Microdialysis of Drug and Drug Metabolite: a Comprehensive In Vitro Analysis for Voriconazole and Voriconazole N-oxide

Purpose Voriconazole is a therapeutically challenging antifungal drug associated with high interindividual pharmacokinetic variability. As a prerequisite to performing clinical trials using the minimally-invasive sampling technique microdialysis, a comprehensive in vitro microdialysis characterization of voriconazole (VRC) and its potentially toxic N-oxide metabolite (NO) was performed. Methods The feasibility of simultaneous microdialysis of VRC and NO was explored in vitro by investigating the relative recovery (RR) of both compounds in the absence and presence of the other. The dependency of RR on compound combination, concentration, microdialysis catheter and study day was evaluated and quantified by linear mixed-effects modeling. Results Median RR of VRC and NO during individual microdialysis were high (87.6% and 91.1%). During simultaneous microdialysis of VRC and NO, median RR did not change (87.9% and 91.1%). The linear mixed-effects model confirmed the absence of significant differences between RR of VRC and NO during individual and simultaneous microdialysis as well as between the two compounds (p > 0.05). No concentration dependency of RR was found (p = 0.284). The study day was the main source of variability (46.3%) while the microdialysis catheter only had a minor effect (4.33%). VRC retrodialysis proved feasible as catheter calibration for both compounds. Conclusion These in vitro microdialysis results encourage the application of microdialysis in clinical trials to assess target-site concentrations of VRC and NO. This can support the generation of a coherent understanding of VRC pharmacokinetics and its sources of variability. Ultimately, a better understanding of human VRC pharmacokinetics might contribute to the development of personalized dosing strategies

Microdialysis of Voriconazole and its N-Oxide Metabolite: Amalgamating Knowledge of Distribution and Metabolism Processes in Humans

Purpose Voriconazole is an essential antifungal drug whose complex pharmacokinetics with high interindividual variability impedes effective and safe therapy. By application of the minimally-invasive sampling technique microdialysis, interstitial space fluid (ISF) concentrations of VRC and its potentially toxic N-oxide metabolite (NO) were assessed to evaluate target-site exposure for further elucidating VRC pharmacokinetics. Methods Plasma and ISF samples of a clinical trial with an approved VRC dosing regimen were analyzed for VRC and NO concentrations. Concentration-time profiles, exposure assessed as area-under-the-curve (AUC) and metabolic ratios of four healthy adults in plasma and ISF were evaluated regarding the impact of multiple dosing and CYP2C19 genotype. Results VRC and NO revealed distribution into ISF with AUC values being ≤2.82- and 17.7-fold lower compared to plasma, respectively. Intraindividual variability of metabolic ratios was largest after the first VRC dose administration while interindividual variability increased with multiple dosing. The CYP2C19 genotype influenced interindividual differences with a maximum 6- and 24-fold larger AUCNO/AUCVRC ratio between the intermediate and rapid metabolizer in plasma and ISF, respectively. VRC metabolism was saturated/auto-inhibited indicated by substantially decreasing metabolic concentration ratios with increasing VRC concentrations and after multiple dosing. Conclusion The feasibility of the simultaneous microdialysis of VRC and NO in vivo was demonstrated and provided new quantitative insights by leveraging distribution and metabolism processes of VRC in humans. The exploratory analysis suggested substantial dissimilarities of VRC and NO pharmacokinetics in plasma and ISF. Ultimately, a thorough understanding of target-site pharmacokinetics might contribute to the optimization of personalized VRC dosing regimens

Towards the Elucidation of the Pharmacokinetics of Voriconazole: A Quantitative Characterization of Its Metabolism

The small-molecule drug voriconazole (VRC) shows a complex and not yet fully understood metabolism. Consequently, its in vivo pharmacokinetics are challenging to predict, leading to therapy failures or adverse events. Thus, a quantitative in vitro characterization of the metabolism and inhibition properties of VRC for human CYP enzymes was aimed for. The Michaelis–Menten kinetics of voriconazole N-oxide (NO) formation, the major circulating metabolite, by CYP2C19, CYP2C9 and CYP3A4, was determined in incubations of human recombinant CYP enzymes and liver and intestine microsomes. The contribution of the individual enzymes to NO formation was 63.1% CYP2C19, 13.4% CYP2C9 and 29.5% CYP3A4 as determined by specific CYP inhibition in microsomes and intersystem extrapolation factors. The type of inhibition and inhibitory potential of VRC, NO and hydroxyvoriconazole (OH–VRC), emerging to be formed independently of CYP enzymes, were evaluated by their effects on CYP marker reactions. Time-independent inhibition by VRC, NO and OH–VRC was observed on all three enzymes with NO being the weakest and VRC and OH–VRC being comparably strong inhibitors of CYP2C9 and CYP3A4. CYP2C19 was significantly inhibited by VRC only. Overall, the quantitative in vitro evaluations of the metabolism contributed to the elucidation of the pharmacokinetics of VRC and provided a basis for physiologically-based pharmacokinetic modeling and thus VRC treatment optimization

Towards the elucidation of voriconazole pharmacokinetics: quantitative insights into distribution and metabolism processes in humans

Invasive fungal infections are an increasing threat to the global public health causing approximately 1.5 million deaths worldwide every year. As the emergence and spread of resistance to antimycotics is expanding and the development of new antifungal agents is lagging behind this epidemiological burden, one important aspect is the rational use of existing drugs, such as voriconazole (VRC). Despite its long-term and frequent application in humans, VRC pharmacokinetics (PK) are still not fully understood revealing large intra- and interindividual variability as well as therapy failures and adverse events. The main source of variability is assumed to derive from the extensive metabolism of VRC involving the cytochrome P450 (CYP) isoenzymes 2C19, 2C9 and 3A4. Furthermore, the target site of VRC, i.e. the interstitial space fluid (ISF), is more relevant for PK investigations in contrast to the usually determined plasma concentrations. A powerful tool for ISF investigations is the minimally-invasive sampling technique microdialysis, which allows the continuous sampling of protein-unbound drug over time. Therefore, the present thesis aimed at contributing to the elucidation of VRC PK by generating mechanistic and quantitative insights into its distribution and metabolism processes in humans to ultimately support the optimisation of VRC dosing strategies. To achieve this objective, research in three main areas was performed. First, a versatile bioanalytical liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed and validated for the quantification of VRC and its metabolites in various biological matrices. Second, in vitro metabolism investigations were performed for a comprehensive characterisation of VRC and its metabolites as substrates and inhibitors of CYP2C19, CYP2C9 and CYP3A4. Third, in vitro and in vivo microdialysis investigations were performed evaluating the application of simultaneous microdialysis of VRC and its potentially toxic metabolite, voriconazole N-oxide (NO), in humans. The developed bioanalytical LC-MS/MS assay enabled the simultaneous quantification of VRC, NO and hydroxyvoriconazole (OH-VRC) in human plasma, ultrafiltrate and microdialysate as well as in the in vitro matrices of human liver/intestine microsomes (HLM/HIM) and recombinant human CYP (rhCYP) isoenzymes. In a first step, the assay was fully validated according to the European Medicines Agency guideline on bioanalytical method validation for the quantification of VRC and NO in plasma and microdialysate. Overall, the quantification was rapid, simple and feasible for clinically relevant concentrations of VRC and NO from 5 to 5000 ng/mL in plasma and ultrafiltrate as well as from 4 to 4000 ng/mL in microdialysate. Due to the high sensitivity of the assay, only 20 µL of plasma or ultrafiltrate and 5 µL of microdialysate were required. For VRC and NO in plasma and microdialysate, between-run accuracy was high with a maximum mean deviation of 7.0% from the nominal concentration and between-run precision was demonstrated by ≤11.8% coefficient of variation. Stability under various conditions was demonstrated for both compounds. In a second step, the assay was successfully adapted for PK analyses in in vitro experiments covering a concentration range of 0.1 to 500 ng/mL for NO and of 1.0 to 500 ng/mL for OH-VRC at a sample volume of 20 µL. Overall, by reducing the required sample volume, the bioanalytical method allowed for an increased number of plasma samples in vulnerable populations and enabled the generation of concentration-time profiles with a higher temporal resolution in microdialysis studies. In vitro metabolism investigations revealed NO formation to follow Michaelis-Menten kinetics and was mediated by the CYP isoenzymes 2C19, 2C9 and 3A4 as determined by incubation of VRC with HLM, HIM and rhCYP. The kinetic parameters of the reaction, i.e. Michaelis-Menten constant (KM), maximum reaction velocity (Vmax) and intrinsic clearance (CLint), were derived and an in vitro in vivo extrapolation using the well-stirred liver model demonstrated their validity by comparison to in vivo data. In contrast, no OH-VRC formation was detected in any of the metabolic systems, suggesting a different metabolic pathway of formation. The contribution of the individual isoenzymes to NO formation was 63.1% for CYP2C19, 13.2% for CYP2C9 and 29.5% for CYP3A4 as determined by specific CYP inhibition in HLM and application of intersystem extrapolation factors to scale the metabolism in rhCYP. The type of inhibition and inhibitory potential of VRC, NO and OH-VRC were evaluated in HLM by their effects on CYP specific marker reactions for CYP2C19, CYP2C9 and CYP3A4. Regarding the half maximum inhibitory concentration (IC50), NO was the weakest and VRC and OH VRC comparably strong inhibitors of CYP2C9 and CYP3A4. CYP2C19 was significantly inhibited by VRC only. Time-independent inhibition by VRC, NO and OH-VRC was demonstrated by the absence of an IC50 shift when a pre-incubation step of inhibitor and enzyme in the absence or presence of NADPH re-generating system was performed. Lastly, the assessment of the inhibitory constant (Ki) confirmed the observations of inhibitory potential from IC50 investigations, as well as revealed competitive inhibition of CYP2C19 by VRC and OH VRC and non-competitive inhibition by NO. Inhibition of CYP2C9 was competitive while inhibition of CYP3A4 was non-competitive for VRC, NO and OH-VRC. As a prerequisite for the assessment of target-site exposure of VRC and NO, the feasibility of simultaneous microdialysis of VRC and NO was first explored in vitro by investigating the relative recovery (RR) of both compounds in the absence and presence of the other. Dependencies of RR on compound combination, compound concentration, microdialysis catheter and study day were evaluated and quantified by a linear mixed-effects model. Median RR of VRC and NO during individual microdialysis were high with 87.6% and 91.1%, respectively. During simultaneous microdialysis of VRC and NO, median RR did not change relevantly being 87.9% and 91.1%, respectively. The linear mixed-effects model confirmed the graphically presumed absence of significant differences between RR of VRC and NO during individual and simultaneous microdialysis as well as between the two compounds (p>0.05). No impact of the investigated clinically relevant compound concentration on RR was found (p=0.284). The study day was the main source of variability (46.3%), while the microdialysis catheter had a minor impact (4.33%). VRC retrodialysis was a feasible catheter calibration method to derive ISF concentrations of VRC and NO simultaneously. Hereinafter, to assess the in vivo feasibility and clinical applicability of the simultaneous microdialysis of VRC and NO, plasma, ultrafiltrate and ISF samples, obtained in a clinical microdialysis trial investigating VRC PK after administration of an approved VRC dosing regimen, were analysed. Concentration-time profiles, exposure assessed as area under the concentration-time curve (AUC) and metabolic ratios (concentrationNO/concentrationVRC) of four healthy male adults in plasma, ultrafiltrate and ISF were evaluated regarding the impact of multiple dosing and the CYP2C19 genotype-predicted phenotype. VRC and NO revealed distribution into ISF with AUC being up to 2.82- and 17.7-fold lower compared to plasma. Intraindividual variability of metabolic ratios was largest after the first VRC dose administration while interindividual differences increased with multiple dosing. The CYP2C19 genotype-predicted phenotype influenced interindividual differences with a maximum 6- and 24 fold larger AUC ratio (AUCNO/AUCVRC) between the intermediate and rapid metaboliser in plasma and ISF, respectively. VRC metabolism was saturated or auto-inhibited, indicated by decreasing metabolic ratios with increasing VRC concentrations. Overall, the present thesis advanced the elucidation of VRC PK by generating mechanistic and quantitative insights into distribution and metabolism processes in humans. The application of the newly established bioanalytical LC-MS/MS assay enabled the thorough characterisation of VRC and its metabolites as substrates and inhibitors of the CYP isoenzymes 2C19, 2C9 and 3A4. Moreover, in vitro microdialysis feasibility investigations provided the basis for the evaluation of simultaneous microdialysis of VRC and NO in vivo. Thus, the exploratory PK analysis highlighted the potential sources of intra- and interindividual differences observed in the context of VRC treatment in humans. Ultimately, a thorough understanding of VRC target-site PK and metabolism is key to the optimisation of personalised VRC dosing regimens.Invasive Pilzinfektionen stellen eine zunehmende Bedrohung für die globale öffentliche Gesundheit dar und verursachen jedes Jahr weltweit etwa 1.5 Millionen Todesfälle. Da die Entstehung und Ausbreitung von Resistenzen gegen Antimykotika zunimmt und die Entwicklung neuer antimykotischer Wirkstoffe hinter dieser epidemiologischen Belastung zurückbleibt, ist ein wichtiger Aspekt der rationale Einsatz vorhandener Arzneimittel wie z. B. Voriconazol (VRC). Trotz der langjährigen und häufigen Anwendung am Menschen ist die Pharmakokinetik (PK) von VRC noch immer nicht vollständig verstanden, was sich in einer großen intra- und interindividuellen Variabilität sowie in Therapieversagen und unerwünschten Arzneimittelwirkungen zeigt. Es wird angenommen, dass die Hauptursache für die Variabilität im komplexen Metabolismus von VRC liegt, an dem die Cytochrom P450 (CYP)-Isoenzyme 2C19, 2C9 und 3A4 beteiligt sind. Darüber hinaus ist der Zielort von VRC, d. h. die interstitielle Flüssigkeit (ISF), für PK-Untersuchungen relevanter als die üblicherweise bestimmten Plasmakonzentrationen. Eine leistungsfähige Methode für ISF-Untersuchungen ist die minimal-invasive Probenahmetechnik der Mikrodialyse, die eine kontinuierliche Probenahme von nicht proteingebundenen Arzneistoffmolekülen über einen bestimmten Zeitraum ermöglicht. Ziel der vorliegenden Arbeit war daher, einen Beitrag zur Aufklärung der VRC PK zu leisten, indem mechanistische und quantitative Einblicke in die Verteilung und die Stoffwechselprozesse beim Menschen gewonnen werden, um letztendlich die Optimierung der Dosierungsstrategien für VRC zu unterstützen. Um dieses Ziel zu erreichen, wurden Untersuchungen in drei Hauptforschungsgebieten durchgeführt. Erstens wurde ein vielseitiger bioanalytischer Flüssigchromatographie-Tandem-Massenspektrometrie-Assay (LC-MS/MS) für die Quantifizierung von VRC und seinen Metaboliten in verschiedenen biologischen Matrices entwickelt und validiert. Zweitens wurden In-vitro-Metabolismusuntersuchungen zur umfassenden Charakterisierung von VRC und seinen Metaboliten als Substrate und Inhibitoren von CYP2C19, CYP2C9 und CYP3A4 durchgeführt. Drittens wurden In-vitro- und In-vivo-Mikrodialyse-Untersuchungen durchgeführt, um die Anwendung der gleichzeitige Mikrodialyse von VRC und dem potenziell toxischen Metaboliten, Voriconazol-N-Oxid (NO), beim Menschen zu bewerten. Der entwickelte bioanalytische LC-MS/MS-Assay ermöglichte die gleichzeitige Quantifizierung von VRC, NO und Hydroxyvoriconazol (OH-VRC) in menschlichem Plasma, Ultrafiltrat und Mikrodialysat sowie in den In-vitro-Matrices menschlicher Leber-/Darm-Mikrosomen (HLM/HIM) und rekombinanter menschlicher CYP-Isoenzyme (rhCYP). In einem ersten Schritt wurde der Assay gemäß der Leitlinie zur „Validierung bioanalytischer Methoden für die Quantifizierung“ der Europäischen Arzneimittelagentur für VRC und NO in Plasma und Mikrodialysat vollständig validiert. Insgesamt war die Quantifizierung schnell, unkompliziert und verwendbar für klinisch relevante Konzentrationen von VRC und NO von 5 bis 5000 ng/mL in Plasma und Ultrafiltrat sowie von 4 bis 4000 ng/mL in Mikrodialysat. Aufgrund der hohen Sensitivität des Assays wurden nur 20 µL Plasma oder Ultrafiltrat und 5 µL Mikrodialysat benötigt. Für VRC und NO in Plasma und Mikrodialysat war die Richtigkeit zwischen den Messsequenzen mit einer maximalen mittleren Abweichung von 7.0 % von der Nennkonzentration hoch und die Präzision zwischen den Messsequenzen wurde mit einem Variationskoeffizienten von ≤11.8 % bestätigt. Beide Substanzen erwiesen sich unter verschiedenen Bedingungen als stabil. In einem zweiten Schritt wurde der Assay erfolgreich für PK-Analysen in In-vitro-Experimenten angepasst, die einen Konzentrationsbereich von 0.1 bis 500 ng/mL für NO und von 1.0 bis 500 ng/mL für OH-VRC bei einem Probenvolumen von nur 20 µL abdeckten. Insgesamt ermöglichte die bioanalytische Methode durch die Verringerung des erforderlichen Probenvolumens eine größere Anzahl von Plasmaproben in vulnerablen Bevölkerungsgruppen und die Erstellung von Konzentrations-Zeitprofilen mit einer höheren zeitlichen Auflösung in Mikrodialysestudien. In-vitro-Metabolismusuntersuchungen ergaben, dass die NO-Bildung einer Michaelis-Menten-Kinetik folgt und durch die CYP-Isoenzyme 2C19, 2C9 und 3A4 vermittelt wird, wie durch Inkubation von VRC mit HLM, HIM und rhCYP festgestellt wurde. Die kinetischen Parameter der Reaktion, d. h. die Michaelis-Menten-Konstante (KM), die maximale Reaktionsgeschwindigkeit (Vmax) und die intrinsische Clearance (CLint), wurden bestimmt und deren Gültigkeit durch eine In-vitro-in-vivo-Extrapolation unter Verwendung des „well-stirred“-Lebermodells und den Vergleich mit In-vivo-Daten gezeigt. Im Gegensatz dazu wurde in keinem der Enzymsysteme eine Bildung von OH-VRC festgestellt, was auf einen anderen Stoffwechselweg der Bildung hindeutet. Der Beitrag der einzelnen Isoenzyme zur NO-Bildung betrug 63.1 % für CYP2C19, 13.2 % für CYP2C9 und 29.5 % für CYP3A4, wie durch spezifische CYP-Hemmung in HLM und Anwendung von Intersystem-Extrapolationsfaktoren zur Skalierung des Metabolismus in rhCYP ermittelt wurde. Die Art der Inhibition und das hemmende Potenzial von VRC, NO und OH-VRC wurden in HLM durch ihre Auswirkungen auf CYP-spezifische Markerreaktionen für CYP2C19, CYP2C9 und CYP3A4 bewertet. Hinsichtlich der halbmaximalen Hemmkonzentration (IC50) waren NO der schwächste und VRC und OH VRC vergleichsweise starke Inhibitoren von CYP2C9 und CYP3A4. CYP2C19 wurde nur durch VRC signifikant gehemmt. Die zeitunabhängige Inhibition durch VRC, NO und OH-VRC wurde durch das Fehlen einer IC50-Verschiebung nachgewiesen, wenn ein Vorinkubationsschritt von Inhibitor und Enzym in Abwesenheit oder Anwesenheit eines NADPH-regenerierenden Systems durchgeführt wurde. Schließlich bestätigte die Bewertung der Inhibitionskonstanten (Ki) die Beobachtungen des inhibitorischen Potenzials aus den IC50-Untersuchungen und zeigte eine kompetitive Hemmung von CYP2C19 durch VRC und OH-VRC sowie eine nicht-kompetitive Hemmung durch NO. Die Hemmung von CYP2C9 war kompetitiv, während die Hemmung von CYP3A4 nicht-kompetitiv für VRC, NO und OH-VRC war. Als Voraussetzung für die Bestimmung der VRC und NO Exposition am Zielort, wurde zunächst die Durchführbarkeit der gleichzeitigen Mikrodialyse von VRC und NO in vitro untersucht, indem die relative Wiederfindung (RR) beider Substanzen in Abwesenheit und Anwesenheit der jeweils anderen bestimmt wurde. Die Abhängigkeiten der RR von der Substanzkombination, der Konzentration der Substanz, dem Mikrodialysekatheter und dem Studientag wurden mit einem linearen gemischten Modell bewertet und quantifiziert. Die mediane RR von VRC und NO während der individuellen Mikrodialyse war mit 87.6 % bzw. 91.1 % hoch. Bei gleichzeitiger Mikrodialyse von VRC und NO änderte sich die mediane RR mit 87.9 % bzw. 91.1 % nicht wesentlich. Das lineare gemischte Modell bestätigte die grafisch vermutete Abwesenheit signifikanter Unterschiede zwischen der RR von VRC und NO bei individueller und gleichzeitiger Mikrodialyse sowie zwischen den beiden Substanzen (p>0.05). Es wurde kein Einfluss der untersuchten klinisch relevanten Konzentrationen der Substanzen auf die RR festgestellt (p=0.284). Der Studientag war die Hauptursache der Variabilität (46.3 %), während der Mikrodialysekatheter einen geringen Einfluss hatte (4.33 %). Die Retrodialyse mit VRC war als Katheterkalibrierungsmethode zur gleichzeitigen Bestimmung der ISF-Konzentrationen von VRC und NO geeignet. Um die In-vivo-Durchführbarkeit und klinische Anwendbarkeit der gleichzeitigen Mikrodialyse von VRC und NO zu bewerten, wurden Plasma-, Ultrafiltrat- und ISF-Proben analysiert, die in einer klinischen Mikrodialysestudie zur Untersuchung der PK von VRC nach Verabreichung eines zugelassenen VRC-Dosierungsschemas gewonnen wurden. Die Konzentrations-Zeitprofile, die als Fläche unter der Konzentrations-Zeitkurve (AUC) bewertete Exposition und die metabolischen Quotienten (KonzentrationNO/KonzentrationVRC) von vier gesunden männlichen Erwachsenen in Plasma, Ultrafiltrat und ISF wurden im Hinblick auf die Auswirkung der Mehrfachdosierung und des vom CYP2C19-Genotyp vorhergesagten Phänotyps bewertet. VRC und NO zeigten eine Verteilung in die ISF, wobei die AUC im Vergleich zum Plasma bis zu 2.82- bzw. 17.7-mal niedriger war. Die intraindividuelle Variabilität der metabolischen Verhältnisse war nach der ersten Verabreichung von VRC am größten, während die interindividuellen Unterschiede bei mehrfacher Verabreichung zunahmen. Der vom CYP2C19-Genotyp vorhergesagte Phänotyp beeinflusste die interindividuellen Unterschiede mit einem maximal 6- bzw. 24-fach größeren AUC-Verhältnis (AUCNO/AUCVRC) zwischen dem intermediären und dem schnellen Metabolisierer im Plasma bzw. in der ISF. Der VRC-Metabolismus war gesättigt oder auto-inhibiert, was durch abnehmende metabolische Verhältnisse bei steigenden VRC-Konzentrationen ersichtlich wurde. Insgesamt hat die vorliegende Arbeit einen Beitrag zur Aufklärung der PK von VRC geleistet, indem sie mechanistische und quantitative Erkenntnisse über die Verteilungs- und Stoffwechselprozesse beim Menschen lieferte. Die Anwendung des neu etablierten bioanalytischen LC-MS/MS-Assays ermöglichte die umfangreiche Charakterisierung von VRC und der Metabolite als Substrate und Inhibitoren der CYP-Isoenzyme 2C19, 2C9 und 3A4. Darüber hinaus formten die In-vitro-Mikrodialyse-Machbarkeitsuntersuchungen die Grundlage für die Bewertung der gleichzeitigen Mikrodialyse von VRC und NO in vivo. Die explorative PK-Analyse hat somit mögliche Ursachen der intra- und interindividuellen Unterschiede aufgezeigt, die im Zusammenhang mit der VRC-Behandlung beim Menschen beobachtet wurden. Letztendlich ist ein umfassendes Verständnis der Zielort-PK und des Metabolismus von VRC der Schlüssel zur Optimierung personalisierter VRC-Dosierungsschemata

Towards the Elucidation of the Pharmacokinetics of Voriconazole: A Quantitative Characterization of Its Metabolism

The small-molecule drug voriconazole (VRC) shows a complex and not yet fully understood metabolism. Consequently, its in vivo pharmacokinetics are challenging to predict, leading to therapy failures or adverse events. Thus, a quantitative in vitro characterization of the metabolism and inhibition properties of VRC for human CYP enzymes was aimed for. The Michaelis–Menten kinetics of voriconazole N-oxide (NO) formation, the major circulating metabolite, by CYP2C19, CYP2C9 and CYP3A4, was determined in incubations of human recombinant CYP enzymes and liver and intestine microsomes. The contribution of the individual enzymes to NO formation was 63.1% CYP2C19, 13.4% CYP2C9 and 29.5% CYP3A4 as determined by specific CYP inhibition in microsomes and intersystem extrapolation factors. The type of inhibition and inhibitory potential of VRC, NO and hydroxyvoriconazole (OH–VRC), emerging to be formed independently of CYP enzymes, were evaluated by their effects on CYP marker reactions. Time-independent inhibition by VRC, NO and OH–VRC was observed on all three enzymes with NO being the weakest and VRC and OH–VRC being comparably strong inhibitors of CYP2C9 and CYP3A4. CYP2C19 was significantly inhibited by VRC only. Overall, the quantitative in vitro evaluations of the metabolism contributed to the elucidation of the pharmacokinetics of VRC and provided a basis for physiologically-based pharmacokinetic modeling and thus VRC treatment optimization

Olfactory transmucosal SARS-CoV-2 invasion as a port of central nervous system entry in individuals with COVID-19

The newly identified severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19, a pandemic respiratory disease. Moreover, thromboembolic events throughout the body, including in the CNS, have been described. Given the neurological symptoms observed in a large majority of individuals with COVID-19, SARS-CoV-2 penetrance of the CNS is likely. By various means, we demonstrate the presence of SARS-CoV-2 RNA and protein in anatomically distinct regions of the nasopharynx and brain. Furthermore, we describe the morphological changes associated with infection such as thromboembolic ischemic infarction of the CNS and present evidence of SARS-CoV-2 neurotropism. SARS-CoV-2 can enter the nervous system by crossing the neural–mucosal interface in olfactory mucosa, exploiting the close vicinity of olfactory mucosal, endothelial and nervous tissue, including delicate olfactory and sensory nerve endings. Subsequently, SARS-CoV-2 appears to follow neuroanatomical structures, penetrating defined neuroanatomical areas including the primary respiratory and cardiovascular control center in the medulla oblongata.Peer Reviewe

Differences in Cell-Intrinsic Inflammatory Programs of Yolk Sac and Bone Marrow Macrophages

Background: Tissue-resident macrophages have mixed developmental origins. They derive in variable extent from yolk sac (YS) hematopoiesis during embryonic development. Bone marrow (BM) hematopoietic progenitors give rise to tissue macrophages in postnatal life, and their contribution increases upon organ injury. Since the phenotype and functions of macrophages are modulated by the tissue of residence, the impact of their origin and developmental paths has remained incompletely understood. Methods: In order to decipher cell-intrinsic macrophage programs, we immortalized hematopoietic progenitors from YS and BM using conditional HoxB8, and carried out an in-depth functional and molecular analysis of differentiated macrophages. Results: While YS and BM macrophages demonstrate close similarities in terms of cellular growth, differentiation, cell death susceptibility and phagocytic properties, they display differences in cell metabolism, expression of inflammatory markers and inflammasome activation. Reduced abundance of PYCARD (ASC) and CASPASE-1 proteins in YS macrophages abrogated interleukin-1β production in response to canonical and non-canonical inflammasome activation. Conclusions: Macrophage ontogeny is associated with distinct cellular programs and immune response. Our findings contribute to the understanding of the regulation and programming of macrophage functions