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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. The aim was to demonstrate that N-deacetylated AZD1981 is responsible for the observed interaction. Methods The excretion and biotransformation of [14C]-AZD1981 were studied in healthy male volunteers, and subsequently in vitro cytochrome P450 (CYP) inhibition and hepatocyte uptake investigations were carried out with metabolites and the parent drug. A clinical DDI study using coadministered twice-daily 100 mg and 400 mg AZD1981 with 25 mg warfarin was performed. Results The excretion balance study showed N-deacetylated AZD1981 to be the most abundant metabolite present in plasma. In vitro data revealed the metabolite to be a weak CYP2C9 time-dependent inhibitor, subject to more active hepatic uptake than the parent molecule. Clinically, the S-warfarin area under the plasma concentration-time curve increased, on average, 1.4-fold [95% confidence interval (CI) 1.22, 1.50] and 2.4-fold (95% CI 2.11, 2.64) after 100 mg ( n = 13) and 400 mg ( n = 11) AZD1981 administration, respectively. In vitro CYP inhibition and hepatocyte uptake data were used to explain the interaction. Conclusions N-deacetylated AZD1981 can be added to the small list of drug metabolites reported as sole contributors to clinical drug-drug interactions, with weak time-dependent inhibition exacerbated by efficient hepatic uptake being the cause. [ABSTRACT FROM AUTHOR]

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. Application of best practice in experimental data generation and predictive methodologies, and a focus on robust mechanistic understanding of the candidate drug disposition properties before nomination to clinical development, have led to maximizing the probability of successful PK predictions so that 83% of AstraZeneca drug development projects progress in the clinic with no PK issues; and 71% of key PK parameter predictions [64% of area under the curve (AUC) predictions; 78% of maximum concentration (C max) predictions; and 70% of half-life predictions] are accurate to within twofold. Here, we discuss methods to predict human PK used by AstraZeneca, how these predictions are assessed and what can be learned from evaluating the predictions for 116 candidate drugs. It is now universally recognized that an acceptable human PK profile increases the probability of a candidate drug becoming a successful therapy. A variety of tools are available to predict human PK behavior in advance of clinical data, including scaling clearance from in vitro metabolic stability data, and physiologically based scaling of volume of distribution. To improve PK prediction methods, it is crucial to continually assess the performance of predictions with first-time-in-human PK data for new candidate drugs. To date, AstraZeneca have compared observed PK to predictions for 116 candidate drugs, and since the launch of our five-dimensional framework in 2011, we have driven sustained improvements in the quality of PK predictions. [ABSTRACT FROM AUTHOR]

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. Methods: Conditioned media was collected and sEVs were isolated from normal human bronchial epithelial cells (NHBEs) and IPF-diseased human bronchial epithelial cells (DHBEs). Results: Increased sEV release from DHBEs compared to NHBEs (n = 4; p < 0.05) was detected by nanoparticle tracking analysis. NHBEs co-cultured with DHBE-derived sEVs for 72 h expressed higher levels of SA-β-Gal and γH2AX protein, p16 and p21 RNA and increased secretion of IL6 and IL8 proteins (all n = 6–8; p < 0.05). sEVs were also co-cultured with healthy air–liquid interface (ALI) cultures and similar results were observed, with increases in p21 and p16 gene expression and IL6 and IL8 (basal and apical) secretion (n = 6; p < 0.05). Transepithelial electrical resistance (TEER) measurements, a reflection of epithelial barrier integrity, were decreased upon the addition of DHBE-derived sEVs (n = 6; p < 0.05). smRNA-sequencing identified nineteen significantly differentially expressed miRNA in DHBE-derived sEVs compared to NHBE-derived sEVs, with candidate miRNAs validated by qPCR (all n = 5; p < 0.05). Four of these miRNAs were upregulated in NHBEs co-cultured with DHBE-derived sEVs and three in healthy ALI cultures co-cultured with DHBE-derived sEVs (n = 3–4; p < 0.05). Conclusions: This data demonstrates that DHBE-derived sEVs transfer senescence to neighbouring healthy cells, promoting the disease state in IPF. [ABSTRACT FROM AUTHOR]

Abstract: This review promotes the value of isolated hepatocytes in modern Drug Discovery programmes and outlines how increased understanding, particularly in the area of in vitro–in vivo extrapolation (IVIVE), has led to more widespread use. The importance of in vitro metabolic intrinsic clearance data for predicting in vivo clearance has been acknowledged for several years and the greater utility of hepatocytes, compared with hepatic microsomes and liver slices, for this application is discussed. The application of hepatocytes in predicting drug–drug interactions (DDIs) resulting from reversible and irreversible (time-dependent) inhibition is relatively novel but affords the potential to study both phase I and phase II processes together with any impact of drug efflux and/or uptake (cellular accumulation). Progress in this area is reviewed along with current opinions on the comparative use of primary hepatocytes and higher throughput reporter gene-based systems for studying cytochrome P450 (CYP) induction. The appreciation of the role of transporter proteins in drug disposition continues to evolve. The study of hepatic uptake using isolated hepatocytes and the interplay between drug transport and metabolism with respect to both clearance and DDIs and subsequent IVIVE is also considered. [Copyright &y& Elsevier]

Abstract: SAR and DMPK studies led to the identification of substituted N-alkyl-N-[1-(3,3-diphenylpropyl)piperidin-4-yl]-2-phenylacetamides as potent and orally bioavailable ligands for the human CCR5 chemokine receptor. [Copyright &y& Elsevier]

Background: The radioligand [11C]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 [11C]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. Methods: PET measurements using [11C]VC-002 and the GE Discovery 710 PET/CT system were performed in seven control subjects at two separate occasions, 2–19 days apart. One subject discontinued the study after the first measurement. Radioligand binding to mAChRs in lung was quantified using an image-derived arterial input function. The total distribution volume (VT) values were obtained on a regional and voxel-by-voxel basis. Kinetic one-tissue and two-tissue compartment models (1TCM, 2TCM), analysis based on linearization of the compartment models (multilinear Logan) and image analysis by data-driven estimation of parametric images based on compartmental theory (DEPICT) were applied. The test-retest repeatability of VT estimates was evaluated by absolute variability (VAR) and intraclass correlation coefficients (ICCs). Results: The 1TCM was the statistically preferred model for description of [11C]VC-002 binding in the lungs. Low VAR (< 10%) across analysis methods indicated good reliability of the PET measurements. The VT estimates were stable after 60 min. Conclusions: The kinetic behaviour and good repeatability of [11C]VC-002 as well as the novel lung image analysis methodology support its application in applied studies on drug-induced mAChR receptor occupancy and the pathophysiology of pulmonary disorders. Trial registration: ClinicalTrials.gov identifier: NCT03097380, registered: 31 March 2017. [ABSTRACT FROM AUTHOR]

Background: 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). Methods: Salmeterol, salbutamol, and FP were simultaneously delivered by inhaled nebulization to rats. In the same animals, salmeterol-d3, salbutamol-d3, and FP-d3 were delivered by intravenous (IV) injection. Samples of lung tissue were obtained at 2- and 30-minute postdosing, and high-resolution MSI was used to study drug distribution and retention. Results: IV delivery resulted in homogeneous lung distribution for all molecules. In comparison, while inhalation also gave rise to drug presence in the entire lung, there were regional chemotype-dependent areas of higher abundance. At the 30-minute time point, inhaled salmeterol and salbutamol were preferentially retained in bronchiolar tissue, whereas FP was retained in all regions of the lungs. Conclusion: This study clearly demonstrates that inhaled small molecule chemotypes are differentially distributed in lung tissue after inhalation, and that high-resolution MSI can be applied to study these retention patterns. [ABSTRACT FROM AUTHOR]

Abstract: 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. In addition, alteration of CYP gene expression has not been fully defined in COPD. We previously established a method to purify ATII cells from the adult human lung using fluorescence‐activated cell sorting. By employing this technique we determined gene expression patterns of 14 CYP enzymes in ATII cells from nonsmokers (n = 4) and smokers (n = 4), both having normal pulmonary function. Although most CYP genes are highly expressed in primary hepatocytes, we found that CYP1B1 mRNA expression was 7.2‐fold higher in ATII compared to hepatocytes (P = .0275). Additionally we noted a 3.0‐fold upregulation of CYP2C19 and 50% reduction in CYP2J2 mRNA expressions in ATII cells isolated from patients with COPD (n = 3) compared to smokers without COPD (n = 4). These data, for the first time, detail a comprehensive set of genes encoding CYP enzymes in human ATII cells and highlights differentially expressed CYP mRNAs of patients with COPD. Such understanding may have important implications for the development of novel inhaled drugs. [ABSTRACT FROM AUTHOR]

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. The model is shown to allow translation between animal species and predicts drug concentrations in human lungs that correlate with the forced expiratory volume for different classes of bronchodilators. Thus, the pulmonary modeling approach has potential to be a key component in the prediction of human PK, efficacy, and safety for future inhaled medicines. [ABSTRACT FROM AUTHOR]

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. This work is a post hoc analysis based on preclinical in vivo pharmacokinetic/pharmacodynamic (PK/PD) data with the aim to evaluate such approaches and provide guidance on clinically effective dose prediction for inhaled medicines. Methods: Five empirically based methodologies were applied on a diverse set of marketed inhaled therapeutics (inhaled corticosteroids and bronchodilators). The approaches include scaling of dose based on body weight or body surface area and variants of PK/PD approaches aiming to predict the therapeutic dose based on having efficacious concentrations of drug in the lung over the dosing interval. Results: The most robust predictions of dose were made by body weight adjustment (90% within 3-fold) and by a specific PK/PD approach aiming for an average predicted 75% effect level during the dosing interval (80% within 3-fold). Scaling of dose based on body surface area consistently under predicted the therapeutic dose. Conclusions: Preclinical in vivo data and empirical scaling to man can be used as a baseline method for clinical dose predictions of inhaled medicines. The development of more sophisticated translational models utilizing free drug concentration and target engagement data is a desirable build. [ABSTRACT FROM AUTHOR]

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 6.5 or pH 7.4, and no concentration-dependent uptake was detected. Montelukast is a carboxylic acid, a relatively potent inhibitor of OATP1B1, OATP1B3, and OATP2B1, and has previously been postulated to be actively transported into human hepatocytes. Using OATP1B1-transfected HEK293 cells and primary human hepatocytes in the presence of OATP inhibitors we demonstrate for the first time that active OATP-dependent transport is unlikely to play a significant role in the human disposition of montelukast. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: A novel series of muscarinic receptor antagonists was developed, with the aim of identifying a compound with high M3 receptor potency and a reduced risk of dose-limiting side effects with potential for the treatment of COPD. Initial compound modifications led to a novel cycloheptyl series, which was improved by focusing on a quinuclidine sub-series. A wide range of N-substituents was evaluated to determine the optimal substituent providing a high M3 receptor potency, high intrinsic clearance and high human plasma protein binding. Compounds achieving in vitro study criteria were selected for in vivo evaluation. Pharmacokinetic half-lives, inhibition of bronchoconstriction and duration of action, as well as systemic side effects, induced by the compounds were assessed in guinea-pig models. Compounds with a long duration of action and good therapeutic index were identified and AZD8683 was selected for progression to the clinic. [Copyright &y& Elsevier]

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. 2. Intrinsic clearance values were determined for 60 new chemical entities using rat hepatocytes freshly isolated at AstraZeneca and rat hepatocytes prepared at the facilities of Abcellute Ltd (SureTran™ purveyors), stored and incubated 24 hours after isolation. A very good correspondence in the intrinsic clearance values underlines the utility of the cell maintenance matrix. 3. For human hepatocytes many of the enzyme activities assayed were well maintained for 7 days of storage but some declined to below 50% of initial values between day 4 and 7 of storage. Human OATP1B1 activity was only determined with one batch and declined to 51% of the initial test value by day 4 and further down to 35% by day 7. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: The optimization of a new series of muscarinic M3 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. [Copyright &y& Elsevier]

Abstract: SAR and PK studies led to the identification of N-(1-{(3R)-3-(3,5-difluorophenyl)-3-[4-methanesulfonylphenyl] propyl}piperidin-4-yl)-N-ethyl-2-[4-methanesulfonylphenyl]acetamide as a highly potent and selective ligand for the human CCR5 chemokine receptor with good oral pharmacokinetic properties. [Copyright &y& Elsevier]

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. Activities for major hepatic and pulmonary human drug-metabolizing enzymes are assessed and the data contextualized towards an in vivo setting using an ex vivo isolated perfused rat lung model. Very low rates of metabolism are observed in incubations with human ATII cells when compared to isolated hepatocytes and fewer of the substrates are found to be metabolized when compared to human lung microsomal incubations. Reactions selective for flavin-containing monooxygenases (FMOs), CYP1B1, CYP2C9, CYP2J2, and CYP3A4 all show significant rates in human lung microsomal incubations, but all activities are higher when rat lung microsomes are used. The work also demonstrates that a lung microsomal intrinsic clearance value towards the lower limit of detection for this parameter (3 µL/min/mg protein) results in a very low level of pulmonary metabolic clearance during the absorption period, for a drug dosed into the lung in vivo. [ABSTRACT FROM AUTHOR]