Time to Change: A Systems Pharmacology Approach to Disentangle Mechanisms of Drug-Induced Mitochondrial Toxicity.

An increasing number of commonly prescribed drugs are known to interfere with mitochondrial function, which is associated with almost half of all Food and Drug Administration black box warnings, a variety of drug withdrawals, and attrition of drug candidates. This can mainly be attributed to a historic lack of sensitive and specific assays to identify the mechanisms underlying mitochondrial toxicity during drug development. In the last decade, a better understanding of drug-induced mitochondrial dysfunction has been achieved by network-based and structure-based systems pharmacological approaches. Here, we propose the implementation of a tiered systems pharmacology approach to detect adverse mitochondrial drug effects during preclinical drug development, which is based on a toolset developed to study inherited mitochondrial disease. This includes phenotypic characterization, profiling of key metabolic alterations, mechanistic studies, and functional in vitro and in vivo studies. Combined with binding pocket similarity comparisons and bottom-up as well as top-down metabolic network modeling, this tiered approach enables identification of mechanisms underlying drug-induced mitochondrial dysfunction. After validation of these off-target mechanisms, drug candidates can be adjusted to minimize mitochondrial activity. Implementing such a tiered systems pharmacology approach could lead to a more efficient drug development trajectory due to lower drug attrition rates and ultimately contribute to the development of safer drugs. SIGNIFICANCE STATEMENT: Many commonly prescribed drugs adversely affect mitochondrial function, which can be detected using phenotypic assays. However, these methods provide only limited insight into the underlying mechanisms. In recent years, a better understanding of drug-induced mitochondrial dysfunction has been achieved by network-based and structure-based system pharmacological approaches. Their implementation in preclinical drug development could reduce the number of drug failures, contributing to safer drug design.

Statins affect human iPSC-derived cardiomyocytes by interfering with mitochondrial function and intracellular acidification.

Statins are effective drugs in reducing cardiovascular morbidity and mortality by inhibiting cholesterol synthesis. These effects are primarily beneficial for the patient's vascular system. A significant number of statin users suffer from muscle complaints probably due to mitochondrial dysfunction, a mechanism that has recently been elucidated. This has raised our interest in exploring the effects of statins on cardiac muscle cells in an era where the elderly and patients with poorer functioning hearts and less metabolic spare capacity start dominating our patient population. Here, we investigated the effects of statins on human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-derived CMs). hiPSC-derived CMs were exposed to simvastatin, atorvastatin, rosuvastatin, and cerivastatin at increasing concentrations. Metabolic assays and fluorescent microscopy were employed to evaluate cellular viability, metabolic capacity, respiration, intracellular acidity, and mitochondrial membrane potential and morphology. Over a concentration range of 0.3-100 µM, simvastatin lactone and atorvastatin acid showed a significant reduction in cellular viability by 42-64%. Simvastatin lactone was the most potent inhibitor of basal and maximal respiration by 56% and 73%, respectively, whereas simvastatin acid and cerivastatin acid only reduced maximal respiration by 50% and 42%, respectively. Simvastatin acid and lactone and atorvastatin acid significantly decreased mitochondrial membrane potential by 20%, 6% and 3%, respectively. The more hydrophilic atorvastatin acid did not seem to affect cardiomyocyte metabolism. This calls for further research on the translatability to the clinical setting, in which a more conscientious approach to statin prescribing might be considered, especially regarding the current shift in population toward older patients with poor cardiac function.

In silico ecotoxicity assessment of pharmaceutical residues in wastewater following oxidative treatment.

Advanced oxidation processes such as thermal plasma activation and UV-C/H2O2 treatment are considered as applications for the degradation of pharmaceutical residues in wastewater complementary to conventional wastewater treatment. It is supposed that direct oxidative treatment can lower the toxicity of hospital sewage water (HSW). The aim of this study was to predict the ecotoxicity for three aquatic species before and after oxidative treatment of 10 quantified pharmaceuticals in hospital sewage water. With the application of oxidative chemistry, pharmaceuticals are degraded into transformation products before reaching complete mineralization. To estimate the potential ecotoxicity for fish, Daphnia and green algae ECOSAR quantitative structure-activity relationship software was used. Structure information from pristine pharmaceuticals and their oxidative transformation products were calculated separately and in a mixture computed to determine the risk quotient (RQ). Calculated mixture toxicities for 10 compounds found in untreated HSW resulted in moderate-high RQ predictions for all three aquatic species. Compared to untreated HSW, 30-min treatment with thermal plasma activation or UV-C/H2O2 resulted in lowered RQs. For the expected transformation products originating from fluoxetine, cyclophosphamide and acetaminophen increased RQs were predicted. Prolongation of thermal plasma oxidation up to 120 min predicted low-moderate toxicity in all target species. It is anticipated that further degradation of oxidative transformation products will end in less toxic aliphatic and carboxylic acid products. Predicted RQs after UV-C/H2O2 treatment turned out to be still moderate-high. In conclusion, in silico extrapolation of experimental findings can provide useful predicted estimates of mixture toxicity. However due to the complex composition of wastewater this in silico approach is a first step to screen for ecotoxicity. It is recommendable to confirm these predictions with ecotoxic bioassays.

A roadmap towards a human-centric safety assessment of advanced therapy medicinal products.

Advanced therapy medicinal products (ATMPs) are among the most complex pharmaceuticals with high human specificity. Species differences severely limit the clinical relevance of in vivo data. We conducted interviews with stakeholders involved in ATMP development about their perspective on the use of in vivo studies, the perceived hurdles and associated potential solutions regarding non-clinical development of ATMPs. In total, 17 stakeholders from 9 different countries were interviewed. A workshop was held with key stakeholders to further discuss major topics identified from the interviews. Conducting in vivo studies remains the status quo for ATMPs development. The hurdles identified included determining the amount of information required before clinical entry and effective use of limited human samples to understand a treatment or for clinical monitoring. A number of key points defined the need for future in vivo studies as well as improved application and implementation of New Approach Methodology (NAM)-based approach for products within a well-known modality or technology platform. These included data transparency, understanding of the added value of in vivo studies, and continuous advancement, evaluation, and qualification of NAMs. Based on the outcome of the discussions, a roadmap with practical steps towards a human-centric safety assessment of ATMPs was established.

In vitro screening model for compound interactions with human and dairy animal BCRP orthologs.

Orthologs of breast cancer resistance protein (BCRP/ABCG2), an ATP-binding cassette (ABC) efflux transmembrane transporter, are present in several species. The list of compounds known to interact with BCRP is growing, and many questions remain concerning species-specific variations in substrate specificity and affinity and the potency of inhibitors. As the most abundant efflux transporter known to be present in the blood-milk barrier, BCRP can increase the elimination of certain xenobiotics to milk, posing a risk for suckling offspring and dairy product consumers. Here we developed a model that can be employed to investigate species-specific differences between BCRP substrates and inhibitors. Membrane vesicles were isolated from transiently transduced human embryonic kidney (HEK) 293 cells, overexpressing BCRP, with human, bovine, caprine, and ovine cDNA sequences. To confirm BCRP transport activity in the transduced cells, D-luciferin efflux was measured and to confirm transport activity in the membrane vesicles, [3H] estrone-3-sulfate ([3H]E1S) influx was measured. We also determined the Michaelis-Menten constant (Km) and Vmax of [3H]E1S for each species. We have developed an in vitro transport model to study differences in compound interactions with BCRP orthologs from milk-producing animal species and humans. BCRP transport activity was demonstrated in the species-specific transduced cells by a reduced accumulation of D-luciferin compared with the control cells, indicating BCRP-mediated efflux of D-luciferin. Functionality of the membrane vesicle model was demonstrated by confirming ATP-dependent transport and by quantifying the kinetic parameters, Km and Vmax for the model substrate [3H]E1S. The values were not significantly different between species for the model substrates tested. This model can be insightful for appropriate inter-species extrapolations and risk assessments of xenobiotics in lactating woman and dairy animals.

Perfluoroalkyl substances (PFASs) are substrates of the renal human organic anion transporter 4 (OAT4).

Poly- and perfluoroalkyl substances (PFASs) are omnipresent in the environment and have been shown to accumulate in humans. Most PFASs are not biotransformed in animals and humans, so that elimination is largely dependent on non-metabolic clearance via bile and urine. Accumulation of certain PFASs in humans may relate to their reabsorption from the pre-urine by transporter proteins in the proximal tubules of the kidney, such as URAT1 and OAT4. The present study assessed the in vitro transport of 7 PFASs (PFHpA, PFOA, PFNA, PFDA, PFBS, PFHxS and PFOS) applying URAT1- or OAT4-transfected human embryonic kidney (HEK) cells. Virtually no transport of PFASs could be measured in URAT1-transfected HEK cells. All PFASs, except PFBS, showed clear uptake in OAT4-transfected HEK cells. In addition, these in vitro results were further supported by in silico docking and molecular dynamic simulation studies assessing transporter-ligand interactions. Information on OAT4-mediated transport may provide insight into the accumulation potential of PFASs in humans, but other kinetic aspects may play a role and should also be taken into account. Quantitative information on all relevant kinetic processes should be integrated in physiologically based kinetic (PBK) models, to predict congener-specific accumulation of PFASs in humans in a more accurate manner.

Metabolic impact of genetic and chemical ADP/ATP carrier inhibition in renal proximal tubule epithelial cells.

Mitochondrial dysfunction is pivotal in drug-induced acute kidney injury (AKI), but the underlying mechanisms remain largely unknown. Transport proteins embedded in the mitochondrial inner membrane form a significant class of potential drug off-targets. So far, most transporter-drug interactions have been reported for the mitochondrial ADP/ATP carrier (AAC). Since it remains unknown to what extent AAC contributes to drug-induced mitochondrial dysfunction in AKI, we here aimed to better understand the functional role of AAC in the energy metabolism of human renal proximal tubular cells. To this end, CRISPR/Cas9 technology was applied to generate AAC3-/- human conditionally immortalized renal proximal tubule epithelial cells. This AAC3-/- cell model was characterized with respect to mitochondrial function and morphology. To explore whether this model could provide first insights into (mitochondrial) adverse drug effects with suspicion towards AAC-mediated mechanisms, wild-type and knockout cells were exposed to established AAC inhibitors, after which cellular metabolic activity and mitochondrial respiratory capacity were measured. Two AAC3-/- clones showed a significant reduction in ADP import and ATP export rates and mitochondrial mass, without influencing overall morphology. AAC3-/- clones exhibited reduced ATP production, oxygen consumption rates and metabolic spare capacity was particularly affected, mainly in conditions with galactose as carbon source. Chemical AAC inhibition was stronger compared to genetic inhibition in AAC3-/-, suggesting functional compensation by remaining AAC isoforms in our knockout model. In conclusion, our results indicate that ciPTEC-OAT1 cells have a predominantly oxidative phenotype that was not additionally activated by switching energy source. Genetic inhibition of AAC3 particularly impacted mitochondrial spare capacity, without affecting mitochondrial morphology, suggesting an important role for AAC in maintaining the metabolic spare respiration.

Brothers in Arms: ABCA1- and ABCG1-Mediated Cholesterol Efflux as Promising Targets in Cardiovascular Disease Treatment.

Atherosclerosis is a leading cause of cardiovascular disease worldwide, and hypercholesterolemia is a major risk factor. Preventive treatments mainly focus on the effective reduction of low-density lipoprotein cholesterol, but their therapeutic value is limited by the inability to completely normalize atherosclerotic risk, probably due to the disease complexity and multifactorial pathogenesis. Consequently, high-density lipoprotein cholesterol gained much interest, as it appeared to be cardioprotective due to its major role in reverse cholesterol transport (RCT). RCT facilitates removal of cholesterol from peripheral tissues, including atherosclerotic plaques, and its subsequent hepatic clearance into bile. Therefore, RCT is expected to limit plaque formation and progression. Cellular cholesterol efflux is initiated and propagated by the ATP-binding cassette (ABC) transporters ABCA1 and ABCG1. Their expression and function are expected to be rate-limiting for cholesterol efflux, which makes them interesting targets to stimulate RCT and lower atherosclerotic risk. This systematic review discusses the molecular mechanisms relevant for RCT and ABCA1 and ABCG1 function, followed by a critical overview of potential pharmacological strategies with small molecules to enhance cellular cholesterol efflux and RCT. These strategies include regulation of ABCA1 and ABCG1 expression, degradation, and mRNA stability. Various small molecules have been demonstrated to increase RCT, but the underlying mechanisms are often not completely understood and are rather unspecific, potentially causing adverse effects. Better understanding of these mechanisms could enable the development of safer drugs to increase RCT and provide more insight into its relation with atherosclerotic risk. SIGNIFICANCE STATEMENT: Hypercholesterolemia is an important risk factor of atherosclerosis, which is a leading pathological mechanism underlying cardiovascular disease. Cholesterol is removed from atherosclerotic plaques and subsequently cleared by the liver into bile. This transport is mediated by high-density lipoprotein particles, to which cholesterol is transferred via ATP-binding cassette transporters ABCA1 and ABCG1. Small-molecule pharmacological strategies stimulating these transporters may provide promising options for cardiovascular disease treatment.

Physiologically based pharmacokinetic/pharmacodynamic model for the prediction of morphine brain disposition and analgesia in adults and children.

Morphine is a widely used opioid analgesic, which shows large differences in clinical response in children, even when aiming for equivalent plasma drug concentrations. Age-dependent brain disposition of morphine could contribute to this variability, as developmental increase in blood-brain barrier (BBB) P-glycoprotein (Pgp) expression has been reported. In addition, age-related pharmacodynamics might also explain the variability in effect. To assess the influence of these processes on morphine effectiveness, a multi-compartment brain physiologically based pharmacokinetic/pharmacodynamic (PB-PK/PD) model was developed in R (Version 3.6.2). Active Pgp-mediated morphine transport was measured in MDCKII-Pgp cells grown on transwell filters and translated by an in vitro-in vivo extrapolation approach, which included developmental Pgp expression. Passive BBB permeability of morphine and its active metabolite morphine-6-glucuronide (M6G) and their pharmacodynamic parameters were derived from experiments reported in literature. Model simulations after single dose morphine were compared with measured and published concentrations of morphine and M6G in plasma, brain extracellular fluid (ECF) and cerebrospinal fluid (CSF), as well as published drug responses in children (1 day- 16 years) and adults. Visual predictive checks indicated acceptable overlays between simulated and measured morphine and M6G concentration-time profiles and prediction errors were between 1 and -1. Incorporation of active Pgp-mediated BBB transport into the PB-PK/PD model resulted in a 1.3-fold reduced brain exposure in adults, indicating only a modest contribution on brain disposition. Analgesic effect-time profiles could be described reasonably well for older children and adults, but were largely underpredicted for neonates. In summary, an age-appropriate morphine PB-PK/PD model was developed for the prediction of brain pharmacokinetics and analgesic effects. In the neonatal population, pharmacodynamic characteristics, but not brain drug disposition, appear to be altered compared to adults and older children, which may explain the reported differences in analgesic effect.

The Role of Efflux Pumps in Tuberculosis Treatment and Their Promise as a Target in Drug Development: Unraveling the Black Box.

Insight into drug transport mechanisms is highly relevant to the efficacious treatment of tuberculosis (TB). Major problems in TB treatment are related to the transport of antituberculosis (anti-TB) drugs across human and mycobacterial membranes, affecting the concentrations of these drugs systemically and locally. Firstly, transporters located in the intestines, liver, and kidneys all determine the pharmacokinetics and pharmacodynamics of anti-TB drugs, with a high risk of drug-drug interactions in the setting of concurrent use of antimycobacterial, antiretroviral, and antidiabetic agents. Secondly, human efflux transporters limit the penetration of anti-TB drugs into the brain and cerebrospinal fluid, which is especially important in the treatment of TB meningitis. Finally, efflux transporters located in the macrophage and Mycobacterium tuberculosis cell membranes play a pivotal role in the emergence of phenotypic tolerance and drug resistance, respectively. We review the role of efflux transporters in TB drug disposition and evaluate the promise of efflux pump inhibition from a novel holistic perspective.

Ontogeny of Small Intestinal Drug Transporters and Metabolizing Enzymes Based on Targeted Quantitative Proteomics.

Most drugs are administered to children orally. An information gap remains on the protein abundance of small intestinal drug-metabolizing enzymes (DMEs) and drug transporters (DTs) across the pediatric age range, which hinders precision dosing in children. To explore age-related differences in DMEs and DTs, surgical leftover intestinal tissues from pediatric and adult jejunum and ileum were collected and analyzed by targeted quantitative proteomics for apical sodium-bile acid transporter, breast cancer resistance protein (BCRP), monocarboxylate transporter 1 (MCT1), multidrug resistance protein 1 (MDR1), multidrug resistance-associated protein (MRP) 2, MRP3, organic anion-transporting polypeptide 2B1, organic cation transporter 1, peptide transporter 1 (PEPT1), CYP2C19, CYP3A4, CYP3A5, UDP glucuronosyltransferase (UGT) 1A1, UGT1A10, and UGT2B7. Samples from 58 children (48 ileums, 10 jejunums, age range: 8 weeks to 17 years) and 16 adults (8 ileums, 8 jejunums) were analyzed. When comparing age groups, BCRP, MDR1, PEPT1, and UGT1A1 abundance was significantly higher in adult ileum as compared with the pediatric ileum. Jejunal BCRP, MRP2, UGT1A1, and CYP3A4 abundance was higher in the adults compared with children 0-2 years of age. Examining the data on a continuous age scale showed that PEPT1 and UGT1A1 abundance was significantly higher, whereas MCT1 and UGT2B7 abundance was lower in adult ileum as compared with the pediatric ileum. Our data contribute to the deeper understanding of the ontogeny of small intestinal drug-metabolizing enzymes and drug transporters and shows DME-, DT-, and intestinal location-specific, age-related changes. SIGNIFICANCE STATEMENT: This is the first study that describes the ontogeny of small intestinal DTs and DMEs in human using liquid chromatography with tandem mass spectrometry-based targeted quantitative proteomics. The current analysis provides a detailed picture about the maturation of DT and DME abundances in the human jejunum and ileum. The presented results supply age-related DT and DME abundance data for building more accurate PBPK models that serve to support safer and more efficient drug dosing regimens for the pediatric population.

Placental disposition of eculizumab, C5 and C5-eculizumab in two pregnancies of a woman with paroxysmal nocturnal haemoglobinuria.

Eculizumab is known to cross the placenta to a limited degree, but recently therapeutic drug levels in cord blood were found in a single case. We report maternal, cord and placental levels of unbound eculizumab, C5 and C5-eculizumab in two pregnancies of a paroxysmal nocturnal haemoglobinuria patient who received 900 mg eculizumab every 2 weeks. In both pregnancies, cord blood concentrations of unbound eculizumab were below 4 µg/mL, while C5-eculizumab levels were 22 and 26 µg/mL, suggesting that a considerable fraction of C5 was blocked in the newborn. Concentrations in each placenta of unbound eculizumab were 41 ± 3 and 45 ± 4 µg/g tissue, of C5-eculizumab 19 ± 2 and 32 ± 3 µg/g, and of C5 20 ± 3 and 30 ± 2 µg/g (mean ± SD, in three tissue samples per placenta). Placental levels of unbound eculizumab were higher than those of C5-eculizumab complexes, while maternal concentrations were approximately equal, suggesting selective transport of unbound eculizumab across the placenta.

Application of proteomics to understand maturation of drug metabolizing enzymes and transporters for the optimization of pediatric drug therapy.

Drug disposition in children is different compared to adults. Growth and developmental change the processes involved in drug disposition and efficacy, including membrane transporters and drug metabolizing enzymes, but for many of these proteins, the exact changes have not been fully elucidated to date. Quantitative proteomics offers a solution to analyze many DME and DT proteins at once and can be performed with very small tissue samples, overcoming many of the challenges previously limiting research in this pediatric field. Liquid chromatography tandem mass spectrometry (LC-MS/MS) based methods for quantification of (membrane) proteins has evolved as a golden standard for proteomic analysis. The last years, big steps have been made in maturation studies of hepatic and renal drug transporters and drug metabolizing enzymes using this method. Protein and organ specific maturation patterns have been identified for the human liver and kidney, which aids pharmacological modelling and predicting drug dosing in the pediatric population. Further research should focus on other organs, like intestine and brain, as well as on innovative methods in which proteomics can be used to further overcome the limited access to pediatric tissues, including liquid biopsies and organoids. In this review there is aimed to provide an overview of available human pediatric proteomics data, discuss its challenges and provide guidance for future research.

Thermal plasma activation and UV/H2O2 oxidative degradation of pharmaceutical residues.

The aquatic environment becomes increasingly contaminated by anthropogenic pollutants such as pharmaceutical residues. Due to poor biodegradation and continuous discharge of persistent compounds in sewage water samples, pharmaceutical residues might end up in surface waters when not removed. To minimize this pollution, onsite wastewater treatment techniques might complement conventional waste water treatment plants (WWTPs). Advanced oxidation processes are useful techniques, since reactive oxygen species (ROS) are used for the degradation of unwanted medicine residues. In this paper we have studied the advanced oxidation in a controlled laboratory setting using thermal plasma and UV/H2O2 treatment. Five different matrices, Milli-Q water, tap water, synthetic urine, diluted urine and synthetic sewage water were spiked with 14 pharmaceuticals with a concentration of 5 µg/L. All compounds were reduced or completely decomposed by both 150 W thermal plasma and UV/H2O2 treatment. Additionally, also hospital sewage water was tested. First the concentrations of 10 pharmaceutical residues were determined by liquid chromatography mass spectrometry (LC-MS/MS). The pharmaceutical concentration ranged from 0.08 up to 2400 µg/L. With the application of 150 W thermal plasma or UV/H2O2, it was found that overall pharmaceutical degradation in hospital sewage water were nearly equivalent to the results obtained in the synthetic sewage water. However, based on the chemical abatement kinetics it was demonstrated that the degree of degradation decreases with increasing matrix complexity. Since reactive oxygen and nitrogen species (RONS) are continuously produced, thermal plasma treatment has the advantage over UV/H2O2 treatment.

Differences in P-glycoprotein activity in human and rodent blood-brain barrier assessed by mechanistic modelling.

Variation in the efficacy and safety of central nervous system drugs between humans and rodents can be explained by physiological differences between species. An important factor could be P-glycoprotein (Pgp) activity in the blood-brain barrier (BBB), as BBB expression of this drug efflux transporter is reportedly lower in humans compared to mouse and rat and subject to an age-dependent increase. This might complicate animal to human extrapolation of brain drug disposition and toxicity, especially in children. In this study, the potential species-specific effect of BBB Pgp activity on brain drug exposure was investigated. An age-dependent brain PBPK model was used to predict cerebrospinal fluid and brain mass concentrations of Pgp substrate drugs. For digoxin, verapamil and quinidine, in vitro kinetic data on their transport by Pgp were derived from literature and used to scale to in vivo parameters. In addition, age-specific digoxin transport was simulated for children with a postnatal age between 25 and 81 days. BBB Pgp activity in the model was optimized using measured CSF data for the Pgp substrates ivermectin, indinavir, vincristine, docetaxel, paclitaxel, olanzapine and citalopram, as no useful in vitro data were available. Inclusion of Pgp activity in the model resulted in optimized predictions of their brain concentration. Total brain-to-plasma AUC values (Kp,brain) in the simulations without Pgp were divided by the Kp,brain values with Pgp. Kp ratios ranged from 1 to 45 for the substrates investigated. Comparison of human with rodent Kp,brain ratios indicated ≥ twofold lower values in human for digoxin, verapamil, indinavir, paclitaxel and citalopram and ≥ twofold higher values for vincristine. In conclusion, BBB Pgp activity appears species-specific. An age-dependent PBPK model-based approach could be useful to extrapolate animal data to human adult and paediatric predictions by taking into account species-specific and developmental BBB Pgp expression.

Toxicity of anticancer drugs in human placental tissue explants and trophoblast cell lines.

The application of anticancer drugs during pregnancy is associated with placenta-related adverse pregnancy outcomes. Therefore, it is important to study placental toxicity of anticancer drugs. The aim of this study was to compare effects on viability and steroidogenesis in placental tissue explants and trophoblast cell lines. Third trimester placental tissue explants were exposed for 72 h (culture day 4-7) to a concentration range of doxorubicin, paclitaxel, cisplatin, carboplatin, crizotinib, gefitinib, imatinib, or sunitinib. JEG-3, undifferentiated BeWo, and syncytialised BeWo cells were exposed for 48 h to the same drugs and concentrations. After exposure, tissue and cell viability were assessed and progesterone and estrone levels were quantified in culture medium. Apart from paclitaxel, all compounds affected both cell and tissue viability at clinically relevant concentrations. Paclitaxel affected explant viability moderately, while it reduced cell viability by 50% or more in all cell lines, at 3-10 nM. Doxorubicin (1 µM) reduced viability in explants to 83 ± 7% of control values, whereas it fully inhibited viability in all cell types. Interference with steroid release in explants was difficult to study due to large variability in measurements, but syncytialised BeWo cells proved suitable for this purpose. We found that 1 µM sunitinib reduced progesterone release to 76 ± 6% of control values, without affecting cell viability. While we observed differences between the models for paclitaxel and doxorubicin, most anticancer drugs affected viability significantly in both placental explants and trophoblast cell lines. Taken together, the placenta should be recognized as a potential target organ for toxicity of anticancer drugs.

A Randomized Trial of Distal Diuretics versus Dietary Sodium Restriction for Hypertension in Chronic Kidney Disease.

BACKGROUND: Distal diuretics are considered less effective than loop diuretics in CKD. However, data to support this perception are limited. METHODS: To investigate whether distal diuretics are noninferior to dietary sodium restriction in reducing BP in patients with CKD stage G3 or G4 and hypertension, we conducted a 6-week, randomized, open-label crossover trial comparing amiloride/hydrochlorothiazide (5 mg/50 mg daily) with dietary sodium restriction (60 mmol per day). Antihypertension medication was discontinued for a 2-week period before randomization. We analyzed effects on BP, kidney function, and fluid balance and related this to renal clearance of diuretics. RESULTS: A total of 26 patients (with a mean eGFR of 39 ml/min per 1.73 m2) completed both treatments. Dietary sodium restriction reduced sodium excretion from 160 to 64 mmol per day. Diuretics produced a greater reduction in 24-hour systolic BP (SBP; from 138 to 124 mm Hg) compared with sodium restriction (from 134 to 129 mm Hg), as well as a significantly greater effect on extracellular water, eGFR, plasma renin, and aldosterone. Both interventions resulted in a similar decrease in body weight and NT-proBNP. Neither approaches decreased albuminuria significantly, whereas diuretics did significantly reduce urinary angiotensinogen and ß2-microglobulin excretion. Although lower eGFR and higher plasma indoxyl sulfate correlated with lower diuretic clearance, the diuretic effects on body weight and BP at lower eGFR were maintained. During diuretic treatment, higher PGE2 excretion correlated with lower free water clearance, and four patients developed mild hyponatremia. CONCLUSIONS: Distal diuretics are noninferior to dietary sodium restriction in reducing BP and extracellular volume in CKD. Diuretic sensitivity in CKD is maintained despite lower diuretic clearance. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: DD-study: Diet or Diuretics for Salt-sensitivity in Chronic Kidney Disease (DD), NCT02875886.

Rifampicin Transport by OATP1B1 Variants.

Single nucleotide polymorphisms in the OATP1B1 transporter have been suggested to partially explain the large interindividual variation in rifampicin exposure. HEK293 cells overexpressing wild-type (WT) or OATP1B1 variants *1b, *4, *5, and *15 were used to determine the in vitro rifampicin intrinsic clearance. For OATP1B1*5 and *15, a 36% and 42% reduction in intrinsic clearance, respectively, compared to WT was found. We consider that these differences in intrinsic clearance most likely have minor clinical implications.

Development of a physiologically-based pharmacokinetic pediatric brain model for prediction of cerebrospinal fluid drug concentrations and the influence of meningitis.

Different pediatric physiologically-based pharmacokinetic (PBPK) models have been described incorporating developmental changes that influence plasma drug concentrations. Drug disposition into cerebrospinal fluid (CSF) is also subject to age-related variation and can be further influenced by brain diseases affecting blood-brain barrier integrity, like meningitis. Here, we developed a generic pediatric brain PBPK model to predict CSF concentrations of drugs that undergo passive transfer, including age-appropriate parameters. The model was validated for the analgesics paracetamol, ibuprofen, flurbiprofen and naproxen, and for a pediatric meningitis population by empirical optimization of the blood-brain barrier penetration of the antibiotic meropenem. Plasma and CSF drug concentrations derived from the literature were used to perform visual predictive checks and to calculate ratios between simulated and observed area under the concentration curves (AUCs) in order to evaluate model performance. Model-simulated concentrations were comparable to observed data over a broad age range (3 months-15 years postnatal age) for all drugs investigated. The ratios between observed and simulated AUCs (AUCo/AUCp) were within 2-fold difference both in plasma (range 0.92-1.09) and in CSF (range 0.64-1.23) indicating acceptable model performance. The model was also able to describe disease-mediated changes in neonates and young children (<3m postnatal age) related to meningitis and sepsis (range AUCo/AUCp plasma: 1.64-1.66, range AUCo/AUCp CSF: 1.43-1.73). Our model provides a new computational tool to predict CSF drug concentrations in children with and without meningitis and can be used as a template model for other compounds that passively enter the CNS.

Safety of drug use in patients with a primary mitochondrial disease: An international Delphi-based consensus.

Clinical guidance is often sought when prescribing drugs for patients with primary mitochondrial disease. Theoretical considerations concerning drug safety in patients with mitochondrial disease may lead to unnecessary withholding of a drug in a situation of clinical need. The aim of this study was to develop consensus on safe medication use in patients with a primary mitochondrial disease. A panel of 16 experts in mitochondrial medicine, pharmacology, and basic science from six different countries was established. A modified Delphi technique was used to allow the panellists to consider draft recommendations anonymously in two Delphi rounds with predetermined levels of agreement. This process was supported by a review of the available literature and a consensus conference that included the panellists and representatives of patient advocacy groups. A high level of consensus was reached regarding the safety of all 46 reviewed drugs, with the knowledge that the risk of adverse events is influenced both by individual patient risk factors and choice of drug or drug class. This paper details the consensus guidelines of an expert panel and provides an important update of previously established guidelines in safe medication use in patients with primary mitochondrial disease. Specific drugs, drug groups, and clinical or genetic conditions are described separately as they require special attention. It is important to emphasise that consensus-based information is useful to provide guidance, but that decisions related to drug prescribing should always be tailored to the specific needs and risks of each individual patient. We aim to present what is current knowledge and plan to update this regularly both to include new drugs and to review those currently included.