestational age [GA]) and postpartum (by PNA and GA). The extrapolated adult (70 kg) population propofol elimination clearance (1.64 L min-1 , estimated relative standard error = 6.02%) is in line with estimates from previous population pharmacokinetic studies. Empirical scaling of BW on the central distribution volume in function of PNA improved the model fit. CONCLUSIONS: It is recommended to describe elimination clearance maturation by GA and PNA instead of PMA on top of size effects when analyzing propofol pharmacokinetics in populations including preterm neonates. Changes in body composition in addition to weight changes or other physio-anatomical changes may explain the changes in central distribution volume. The developed model may serve as a prior for propofol dose finding and target-controlled infusion in (preterm) neonates.

A Population Pharmacokinetic Modeling and Simulation Study of Posaconazole Oral Suspension in Immunocompromised Pediatric Patients: A Short Communication.

BACKGROUND: Posaconazole oral suspension emerged as a promising candidate for prophylaxis of invasive fungal infections in immunocompromised children. Its pharmacodynamic advantages include a broad-spectrum activity and a favorable safety profile; however, they are overshadowed by its large pharmacokinetic (PK) variability, which might cause subtherapeutic exposure. The aim of this study was to develop a population (pop) PK model based on rich sampling data to better understand the PK of posaconazole oral suspension in pediatric patients. METHODS: Data were obtained from a prospective interventional study involving hospitalized pediatric patients with a hematologic malignancy and prophylactically treated with posaconazole oral suspension. After constructing the popPK model, the probability of target attainment (PTA; 100% T ≥ 0.7 mg/L) for prophylaxis under fixed, body weight-based, and body surface area-based dosing was evaluated using Monte Carlo simulation. RESULTS: Fourteen patients contributed 112 posaconazole plasma concentrations. The PK of posaconazole was adequately described by a 1-compartment model with lag time 2.71 hours [13%]; nonlinear bioavailability ED50 99.1 mg/m2 (fixed); first-order absorption rate constant 0.325 hour-1 [27%]; apparent volume of distribution 1150 L [34%]; and apparent clearance 15.4 L/h [24%] (∼70-kg individual). The bioavailability decreased in the presence of diarrhea and co-treatment with a proton pump inhibitor (PPI). The unexplained interindividual variability in posaconazole PK remained large. The PTA was <85%, irrespective of the simulated dosing strategy. Patients without diarrhea and not administered a PPI had the highest PTA (85% under the fixed 300-mg dosing 4 times per day). CONCLUSIONS: Therapeutic drug monitoring is recommended during prophylactic posaconazole therapy in immunocompromised pediatric patients. Large-scale comparative studies are needed to characterize the PK variability between different posaconazole formulations in this cohort.

Connexin and Pannexin (Hemi)Channels: Emerging Targets in the Treatment of Liver Disease.

Connexin proteins are the building blocks of hemichannels, which dock further between adjacent cells to form gap junctions. Gap junctions control the intercellular exchange of critical homeostasis regulators. By doing so, gap junctions control virtually all aspects of the hepatic life cycle. In the last decade, it has become clear that connexin hemichannels also provide a pathway for cellular communication on their own independent of their role as structural precursors of gap junctions, namely between the cytosol of an individual cell and its extracellular environment. In contrast to gap junctions, connexin hemichannels become particularly active in liver disease by facilitating inflammation and cell death. This equally holds true for cellular channels composed of pannexins, being connexin-like proteins recently identified in the liver that gather in structures reminiscent of hemichannels. This paper gives an overview of the involvement of connexin-based and pannexin-based channels in noncancerous liver disease.

Robustness testing and optimization of an adverse outcome pathway on cholestatic liver injury.

Adverse outcome pathways (AOPs) have been recently introduced as tools to map the mechanisms underlying toxic events relevant for chemical risk assessment. AOPs particularly depict the linkage between a molecular initiating event and an adverse outcome through a number of intermediate key events. An AOP has been previously introduced for cholestatic liver injury. The objective of this study was to test the robustness of this AOP for different types of cholestatic insult and the in vitro to in vivo extrapolation. For this purpose, in vitro samples from human hepatoma HepaRG cell cultures were exposed to cholestatic drugs (i.e. intrahepatic cholestasis), while in vivo samples were obtained from livers of cholestatic mice (i.e. extrahepatic cholestasis). The occurrence of cholestasis in vitro was confirmed through analysis of bile transporter functionality and bile acid analysis. Transcriptomic analysis revealed inflammation and oxidative stress as key events in both types of cholestatic liver injury. Major transcriptional differences between intrahepatic and extrahepatic cholestatic liver insults were observed at the level of cell death and metabolism. Novel key events identified by pathway analysis included endoplasmic reticulum stress in intrahepatic cholestasis, and autophagy and necroptosis in both intrahepatic as extrahepatic cholestasis. This study demonstrates that AOPs constitute dynamic tools that should be frequently updated with new input information.

A sensitive liquid chromatography method for analysis of propofol in small volumes of neonatal blood.

WHAT IS KNOWN AND OBJECTIVE: Sampling volumes of blood from neonates is necessarily limited. However, most of the published propofol analysis assays require a relatively large blood sample volume (typically ≥0.5 mL). Therefore, the aim of the present study was to develop and validate a sensitive method requiring a smaller sample volume (0.2 mL) to fulfill clinically relevant research requirements. METHODS: Following simple protein precipitation and centrifugation, the supernatant was injected into the HPLC-fluorescence system and separated with a reverse phase column. Propofol and the internal standard (thymol) were detected and quantified using fluorescence at excitation and emission wavelengths of 270 nm and 310 nm, respectively. The method was validated with reference to the Food and Drug Administration (FDA) guidance for industry. Accuracy (CV, %) and precision (RSD, %) were evaluated at three quality control concentration levels (0.05, 0.5 and 5 µg/mL). RESULTS AND DISCUSSION: Calibration curves were linear in the range of 0.005-20 µg/mL. Intra- and interday accuracy (-4.4%-13.6%) and precision (0.2%-5.8%) for propofol were below 15%. The calculated LOD (limit of detection) and LLOQ (lower limit of quantification) were 0.0021 µg/mL and 0.0069 µg/mL, respectively. Propofol samples were stable for 4 months at -20°C after the sample preparation. This method was applied for analyzing blood samples from 41 neonates that received propofol, as part of a dose-finding study. The measured median (range) concentration was 0.14 (0.03-1.11) µg/mL, which was in the range of the calibration curve. The calculated median (range) propofol half-life of the gamma elimination phase was 10.4 (4.7-26.7) hours. WHAT IS NEW AND CONCLUSION: A minimal volume (0.2 mL) of blood from neonates is required for the determination of propofol with this method. The method can be used to support the quantification of propofol drug concentrations for pharmacokinetic studies in the neonatal population.

Cholestasis Differentially Affects Liver Connexins.

Connexins are goal keepers of tissue homeostasis, including in the liver. As a result, they are frequently involved in disease. The current study was set up to investigate the effects of cholestatic disease on the production of connexin26, connexin32 and connexin43 in the liver. For this purpose, bile duct ligation, a well-known trigger of cholestatic liver injury, was applied to mice. In parallel, human hepatoma HepaRG cell cultures were exposed to cholestatic drugs and bile acids. Samples from both the in vivo and in vitro settings were subsequently subjected to assessment of mRNA and protein quantities as well as to in situ immunostaining. While the outcome of cholestasis on connexin26 and connexin43 varied among experimental settings, a more generalized repressing effect was seen for connexin32. This has also been observed in many other liver pathologies and could suggest a role for connexin32 as a robust biomarker of liver disease and toxicity.

Transplacental transport of paracetamol and its phase II metabolites using the ex vivo placenta perfusion model.

In Europe, 50-60% of pregnant women uses paracetamol (PCM), also known as acetaminophen. While it was considered to be safe, recent studies have shown an association between prenatal exposure to PCM and increased incidences of autism, cryptorchidism, asthma and ADHD. In this study the transplacental transfer of PCM and its metabolites was investigated using an ex vivo human placenta perfusion model (closed circuit; n = 38). Maternal-to-foetal (M-F) and foetal-to-maternal (F-M) transplacental transfer was determined at a concentration correlating with the maximum and steady state concentration in normal clinical use. Antipyrine (AP) was added as reference compound. Samples of the foetal and maternal perfusion medium were taken until 210 (PCM) or 360 min (paracetamol sulphate (PCM-S) and paracetamol glucuronide (PCM-G). PCM and AP concentrations reached an equilibrium between foetal and maternal compartments within the duration of the perfusion experiment and irrespective of the transfer direction. The percentage placental transfer of PCM was 45% (M-F and F-M). For PCM-S, transfer was 39% (M-F) and 28% (F-M), while the PCM-G transfer was 34% (M-F) and 25% (F-M). During placenta perfusions with the metabolites slight conversion (3.5-4.1%) to PCM was observed. In conclusion, PCM crosses the placental barrier rapidly via passive diffusion. Differences in flow rate and villous placental structure explain the significantly faster M-F transfer than F-M transfer of PCM. The larger and more hydrophilic molecules PCM-S and PCM-G cross the placenta at a significantly lower rate. Moreover, their F-M transport is about 40% slower than M-F transport, suggesting involvement of a transporter.

Current insights in the complexities underlying drug-induced cholestasis.

Drug-induced cholestasis (DIC) poses a major challenge to the pharmaceutical industry and regulatory agencies. It causes both drug attrition and post-approval withdrawal of drugs. DIC represents itself as an impaired secretion and flow of bile, leading to the pathological hepatic and/or systemic accumulation of bile acids (BAs) and their conjugate bile salts. Due to the high number of mechanisms underlying DIC, predicting a compound's cholestatic potential during early stages of drug development remains elusive. A profound understanding of the different molecular mechanisms of DIC is, therefore, of utmost importance. Although many knowledge gaps and caveats still exist, it is generally accepted that alterations of certain hepatobiliary membrane transporters and changes in hepatocellular morphology may cause DIC. Consequently, liver models, which represent most of these mechanisms, are valuable tools to predict human DIC. Some of these models, such as membrane-based in vitro models, are exceptionally well-suited to investigate specific mechanisms (i.e. transporter inhibition) of DIC, while others, such as liver slices, encompass all relevant biological processes and, therefore, offer a better representation of the in vivo situation. In the current review, we highlight the principal molecular mechanisms associated with DIC and offer an overview and critical appraisal of the different liver models that are currently being used to predict the cholestatic potential of drugs.