Epithelial senescence in idiopathic pulmonary fibrosis is propagated by small extracellular vesicles.

Background: Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease that affects 3 million people worldwide. Senescence and small extracellular vesicles (sEVs) have been implicated in the pathogenesis of IPF, although how sEVs promote disease remains unclear. Here, we profile sEVs from bronchial epithelial cells and determine small RNA (smRNA) content.

Proof of lung muscarinic receptor occupancy by tiotropium: Translational Positron Emission Tomography studies in non-human primates and humans.

Introduction: Molecular imaging has not been used to support the development of drugs for the treatment of pulmonary disorders. The aim of the present translational study was to advance quantitative pulmonary PET imaging by demonstrating occupancy of the reference asthma drug tiotropium at muscarinic acetylcholine receptors (mAChR).

Methods: PET imaging was performed using the muscarinic radioligand [C]VC-002.

Quantification and reliability of [C]VC - 002 binding to muscarinic acetylcholine receptors in the human lung - a test-retest PET study in control subjects.

Background: The radioligand [C]VC-002 was introduced in a small initial study long ago for imaging of muscarinic acetylcholine receptors (mAChRs) in human lungs using positron emission tomography (PET). The objectives of the present study in control subjects were to advance the methodology for quantification of [C]VC-002 binding in lung and to examine the reliability using a test-retest paradigm. This work constituted a self-standing preparatory step in a larger clinical trial aiming at estimating mAChR occupancy in the human lungs following inhalation of mAChR antagonists.

Improving the Accuracy of Predicted Human Pharmacokinetics: Lessons Learned from the AstraZeneca Drug Pipeline Over Two Decades.

During drug discovery and prior to the first human dose of a novel candidate drug, the pharmacokinetic (PK) behavior of the drug in humans is predicted from preclinical data. This helps to inform the likelihood of achieving therapeutic exposures in early clinical development. Once clinical data are available, the observed human PK are compared with predictions, providing an opportunity to assess and refine prediction methods.

Pulmonary Metabolism of Substrates for Key Drug-Metabolizing Enzymes by Human Alveolar Type II Cells, Human and Rat Lung Microsomes, and the Isolated Perfused Rat Lung Model.

Significant pulmonary metabolism of inhaled drugs could have drug safety implications or influence pharmacological effectiveness. To study this in vitro, lung microsomes or S9 are often employed. Here, we have determined if rat and human lung microsomes are fit for purpose or whether it is better to use specific cells where drug-metabolizing enzymes are concentrated, such as alveolar type II (ATII) cells.

Revealing the Regional Localization and Differential Lung Retention of Inhaled Compounds by Mass Spectrometry Imaging.

For the treatment of respiratory disease, inhaled drug delivery aims to provide direct access to pharmacological target sites while minimizing systemic exposure. Despite this long-held tenet of inhaled therapeutic advantage, there are limited data of regional drug localization in the lungs after inhalation. The aim of this study was to investigate the distribution and retention of different chemotypes typifying available inhaled drugs [slowly dissolving neutral fluticasone propionate (FP) and soluble bases salmeterol and salbutamol] using mass spectrometry imaging (MSI).

Plasma Protein Binding as an Optimizable Parameter for Acidic Drugs.

The low volume of distribution associated with acidic molecules means that clearance (CL) must also be very low to achieve an effective half-life commensurate with once or twice daily dosing. Plasma protein binding (PPB) should not usually be considered a parameter for optimization, but in the particular case of acidic molecules, raising the PPB above a certain level can result in distribution volume becoming a constant low value equal to the distribution volume of albumin while acting to reduce CL through restricting hepatic and renal access of unbound drug. Thus effective half-life can be increased.

Expression of cytochrome P450 mRNAs in Type II alveolar cells from subjects with chronic obstructive pulmonary disease.

Inhaled drugs are critical for the treatment of inflammatory airway diseases such as chronic obstructive pulmonary disease (COPD). To develop better therapeutics for pulmonary disease it is of potential importance to understand molecular mechanisms of local biotransformation in the lung. Alveolar epithelial type II (ATII) cells have a key role in homeostasis in the lung, but little is known about expression patterns of genes encoding cytochrome P450 (CYP) enzymes in ATII cells.

Translational model to predict pulmonary pharmacokinetics and efficacy in man for inhaled bronchodilators.

Translational pharmacokinetic (PK) models are needed to describe and predict drug concentration-time profiles in lung tissue at the site of action to enable animal-to-man translation and prediction of efficacy in humans for inhaled medicines. Current pulmonary PK models are generally descriptive rather than predictive, drug/compound specific, and fail to show successful cross-species translation. The objective of this work was to develop a robust compartmental modeling approach that captures key features of lung and systemic PK after pulmonary administration of a set of 12 soluble drugs containing single basic, dibasic, or cationic functional groups.

Benchmarking of Human Dose Prediction for Inhaled Medicines from Preclinical In Vivo Data.

Purpose: A scientifically robust prediction of human dose is important in determining whether to progress a candidate drug into clinical development. A particular challenge for inhaled medicines is that unbound drug concentrations at the pharmacological target site cannot be easily measured or predicted. In the absence of such data, alternative empirical methods can be useful.

Intrinsic Clearance Assay Incubational Binding: A Method Comparison.

The fraction of unbound drug (fu) in in vitro intrinsic clearance (CL) incubation is an important parameter in the pursuit of accurate clearance predictions and is often predicted using algorithms based on drug lipophilicity measures. However, analysis of an AstraZeneca database suggests that simple lipophilicity alone is a relatively poor predictor of fu measured using equilibrium dialysis. He fu value can also be measured directly in CL assays using multiple concentrations of hepatocytes or microsomal protein.

An S-warfarin and AZD1981 interaction: in vitro and clinical pilot data suggest the N-deacetylated amino acid metabolite as the primary perpetrator.

Aim: AZD1981 is an orally bioavailable chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTh2) receptor antagonist progressed to phase II trials for the treatment of allergic asthma. Previously performed in vitro human hepatocyte incubations identified N-deacetylated AZD1981 as a primary metabolite. We report on metabolite exposure from a clinical excretion balance, on in vitro studies performed to determine the likelihood of a metabolite-dependent drug-drug interaction (DDI) and on a clinical warfarin DDI study.

Determination of Human Hepatocyte Intrinsic Clearance for Slowly Metabolized Compounds: Comparison of a Primary Hepatocyte/Stromal Cell Co-culture with Plated Primary Hepatocytes and HepaRG.

A key requirement in drug discovery is to accurately define intrinsic clearance (CL(int)) values of less than 1 µl/min/10(6) hepatocytes, which requires assays that allow for longer incubation time as a complement to suspended hepatocytes. This study assessed the effectiveness of plated HepaRG cells, plated primary human hepatocytes (PHHs), and the HµREL human hepatocyte/stromal cell co-cultures for determination of low CL(int) values. The investigation demonstrated that the systems were capable of providing statistically significant CL(int) estimations down to 0.

Montelukast Disposition: No Indication of Transporter-Mediated Uptake in OATP2B1 and OATP1B1 Expressing HEK293 Cells.

Clinical studies with montelukast show variability in effect and polymorphic OATP2B1-dependent absorption has previously been implicated as a possible cause. This claim has been challenged with conflicting data and here we used OATP2B1-transfected HEK293 cells to clarify the mechanisms involved. For montelukast, no significant difference in cell uptake between HEK-OATP2B1 and empty vector cell lines was observed at pH 6.

Discovery and evaluation of a novel monocyclic series of CXCR2 antagonists.

Antagonism of the chemokine receptor CXCR2 has been proposed as a strategy for the treatment of inflammatory diseases such as arthritis, chronic obstructive pulmonary disease and asthma. Earlier series of bicyclic CXCR2 antagonists discovered at AstraZeneca were shown to have low solubility and poor oral bioavailability. In this Letter we describe the design, synthesis and characterisation of a new series of monocyclic CXCR2 antagonists with improved solubility and good pharmacokinetic profiles.

HepaRG cells as human-relevant in vitro model to study the effects of inflammatory stimuli on cytochrome P450 isoenzymes.

The suppression of hepatic cytochrome P450 (P450) expression during inflammatory and infectious diseases and the relief of this suppression by successful disease treatment have been previously demonstrated to impact drug disposition. To address this clinically relevant phenomenon preclinically, the effect of proinflammatory cytokines on P450 isoenzymes in human hepatocytes has been examined by several researchers. In the present study we used the human hepatoma cell line (HepaRG) and cryopreserved primary human hepatocytes to investigate the effects of various inflammatory stimuli on P450 levels with the aim of further characterizing HepaRG cells as a useful surrogate for primary hepatocytes.

Predicting metabolic clearance for drugs that are actively transported into hepatocytes: incubational binding as a consequence of in vitro hepatocyte concentration is a key factor.

Incubational binding or the fraction of drug unbound in an in vitro incubation, fuinc, is an important parameter to predict or measure in the pursuit of accurate clearance predictions from in vitro data. Here we describe a method for fuinc determination directly in the hepatocyte intrinsic clearance (CLint) assay with emphasis on compounds that are actively transported into hepatocytes, hypothesizing that for such compounds the typical protocol of 1 million hepatocytes/ml systematically underestimates the maximum attainable unbound intracellular drug concentration. Using the transporter substrate atorvastatin as a test compound, incubations were performed and a mathematical model applied to describe metabolism, distribution, and binding at different hepatocyte concentrations.

Species differences in biliary clearance and possible relevance of hepatic uptake and efflux transporters involvement.

From a search of the available literature, a database of 22 drugs of all charge types and several different therapeutic classes was compiled to compare rat and human biliary clearance data. Dog biliary excretion data were also found for nine of the drugs. For 19 of the 22 drugs (86%), rat unbound biliary clearance values, when normalized for body weight, exceeded those for humans by factors ranging from 9 to over 2500-fold, whereas human/dog differences were much less dramatic.

A novel matrix for the short-term storage of cells: utility in drug metabolism and drug transporter studies with rat, dog and human hepatocytes.

1. The SureTran matrix is a novel method facilitating short-term maintenance of fresh primary hepatocyte cellular function and offers the potential use of primary cells "as fresh" for several days post isolation. In the study presented, the maintenance of several key phase I and II drug metabolizing enzyme and drug transporter activities is demonstrated with rat and dog hepatocytes preserved for up to 7 days after cell isolation.

Optimisation of DMPK by the inhaled route: challenges and approaches.

The renewed interest in inhalation delivery over recent years has led to an expansion in the understanding of lung pharmacokinetics. Historically optimisation of inhaled drugs focused largely on development of material properties, consistent with achieving a good lung deposition, alongside demonstrating appropriate in vivo efficacy with little understanding of the relationship to pharmacokinetics in the lung. Recent efforts have led to an increased understanding of lung concentrations and how to maximise exposure in order to achieve the desired pharmacological response at a dose consistent with development of an inhaled product.

Lead optimisation of pyrazoles as novel FPR1 antagonists.

Optimisation of a series of pyrazole inhibitors of the human FPR1 receptor has been achieved. The use of an in vitro media loss assay was utilised to identify sub-series with more robust DMPK profiles. These were subsequently improved to generate analogues with attractive overall profiles.

The discovery of AZD9164, a novel muscarinic M3 antagonist.

The optimization of a new series of muscarinic M(3) antagonists is described, leading to the identification of AZD9164 which was progressed into the clinic for evaluation of its potential as a treatment for COPD.

The use of HepaRG and human hepatocyte data in predicting CYP induction drug-drug interactions via static equation and dynamic mechanistic modelling approaches.

The method of predicting CYP induction drug-drug interactions (DDIs) from a relative induction score (RIS) calibration has been developed to provide a novel model facilitating predictions for any CYP-inducer substrate combination by inclusion of parameters such as the fraction of hepatic clearance mediated by a specific CYP and fraction of the dose escaping intestinal extraction. In vitro HepaRG CYP3A4 induction data were used as a basis for the approach and a large number of DDIs were well predicted. Primary human hepatocyte data were also used to make predictions, using the HepaRG calibration as a foundation.

Functional consequences of active hepatic uptake on cytochrome P450 inhibition in rat and human hepatocytes.

A series of cytochrome P450 (P450) inhibition experiments were conducted with four hepatic uptake substrates (AZ3, AZ25, atorvastatin, and pitavastatin) using hepatocytes and recombinant P450s. The uptake was shown to be temperature-dependent and was inhibited by estrone sulfate, signifying an active component. At the lowest concentrations tested, the inhibitors concentrated up to 1000-fold in rat hepatocytes, but demonstrated only 5-fold greater P450 inhibition relative to recombinant rat P450s, indicating high intracellular binding.