Innovative approaches and recent advances in the study of ontogeny of drug metabolism and transport.

The disposition of a drug is driven by various processes, such as drug metabolism, drug transport, glomerular filtration and body composition. These processes are subject to developmental changes reflecting growth and maturation along the paediatric continuum. However, knowledge gaps exist on these changes and their clinical impact. Filling these gaps may aid better prediction of drug disposition and creation of age-appropriate dosing guidelines. We present innovative approaches to study these developmental changes in relation to drug metabolism and transport. First, analytical methods such as including liquid chromatography-mass spectrometry for proteomic analyses allow quantitation of the expressions of a wide variety of proteins, e.g. membrane transporters, in a small piece of organ tissue. The latter is specifically important for paediatric research, where tissues are scarcely available. Second, innovative study designs using radioactive labelled microtracers allowed study-without risk for the child-of the oral bioavailability of compounds used as markers for certain drug metabolism pathways. Third, the use of modelling and simulation to support dosing recommendations for children is supported by both the European Medicines Agency and the US Food and Drug Administration. This may even do away with the need for a paediatric trial. Physiologically based pharmacokinetics models, which include age-specific physiological information are, therefore, increasingly being used, not only to aid paediatric drug development but also to improve existing drug therapies.

Prospective Investigation of the Performance of 2 Gamma-Hydroxybutyric Acid Tests: DrugCheck GHB Single Test and Viva-E GHB Immunoassay.

BACKGROUND: Gamma-hydroxybutyric acid (GHB) is a recreational drug with central nervous system depressing effects that is often abused. A urine GHB point-of-care test can be of great diagnostic value. The objective of this prospective study was to determine the performance of the new DrugCheck GHB Single Test and the Viva-E GHB immunoassay for urine samples in emergency department patients. METHODS: Patients presented to the emergency department of the OLVG hospital in Amsterdam with a Glasgow Coma Scale score <15 and potential drug of abuse intoxication were included in the study. Between June 2016 and October 2017, 375 patients were included. Using the DrugCheck GHB Single Test (Express Diagnostics Int'l, Blue Earth, MN) and the Viva-E GHB immunoassay (Siemens Healthineers, The Hague, the Netherlands), patients' urine samples were tested for GHB (cutoff for a positive result, 10 or 50 mcg/mL GHB). To ensure quality, the results obtained were compared with those generated using a validated gas chromatography method. The tests were considered reliable if specificity and sensitivity were both >90%. Possible cross-reactivity with ethanol was investigated by analyzing ethanol concentrations in patients' samples. RESULTS: Seventy percentage of the included patients was men, and the median age was 34 years old. The DrugCheck GHB Single Test's specificity and sensitivity were 90.0% and 72.9%, respectively, and using 50 mcg/mL as a cutoff value, its specificity and sensitivity improved to 96.7% and 75.0%, respectively. Serum and urine ethanol levels in the false-positive group were significantly higher compared with those in the true-negative group. The specificity and sensitivity of the Viva-E GHB immunoassay (cutoff value of 50 mcg/mL and excluding samples with ethanol levels ≥2.0 g/L) were 99.4% and 93.5%, respectively. CONCLUSIONS: The DrugCheck GHB Single Test's specificity was sufficient, whereas its sensitivity was poor, making it unsuitable for use at point-of-care. Contrarily, using 50 mcg/mL as the cutoff value and excluding samples with ethanol levels ≥2.0 g/L, the Viva-E GHB immunoassay showed acceptable results to detect clinically relevant GHB intoxications.

Dose-linearity of the pharmacokinetics of an intravenous [14 C]midazolam microdose in children.

AIMS: Drug disposition in children may vary from adults due to age-related variation in drug metabolism. Microdose studies present an innovation to study pharmacokinetics (PK) in paediatrics; however, they should be used only when the PK is dose linear. We aimed to assess dose linearity of a [14 C]midazolam microdose, by comparing the PK of an intravenous (IV) microtracer (a microdose given simultaneously with a therapeutic midazolam dose), with the PK of a single isolated microdose. METHODS: Preterm to 2-year-old infants admitted to the intensive care unit received [14 C]midazolam IV as a microtracer or microdose, followed by dense blood sampling up to 36 hours. Plasma concentrations of [14 C]midazolam and [14 C]1-hydroxy-midazolam were determined by accelerator mass spectrometry. Noncompartmental PK analysis was performed and a population PK model was developed. RESULTS: Of 15 infants (median gestational age 39.4 [range 23.9-41.4] weeks, postnatal age 11.4 [0.6-49.1] weeks), 6 received a microtracer and 9 a microdose of [14 C]midazolam (111 Bq kg-1 ; 37.6 ng kg-1 ). In a 2-compartment PK model, bodyweight was the most significant covariate for volume of distribution. There was no statistically significant difference in any PK parameter between the microdose and microtracer, nor in the area under curve ratio [14 C]1-OH-midazolam/[14 C]midazolam, showing the PK of midazolam to be linear within the range of the therapeutic and microdoses. CONCLUSION: Our data support the dose linearity of the PK of an IV [14 C]midazolam microdose in children. Hence, a [14 C]midazolam microdosing approach may be used as an alternative to a therapeutic dose of midazolam to study developmental changes in hepatic CYP3A activity in young children.

Proteomic Analysis of the Developmental Trajectory of Human Hepatic Membrane Transporter Proteins in the First Three Months of Life.

Human hepatic membrane-embedded transporter proteins are involved in trafficking endogenous and exogenous substrates. Even though impact of transporters on pharmacokinetics is recognized, little is known on maturation of transporter protein expression levels, especially during early life. We aimed to study the protein expression of 10 transporters in liver tissue from fetuses, infants, and adults. Transporter protein expression levels [ATP-binding cassette transporter (ABC)B1, ABCG2, ABCC2, ABCC3, bile salt efflux pump, glucose transporter 1, monocarboxylate transporter 1, organic anion transporter polypeptide (OATP)1B1, OATP2B1, and organic cation/carnitine transporter 2) were quantified using ultraperformance liquid chromatography tandem mass spectrometry in snap-frozen postmortem fetal, infant, and adult liver samples. Protein expression was quantified in isolated crude membrane fractions. The possible association between postnatal and postmenstrual age versus protein expression was studied. We studied 25 liver samples, as follows: 10 fetal [median gestational age 23.2 wk (range 16.4-37.9)], 12 infantile [gestational age at birth 35.1 wk (27.1-41.0), postnatal age 1 wk (0-11.4)], and 3 adult. The relationship of protein expression with age was explored by comparing age groups. Correlating age within the fetal/infant age group suggested four specific protein expression patterns, as follows: stable, low to high, high to low, and low-high-low. The impact of growth and development on human membrane transporter protein expression is transporter-dependent. The suggested age-related differences in transporter protein expression may aid our understanding of normal growth and development, and also may impact the disposition of substrate drugs in neonates and young infants.

Human Intestinal PEPT1 Transporter Expression and Localization in Preterm and Term Infants.

The intestinal influx oligopeptide transporter peptide transporter 1 (PEPT1) (SLC15A1) is best known for nutrient-derived di- and tripeptide transport. Its role in drug absorption is increasingly recognized. To better understand the disposition of PEPT1 substrate drugs in young infants, we studied intestinal PEPT1 mRNA expression and tissue localization across the pediatric age range. PEPT1 mRNA expression was determined using real-time reverse-transcription polymerase chain reaction in small intestinal tissues collected from surgical procedures (neonates and infants) or biopsies (older children and adolescents). PEPT1 mRNA relative to villin mRNA expression was compared between neonates/infants and older children/adolescents. PEPT1 was visualized in infant tissue using immunohistochemical staining. Other transporters [multidrug resistance protein 1 (MDR1), multidrug resistance-like protein 2 (MRP2), and organic anion transporter polypeptide 2B1 (OATP2B1)] were also stained to describe the localization in relation to PEPT1. Twenty-six intestinal samples (n = 20 neonates/infants, n = 2 pediatric, n = 4 adolescents) were analyzed. The young infant samples were collected at a median (range) gestational age at birth of 29.2 weeks (24.7-40) and postnatal age of 2.4 weeks (0-16.6). The PEPT1 mRNA expression of the neonates/infants was only marginally lower (0.8-fold) than the older children (P < 0.05). Similar and clear apical PEPT1 and MRP2 staining, apical and lateral MDR1 staining, and intraepithelial OATP2B1 staining at the basolateral membrane of the enterocyte were detected in 12 infant and 2 adolescent samples. Although small intestinal PEPT1 expression tended to be lower in neonates than in older children, this difference is small and tissue distribution is similar. This finding suggests similar oral absorption of PEPT1 substrates across the pediatric age range.

Maximum likelihood estimation of renal transporter ontogeny profiles for pediatric PBPK modeling.

Optimal treatment of infants with many renally cleared drugs must account for maturational differences in renal transporter (RT) activity. Pediatric physiologically-based pharmacokinetic (PBPK) models may incorporate RT activity, but this requires ontogeny profiles for RT activity in children, especially neonates, to predict drug disposition. Therefore, RT expression measurements from human kidney postmortem cortical tissue samples were normalized to represent a fraction of mature RT activity. Using these data, maximum likelihood estimated the distributions of RT activity across the pediatric age spectrum, including preterm and term neonates. PBPK models of four RT substrates (acyclovir, ciprofloxacin, furosemide, and meropenem) were evaluated with and without ontogeny profiles using average fold error (AFE), absolute average fold error (AAFE), and proportion of observations within the 5-95% prediction interval. Novel maximum likelihood profiles estimated ontogeny distributions for the following RT: OAT1, OAT3, OCT2, P-gp, URAT1, BCRP, MATE1, MRP2, MRP4, and MATE-2 K. Profiles for OAT3, P-gp, and MATE1 improved infant furosemide and neonate meropenem PBPK model AFE from 0.08 to 0.70 and 0.53 to 1.34 and model AAFE from 12.08 to 1.44 and 2.09 to 1.36, respectively, and improved the percent of data within the 5-95% prediction interval from 48% to 98% for neonatal ciprofloxacin simulations, respectively. Even after accounting for other critical population-specific maturational differences, novel RT ontogeny profiles substantially improved neonatal PBPK model performance, providing validated estimates of maturational differences in RT activity for optimal dosing in children.

Considerations for Human ADME Strategy and Design Paradigm Shift(s) - An Industry White Paper.

The human absorption, distribution, metabolism, and excretion (hADME) study is the cornerstone of the clinical pharmacology package for small molecule drugs, providing comprehensive information on the rates and routes of disposition and elimination of drug-related material in humans through the use of 14 C-labeled drug. Significant changes have already been made in the design of the hADME study for many companies, but opportunity exists to continue to re-think both the design and timing of the hADME study in light of the potential offered by newer technologies, that enable flexibility in particular to reducing the magnitude of the radioactive dose used. This paper provides considerations on the variety of current strategies that exist across a number of pharmaceutical companies and on some of the ongoing debates around a potential move to the so called "human first/human only" approach, already adopted by at least one company. The paper also provides a framework for continuing the discussion in the application of further shifts in the paradigm.

Pediatric Pharmacokinetics and Dose Predictions: A Report of a Satellite Meeting to the 10th Juvenile Toxicity Symposium.

On April 24, 2019, a symposium on Pediatric Pharmacokinetics and Dose Predictions was held as a satellite meeting to the 10th Juvenile Toxicity Symposium. This symposium brought together scientists from academia, industry, and clinical research organizations with the aim to update each other on the current knowledge on pediatric drug development. Through more knowledge on specific ontogeny profiles of drug metabolism and transporter proteins, integrated into physiologically-based pharmacokinetic (PBPK) models, we have gained a more integrated understanding of age-related differences in pharmacokinetics (PKs), Relevant examples were presented during the meeting. PBPK may be considered the gold standard for pediatric PK prediction, but still it is important to know that simpler methods, such as allometry, allometry combined with maturation function, functions based on the elimination pathway, or linear models, also perform well, depending on the age range or the mechanisms involved. Knowledge from different methods and information sources should be combined (e.g., microdosing can reveal early read-out of age-related differences in exposure), and such results can be a value to verify models. To further establish best practices for dose setting in pediatrics, more in vitro and in vivo research is needed on aspects such as age-related changes in the exposure-response relationship and the impact of disease on PK. New information coupled with the refining of model-based drug development approaches will allow faster targeting of intended age groups and allow more efficient design of pediatric clinical trials.

The Oral Bioavailability and Metabolism of Midazolam in Stable Critically Ill Children: A Pharmacokinetic Microtracing Study.

Midazolam is metabolized by the developmentally regulated intestinal and hepatic drug-metabolizing enzyme cytochrome P450 (CYP) 3A4/5. It is frequently administered orally to children, yet knowledge is lacking on the oral bioavailability in term neonates up until 1 year of age. Furthermore, the dispositions of the major metabolites 1-OH-midazolam (OHM) and 1-OH-midazolam-glucuronide (OHMG) after oral administration are largely unknown for the entire pediatric age span. We aimed to fill these knowledge gaps with a pediatric [14 C]midazolam microtracer population pharmacokinetic study. Forty-six stable, critically ill children (median age 9.8 (range 0.3-276.4) weeks) received a single oral [14 C]midazolam microtracer (58 (40-67) Bq/kg) when they received a therapeutic continuous intravenous midazolam infusion and had an arterial line in place enabling blood sampling. For midazolam, in a one-compartment model, bodyweight was a significant predictor for clearance (0.98 L/hour) and volume of distribution (8.7 L) (values for a typical individual of 5 kg). The typical oral bioavailability in the population was 66% (range 25-85%). The exposures of OHM and OHMG were highest for the youngest age groups and significantly decreased with postnatal age. The oral bioavailability of midazolam, largely reflective of intestinal and hepatic CYP3A activity, was on average lower than the reported 49-92% for preterm neonates, and higher than the reported 21% for children> 1 year of age and 30% for adults. As midazolam oral bioavailability varied widely, systemic exposure of other CYP3A-substrate drugs after oral dosing in this population may also be unpredictable, with risk of therapy failure or toxicity.

Alternative Splicing of the SLCO1B1 Gene: An Exploratory Analysis of Isoform Diversity in Pediatric Liver.

The hepatic influx transporter OATP1B1 (SLCO1B1) plays an important role in the disposition of endogenous substrates and drugs prescribed to children. Alternative splicing increases the diversity of protein products from > 90% of human genes and may be triggered by developmental signals. As concentrations of several endogenous OATP1B1 substrates change during growth and development, with this exploratory study we investigated age-dependent alternative splicing of SLCO1B1 mRNA in 97 postmortem livers (fetus-adolescents). Twenty-seven splice variants were detected; 10 were confirmed by additional bioinformatic analyses and verified by quantitative polymerase chain reaction, and selected for detailed analysis based on relative abundance, association with age, and overlap with an adjacent gene. Two splice variants code for reference OATP1B1 protein, and eight code for truncated proteins. The expression of eight isoforms was associated with age. We conclude that alternative splicing of SLCO1B1 occurs frequently in children; although the functional consequences remain unknown, the data raise the possibility of a regulatory role for alternative splicing in mediating developmental changes in drug disposition.

A New Framework to Implement Model-Informed Dosing in Clinical Guidelines: Piperacillin and Amikacin as Proof of Concept.

Background: Modeling and simulation is increasingly used to study pediatric pharmacokinetics, but clinical implementation of age-appropriate doses lags behind. Therefore, we aimed to develop model-informed doses using published pharmacokinetic data and a decision framework to adjust dosing guidelines based on these doses, using piperacillin and amikacin in critically ill children as proof of concept. Methods: Piperacillin and amikacin pharmacokinetic models in critically ill children were extracted from literature. Concentration-time profiles were simulated for various dosing regimens for a virtual PICU patient dataset, including the current DPF dose and doses proposed in the studied publications. Probability of target attainment (PTA) was compared between the different dosing regimens. Next, updated dosing recommendations for the DPF were proposed, and evaluated using a new framework based on PK study quality and benefit-risk analysis of clinical implementation. Results: Three studies for piperacillin (critically ill children) and one for amikacin (critically ill pediatric burn patients) were included. Simulated concentration-time profiles were performed for a virtual dataset of 307 critically ill pediatric patients, age range 0.1-17.9 y. PTA for unbound piperacillin trough concentrations >16 mg/L was >90% only for continuous infusion regimens of 400 mg/kg/day vs. 9.7% for the current DPF dose (80 mg/kg/6 h, 30 min infusion). Amikacin PTA was >90% with 20 mg/kg/d, higher than the PTA of the DPF dose of 15 mg/kg/d (63.5%). Using our new decision framework, altered DPF doses were proposed for piperacillin (better PTA with loading dose plus continuous infusion), but not for amikacin (studied and target population were not comparable and risk for toxicity with higher dose). Conclusions: We show the feasibility to develop model-informed dosing guidelines for clinical implementation using existing pharmacokinetic data. This approach could complement literature and consensus-based dosing guidelines for off-label drugs in the absence of stronger evidence to support pediatricians in daily practice.

Rectal Omeprazole in Infants With Gastroesophageal Reflux Disease: A Randomized Pilot Trial.

BACKGROUND AND OBJECTIVE: Omeprazole is a proton pump inhibitor that is used in acid suppression therapy in infants. Infants cannot swallow the oral tablets or capsules. Since, infants require a non-standard dose of omeprazole, the granules or tablets are often crushed or suspended in water or sodium bicarbonate, which may destroy the enteric coating. In this study we explore the efficacy and pharmacokinetics of rectally administered omeprazole in infants with gastroesophageal reflux disease (GERD) due to esophageal atresia (EA) or congenital diaphragmatic hernia (CDH) and compare these with orally administered omeprazole. METHODS: Infants (6-12 weeks postnatal and bodyweight > 3 kg) with EA or CDH and GERD were randomized to receive a single dose of 1 mg/kg omeprazole rectally or orally. The primary outcome was the percentage of infants for whom omeprazole was effective according to predefined criteria for 24-h intraesophageal pH. Secondary outcomes were the percentages of time that gastric pH was < 3 or < 4, as well as the pharmacokinetic parameters. RESULTS: Seventeen infants, 4 with EA and 13 with CDH, were included. The proportion of infants for whom omeprazole was effective was 56% (5 of 9 infants) after rectal administration and 50% (4 of 8 infants) after oral administration. The total reflux time in minutes and percentages and the number of reflux episodes of pH < 4 decreased statistically significantly after both rectal and oral omeprazole administration. Rectal and oral administration of omeprazole resulted in similar serum exposure. CONCLUSIONS: A single rectal omeprazole dose (1 mg/kg) results in consistent increases in intraesophageal and gastric pH in infants with EA- or CDH-related GERD, similar to an oral dose. Considering the challenges with existing oral formulations, rectal omeprazole presents as an innovative, promising alternative for infants with pathological GERD. CLINICAL TRIAL REGISTER: ClinicalTrials.gov Identifier: NCT00226044.

Proof of Concept: First Pediatric [14 C]microtracer Study to Create Metabolite Profiles of Midazolam.

Growth and development affect drug-metabolizing enzyme activity thus could alter the metabolic profile of a drug. Traditional studies to create metabolite profiles and study the routes of excretion are unethical in children due to the high radioactive burden. To overcome this challenge, we aimed to show the feasibility of an absorption, distribution, metabolism, and excretion (ADME) study using a [14 C]midazolam microtracer as proof of concept in children. Twelve stable, critically ill children received an oral [14 C]midazolam microtracer (20 ng/kg; 60 Bq/kg) while receiving intravenous therapeutic midazolam. Blood was sampled up to 24 hours after dosing. A time-averaged plasma pool per patient was prepared reflecting the mean area under the curve plasma level, and subsequently one pool for each age group (0-1 month, 1-6 months, 0.5-2 years, and 2-6 years). For each pool [14 C]levels were quantified by accelerator mass spectrometry, and metabolites identified by high resolution mass spectrometry. Urine and feces (n = 4) were collected up to 72 hours. The approach resulted in sufficient sensitivity to quantify individual metabolites in chromatograms. [14 C]1-OH-midazolam-glucuronide was most abundant in all but one age group, followed by unchanged [14 C]midazolam and [14 C]1-OH-midazolam. The small proportion of unspecified metabolites most probably includes [14 C]midazolam-glucuronide and [14 C]4-OH-midazolam. Excretion was mainly in urine; the total recovery in urine and feces was 77-94%. This first pediatric pilot study makes clear that using a [14 C]midazolam microtracer is feasible and safe to generate metabolite profiles and study recovery in children. This approach is promising for first-in-child studies to delineate age-related variation in drug metabolite profiles.

Incorporating Ontogeny in Physiologically Based Pharmacokinetic Modeling to Improve Pediatric Drug Development: What We Know About Developmental Changes in Membrane Transporters.

Developmental changes in the biological processes involved in the disposition of drugs, such as membrane transporter expression and activity, may alter the drug exposure and clearance in pediatric patients. Physiologically based pharmacokinetic (PBPK) models take these age-dependent changes into account and may be used to predict drug exposure in children. As a result, this mechanistic-based tool has increasingly been applied to improve pediatric drug development. Under the Prescription Drug User Fee Act VI, the US Food and Drug Administration has committed to facilitate the advancement of PBPK modeling in the drug application review process. Yet, significant knowledge gaps on developmental biology still exist, which must be addressed to increase the confidence of prediction. Recently, more data on ontogeny of transporters have emerged and supplied a missing piece of the puzzle. This article highlights the recent findings on the ontogeny of transporters specifically in the intestine, liver, and kidney. It also provides a case study that illustrates the utility of incorporating this information in predicting drug exposure in children using a PBPK approach. Collaborative work has greatly improved the understanding of the interplay between developmental physiology and drug disposition. Such efforts will continue to be needed to address the remaining knowledge gaps to enhance the application of PBPK modeling in drug development for children.

A Comprehensive Analysis of Ontogeny of Renal Drug Transporters: mRNA Analyses, Quantitative Proteomics, and Localization.

Human renal membrane transporters play key roles in the disposition of renally cleared drugs and endogenous substrates, but their ontogeny is largely unknown. Using 184 human postmortem frozen renal cortical tissues (preterm newborns to adults) and a subset of 62 tissue samples, we measured the mRNA levels of 11 renal transporters and the transcription factor pregnane X receptor (PXR) with quantitative real-time polymerase chain reaction, and protein abundance of nine transporters using liquid chromatography tandem mass spectrometry selective reaction monitoring, respectively. Expression levels of p-glycoprotein, urate transporter 1, organic anion transporter 1, organic anion transporter 3, and organic cation transporter 2 increased with age. Protein levels of multidrug and toxin extrusion transporter 2-K and breast cancer resistance protein showed no difference from newborns to adults, despite age-related changes in mRNA expression. Multidrug and toxin extrusion transporter 1, glucose transporter 2, multidrug resistance-associated protein 2, multidrug resistance-associated protein 4 (MRP4), and PXR expression levels were stable. Using immunohistochemistry, we found that MRP4 localization in pediatric samples was similar to that in adult samples. Collectively, our study revealed that renal drug transporters exhibited different rates and patterns of maturation, suggesting that renal handling of substrates may change with age.

Proteomics of human liver membrane transporters: a focus on fetuses and newborn infants.

BACKGROUND: Hepatic membrane transporters are involved in the transport of many endogenous and exogenous compounds, including drugs. We aimed to study the relation of age with absolute transporter protein expression in a cohort of 62 mainly fetus and newborn samples. METHODS: Protein expressions of BCRP, BSEP, GLUT1, MCT1, MDR1, MRP1, MRP2, MRP3, NTCP, OCT1, OATP1B1, OATP1B3, OATP2B1 and ATP1A1 were quantified with LC-MS/MS in isolated crude membrane fractions of snap-frozen post-mortem fetal and pediatric, and surgical adult liver samples. mRNA expression was quantified using RNA sequencing, and genetic variants with TaqMan assays. We explored relationships between protein expression and age (gestational age [GA], postnatal age [PNA], and postmenstrual age); between protein and mRNA expression; and between protein expression and genotype. RESULTS: We analyzed 36 fetal (median GA 23.4 weeks [range 15.3-41.3]), 12 premature newborn (GA 30.2 weeks [24.9-36.7], PNA 1.0 weeks [0.14-11.4]), 10 term newborn (GA 40.0 weeks [39.7-41.3], PNA 3.9 weeks [0.3-18.1]), 4 pediatric (PNA 4.1 years [1.1-7.4]) and 8 adult liver samples. A relationship with age was found for BCRP, BSEP, GLUT1, MDR1, MRP1, MRP2, MRP3, NTCP, OATP1B1 and OCT1, with the strongest relationship for postmenstrual age. For most transporters mRNA and protein expression were not correlated. No genotype-protein expression relationship was detected. DISCUSSION AND CONCLUSION: Various developmental patterns of protein expression of hepatic transporters emerged in fetuses and newborns up to four months of age. Postmenstrual age was the most robust factor predicting transporter expression in this cohort. Our data fill an important gap in current pediatric transporter ontogeny knowledge.