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An acceptable human pharmacokinetic profile increases the probability of a promising candidate drug becoming a successful therapy. The development of a mechanistic understanding of the disposition of candidate drugs towards confident predictions of key properties associated with human drug absorption, metabolism, distribution and excretion (ADME) is critical. This is achievable from a consideration of physicochemical properties, data from in vitro assays with human-derived material and from in vivo animal studies. This chapter is targeted at medicinal chemists and drug design teams and provides general definitions of the fundamental PK parameters together with best practice characterization, optimization and prediction strategies employed in the authors' laboratory.

IntroductionMolecular 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).MethodsPET imaging was performed using the muscarinic radioligand [11C]VC-002. The key methodological step involved estimating muscarinic receptor binding while disentangling it from the background of non-specific binding. The relationship between tiotropium exposure and receptor occupancy (RO) was assessed in non-human primates (NHPs) after intravenous injection of tiotropium doses at a broad dose interval (0.03–1 µg/kg). The feasibility of measuring RO in the human lung was then confirmed in seven healthy human subjects after inhalation of a single therapeutic dose of tiotropium (18 µg).ResultsThere was an evident effect of tiotropium on [11C]VC-002 binding to mAChRs in lungs in both NHPs and humans. In NHPs, RO was 11 to 78% and increased in a dose dependent manner. Non-displaceable binding in NHPs was about 10% of total binding. In humans, RO was 6%–65%, and non-displaceable binding was about 20% of total binding at baseline.DiscussionThe results demonstrate that [11C]VC-002 binds specifically to mAChRs in the lungs enabling the assessment of RO following administration of muscarinic antagonist drugs. Furthermore, the methodology has potential not only for dose finding and comparison of drug formulations in future applied studies, but also for evaluating changes in lung receptor distribution during disease or in response to therapy.Clinical Trial RegistrationClinicalTrials.gov, identifier: NCT03097380.

Purpose 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). Materials and methods PET imaging was performed using the muscarinic radioligand [11C]VC-002. The key methodological step involved estimating muscarinic receptor binding while disentangling it from the background of non-specific binding. The relationship between tiotropium exposure and receptor occupancy (RO) was assessed in non-human primates (NHPs) after intravenous injection of tiotropium doses at a broad dose interval (0.1-1mg/kg). The feasibility of measuring RO in the human lung was then confirmed in seven healthy human subjects after inhalation of a single therapeutic dose of tiotropium (18mg). Results There was an evident effect of tiotropium on [11C]VC-002 binding to mAChRs in lungs in both NHPs and humans. In NHPs, RO was 11 to 78% and increased in a dose dependent manner. Non-displaceable binding in NHPs was about 10% of total binding. In humans, RO was 6-65%, and non-displaceable binding was about 20% of total binding at baseline. Conclusions The results demonstrate that [11C]VC-002 binds specifically to mAChRs in the lungs enabling the assessment of RO following administration of muscarinic antagonist drugs. Furthermore, the methodology has potential not only to support early drug development, but also for evaluating changes in lung receptor distribution in a diagnostic setting, during disease or in response to therapy.

BackgroundThe aim of the present translational PET study was to estimate occupancy of tiotropium at muscarinic acetylcholine receptors (mAChR) in the lungs in vivo. The relationship between the tiotropium exposure and receptor occupancy (RO) in the lung was assessed in non-human primates (NHPs) after intravenous injection of tiotropium doses at a broad dose interval (0.1-1mg/kg). The feasibility of measuring mAChR occupancy in the human lung was then confirmed in seven healthy human subjects after inhalation of a single therapeutic dose of tiotropium (18mg). PET examinations were performed using radioligand [11C]VC-002. Occupancy in lungs was estimated using Lassen plot analysis of parametric images showing total radioligand binding (total distribution volume, VT).ResultsThere was an evident effect of tiotropium on [11C]VC-002 binding to mAChRs in lungs in both NHPs and humans. In NHPs, the occupancy was 11 to 78%, increasing in a dose dependent manner. The Lassen-plot based estimate of non-displaceable binding in NHPs was about 10% of the VT. In humans, occupancy was 6-65%, and non-displaceable binding (VND) was about 20% of total binding, VT at baseline. ConclusionsThe results demonstrated that [11C]VC-002 binds specifically to mAChRs in the lungs such that it allows for the detection and quantification of lung muscarinic receptor occupancy following administration of an intravenously administered or inhaled muscarinic antagonist drugs. The methodology has potential for dose finding and comparison of drug formulations in future applied studies. Clinical Trial Registration: ClinicalTrials.gov identifier: NCT03097380, registered: 31 March 2017, url: https://clinicaltrials.gov/ct2/show/NCT03097380

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.

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. Here we detail the approaches and lessons learned at AstraZeneca during the optimization of acidic CXC chemokine receptor 2 (CXCR2) antagonists for the oral drug treatment of inflammatory diseases, resulting in discovery and clinical testing of N-[2-[(2,3-difluorophenyl)methylsulfanyl]-6-[(2R,3S)-3,4-dihydroxybutan-2-yl]oxypyrimidin-4-yl]azetidine-1-sulfonamide (AZD5069) and AZD4721, orally bioavailable acidic molecules with PPB of Significance Statement Provided that the pharmacologic potency is high enough, modulation of plasma protein binding can form part of a viable strategy in drug discovery to optimize the effective half-life of drug candidates in humans.

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.

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 (Cmax) 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.

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.

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 0.2 µl/min/10(6) cells. The HµREL assay provided a higher level of reproducibility, with repeat significant CL(int) values being defined in a minimum of triplicate consecutive assays for six of seven of the low CL(int) compounds compared with four of seven for PHHs and two of seven for HepaRG. The assays were also compared with a suspension assay using drugs with higher CL(int) values and diverse enzymology. The CL(int) values from the PHH and HµREL assays were similar to those defined by a hepatocyte suspension assay, indicating that they can be used interchangeably alongside a standard assay. Finally, data from these two assays could also predict in vivo hepatic metabolic CL(int) to within 3-fold for greater than 70% of the compounds tested, with average fold errors (AFE) of 1.6 and 2.3, respectively, whereas the HepaRG data were predictive to within 3-fold for only 50% of compounds (AFE 2.9). In summary, all systems have utility for low CL(int) determination, but the HµREL co-culture appears slightly superior regarding overall assay performance.

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.

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. 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. 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. 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.

The fraction of unbound drug (fuinc) in in vitro intrinsic clearance (CLint) 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 fuinc measured using equilibrium dialysis. He fuinc value can also be measured directly in CLint assays using multiple concentrations of hepatocytes or microsomal protein. Since this approach informs of the unbound drug concentration in the assay used to predict in vivo clearance, it should be considered the gold standard method. As a starting point for building better predictive algorithms we aimed to determine if equilibrium dialysis really is an appropriate assay for assessing fuinc Employing a large number of compounds with a wide range of lipophilicities, experiments were performed to measure fuinc using rat hepatocytes (RH) and human liver microsomes (HLM) in both assay formats. A high percentage (94% and 93% for HLM and RH, respectively) of the fuinc values were within 2-fold when the compound distribution coefficient describing the ratio of compound concentration in octanol and pH 7.4 buffer when the test system is at equilibrium (lipophilicity measure) (logD7.4) values were less than 3.5. However, with logD7.4 values greater than these, the agreement was considerably worse. Additional experimental data generated indicated that this discrepancy was likely due to failings in the direct method when drug binding is high. Thus, we conclude that unbound CLint can be indeed calculated indirectly by incorporating equilibrium dialysis data with measured CLint but that simple lipophilicity descriptors alone may be inadequate for predicting fuinc.

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.

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.The excretion and biotransformation of [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.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.