Challenges in inhaled product development and opportunities for open innovation

Advanced Drug Delivery Reviews 63 (2011) 69–87

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Advanced Drug Delivery Reviews j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a d d r

Challenges in inhaled product development and opportunities for open innovation☆ Ben Forbes a,⁎, Bahman Asgharian b, Lea Ann Dailey a, Douglas Ferguson c, Per Gerde d,e, Mark Gumbleton f, Lena Gustavsson g, Colin Hardy h, David Hassall i, Rhys Jones j, Ruth Lock k, Janet Maas k, Tim McGovern l, Gary R. Pitcairn j, Graham Somers i, Ron K. Wolff k a

King's College London, Pharmaceutical Science Division, 150 Stamford St, London SE1 9NH, UK Applied Research Associates, 8537 Six Forks Road, Raleigh NC 27615, USA AstraZeneca R & D, Bakewell Road, Loughborough LE11 5RH, UK d Karolinska Institutet, Stockholm SE-171 77, Sweden e Inhalation Sciences Sweden AB, Stockholm SE-171 77, Sweden f University of Cardiff, Welsh School of Pharmacy, King Edward VII Avenue, Cardiff CF10 3NB, UK g AstraZeneca R & D, Lund SE-221-87, Sweden h Huntingdon Life Sciences, Woolley Road, Huntingdon PE28 4HS, UK i GlaxoSmithKline R & D, Gunnels Wood Road, Stevenage SG1 2NY, UK j Pfizer R & D, Sandwich, Kent CT13 9NJ, UK k Novartis Horsham Research Centre, Wimblehurst Road, Horsham, RH12 5AB, UK l SciLucent LLC, 585 Grove Street, Herndon, VA 20170, USA b c

a r t i c l e

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Article history: Received 5 October 2010 Accepted 25 November 2010 Available online 6 December 2010 Keywords: Aerosol dosimetry Deposition Inhalation toxicology ADME Isolated perfused lung Transporter Pharmacokinetics Pharmacodynamics

a b s t r a c t Dosimetry, safety and the efficacy of drugs in the lungs are critical factors in the development of inhaled medicines. This article considers the challenges in each of these areas with reference to current industry practices for developing inhaled products, and suggests collaborative scientific approaches to address these challenges. The portfolio of molecules requiring delivery by inhalation has expanded rapidly to include novel drugs for lung disease, combination therapies, biopharmaceuticals and candidates for systemic delivery via the lung. For these drugs to be developed as inhaled medicines, a better understanding of their fate in the lungs and how this might be modified is required. Harmonised approaches based on ‘best practice’ are advocated for dosimetry and safety studies; this would provide coherent data to help product developers and regulatory agencies differentiate new inhaled drug products. To date, there are limited reports describing full temporal relationships between pharmacokinetic (PK) and pharmacodynamic (PD) measurements. A better understanding of pulmonary PK and PK/PD relationships would help mitigate the risk of not engaging successfully or persistently with the drug target as well as identifying the potential for drug accumulation in the lung or excessive systemic exposure. Recommendations are made for (i) better industry-academiaregulatory co-operation, (ii) sharing of pre-competitive data, and (iii) open innovation through collaborative research in key topics such as lung deposition, drug solubility and dissolution in lung fluid, adaptive responses in safety studies, biomarker development and validation, the role of transporters in pulmonary drug disposition, target localisation within the lung and the determinants of local efficacy following inhaled drug administration. © 2010 Elsevier B.V. All rights reserved.

Contents 1.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Drugs in the lungs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Inhaled product development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

70 70 70

☆ This article is based upon an international workshop held by the Academy of Pharmaceutical Sciences Great Britain on 10 June 2010 at GlaxoSmithKline, Stevenage, UK, to launch the Drugs in the Lungs Network. The meeting aimed to identify common challenges facing those undertaking inhaled product development. Details of the Workshop participants, presentations, discussions and the consensus achieved are freely available on the APSGB website [1]. This article by the meeting organisers and expert speakers aims to deliver a more detailed perspective on the topics discussed and conclusions reached at the meeting. ⁎ Corresponding author. E-mail address: [email protected] (B. Forbes). 0169-409X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.addr.2010.11.004

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1.3. Common challenges in developing inhaled medicines . 1.4. Open innovation . . . . . . . . . . . . . . . . . . . 2. Dosimetry in inhaled product development . . . . . . . . . 2.1. Dose calculations . . . . . . . . . . . . . . . . . . 2.2. Estimating the deposited dose . . . . . . . . . . . . 2.3. Measuring the deposited dose . . . . . . . . . . . . 2.4. Modelling pharmaceutical aerosol deposition . . . . . 2.5. Current practice and opportunities . . . . . . . . . . 3. Inhalation safety studies . . . . . . . . . . . . . . . . . . 3.1. Regulatory requirements . . . . . . . . . . . . . . . 3.2. Biomarkers of toxicity . . . . . . . . . . . . . . . . 3.3. Interpretation of adverse effects . . . . . . . . . . . 3.4. Toxicokinetics . . . . . . . . . . . . . . . . . . . . 3.5. Developments in safety science . . . . . . . . . . . . 4. Pulmonary drug disposition . . . . . . . . . . . . . . . . . 4.1. Intrinsic and formulation-driven pharmacokinetics (PK) 4.2. Experimental models for PK studies . . . . . . . . . . 4.3. The influence of drug transporters . . . . . . . . . . 4.4. Current research activity . . . . . . . . . . . . . . . 5. Pharmacokinetic-pharmacodynamic relationships in the lung . 5.1. Pharmacodynamics (PD) in the lungs . . . . . . . . . 5.2. Measuring pulmonary drug concentrations . . . . . . 5.2.1. Lung tissue homogenates . . . . . . . . . . 5.2.2. Bronchoscopic tissue biopsy . . . . . . . . . 5.2.3. Microdialysis . . . . . . . . . . . . . . . . 5.2.4. Epithelial lining fluid . . . . . . . . . . . . 5.2.5. Induced sputum . . . . . . . . . . . . . . . 5.2.6. Imaging. . . . . . . . . . . . . . . . . . . 5.3. Pulmonary PD endpoints . . . . . . . . . . . . . . . 5.4. PK/PD for locally acting inhaled drugs . . . . . . . . . 6. Challenges and opportunities . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction This article considers inhaled product development with an emphasis on dosimetry, safety and efficacy. A commentary on current industry practices in these areas is provided based on the experience of the authors together with the consensus views of the APSGB Drugs in the Lungs Workshop on 10 June 2010 where these topics were discussed in a series of structured debates [1]. Furthermore, consideration is given to the scientific developments and collaborative approaches required for industry to move towards a more efficient paradigm for developing inhaled medicines. 1.1. Drugs in the lungs The successful integration of novel drugs with devices capable of delivering defined doses to the respiratory tract has resulted in a proven track record for inhalation as a route of administration that limits systemic exposure and provides localised topical delivery. Thus, a number of orally inhaled products have been developed successfully over the last 50 years, providing symptomatic relief to millions of patients with asthma and chronic obstructive pulmonary disease (COPD) [2]. Inhalation is also a proven means of systemic delivery for drugs that have limited bioavailability by other routes or would benefit from rapid onset of action and a variety of products are in development for this purpose [3,4]. In recent decades, advances in device design and formulation science have addressed the need for more efficient inhalers that are capable of delivering larger doses to the lung with low extrathoracic deposition [2,5]. Once deposited in the lungs, drug disposition (dissolution, absorption, distribution, metabolism and elimination) and the influence of pulmonary pharmacokinetics (PK) on drug efficacy and safety are the critical determinants of clinical outcomes. Pulmonary disposition remains poorly understood despite modern capabilities in imaging, analytical and biological

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science which make measurement of drug disposition and mode of action more accessible. This raises the question, how can the fate of drugs in the lungs be understood better to allow improvements in current therapy and expedite the development of new inhaled medicines?

1.2. Inhaled product development In the early stages of drug discovery a sound scientific case is built to rationalise and validate a potential biological target. As a whole, the industry is well versed and able to undertake these tasks in an efficient manner. Once a drug target has been accepted as part of a wider portfolio of mechanisms, the intellectual property around know-how and tractability grows. However, the development of new medicines depends not only upon understanding the disease and target, but also how amenable the drug molecule is to pharmaceutical development. Regulatory guidelines dictate well-defined non-clinical and clinical phases of medicine development, but escalating costs, high attrition (failure to reach market) for novel therapies, poor product differentiation for reimbursement and generic competition are increasingly severe challenges to bringing novel medicines to market. The success stories in inhaled therapy of lung diseases are restricted to a small number of target classes, notably β2 receptor agonists, antimuscarinic drugs and corticosteroids [6] which for β2 receptor agonists and antimuscarinic drugs are associated with the muscular bronchioles and the airways of the proximal lung. In addition, most inhaled therapies do not modify to any great extent the underlying diseases, although steroids may impart some beneficial effects. In this regard, there is considerable scope to develop novel new medicines which seek to modify respiratory disease processes directly and to embrace respiratory disease areas for which therapy is inadequate or non-existent.

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1.3. Common challenges in developing inhaled medicines The inhaled route of delivery has always been associated with a considerable challenge in getting the drug to its target. The lungs are a highly complex organ designed to filter inspired air with many different cell types contributing to their function. Furthermore, the lungs may change dramatically when afflicted by disease resulting in an internal environment that works against the drug reaching and interacting successfully with the target. For targets in the upper airways this may have lesser significance, but drug delivery to the deep lung may be impeded by changes such as mucus hypersecretion or thickening, airway narrowing or collapse, fibrosis and poor blood circulation. To mitigate the risk of failing to deliver an inhaled molecule to its site of action, a far greater understanding of the impact of disease on lung pathophysiology is required. The health and economic burden of respiratory disease [7] not only provides a huge market for inhaled therapy, but also invokes a need to evaluate current practices and identify ways to develop new and better inhaled medicines. In contrast to precedented mechanisms, future drug targets in the lung are likely to be novel and necessitate new classes of molecules, engaging unprecedented mechanisms for which limited biological information is available [7–9]. For example, new approaches to disease modification will require pulmonary delivery of biopharmaceuticals, genes and small interfering RNA. The pharmaceutical portfolio for delivery by inhalation will be increased further by the emergence of drugs for systemic delivery via inhalation [3,4] and combination therapies (9). This expansion in the number and classes of drugs for delivery to the lungs will bring new challenges in establishing their safety and efficacy. This is likely to require new methods and collaborative approaches to the way that industry currently develops inhaled medicines, as the achievements of the past will be no guarantee of success in the future. Development of best practice may require new ways of cross company collaboration that break current conventions. Pertinent questions include: • How consistent is the industry approach to developing inhaled medicines? • What are the molecular characteristics that make a good respiratory drug? • Are standardised validated methods used for drug administration in non-clinical settings? • Do pharmaceutical companies measure similar parameters and at what stage in the discovery and development cycle are safety and efficacy studies conducted? • Are toxicological data obtained and reported similarly between companies and is inconsistent reporting of pathology creating a more complex picture for the industry and regulators than is necessary? • Do the endpoints of clinical trials show sufficient commonality across companies to demonstrate the true value of a new medicine? 1.4. Open innovation Given the considerable challenges outlined above, can pharmaceutical companies continue to work in isolation to develop inhaled medicines or is it possible for cross company collaborations in a precompetitive environment to increase the future chances of success for all? The latter will depend upon what information can aid not only the progression of targets through the drug development pipeline, but also guide regulatory authorities, payers and medical practitioners to a better understanding of the potential of a new drug. If common barriers and bottlenecks to progression are identified it may be possible to find better ways to develop understanding of inhaled medicines through wider collaborations between industry, academia and contract research organisations. This will need to encompass areas of mutual benefit,

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while maintaining intellectual property rights which so often tend to stifle innovative approaches. While intellectual property is of great importance to each pharmaceutical company, this need not prevent wider collaboration between companies to develop such assets where the nature of the challenges facing the industry is common. Pre-competitive collaborations in the pharmaceutical sciences are emerging and being advocated, for example, in the bioinformatics field [10]. In inhalation science, initiatives include the recent publication by the Association of Inhalation Toxicologists (AIT) encouraging a harmonised data-driven approach to calculating delivered dose in non-clinical toxicology studies [11] and the recent U-BIOPRED consortium (unbiased markers for the prediction of respiratory disease outcomes; part of the European Innovative Medicines Initiative), which is seeking to improve the diagnosis of asthma to aid better treatment [12,13]. In addition, the Cross Company Animal Models group (CCAMS; incorporating pharmaceutical companies, academia and CROs) is seeking to unify models for chronic respiratory diseases and methods used during drug discovery by gaining consensus on those techniques best able to predict a drug effect in vivo. Adoption of best practice would allow common models to be used, reduce the number of animals used overall and aid regulatory authorities by providing comparable data across licensing submissions. At a time when development costs are rising and payers are seeking greater differentiation as well as value for money for new drugs, the time of stand-alone pharmaceutical companies may be coming to an end. Sharing best practice and undertaking pre-competitive approaches may help to reduce the pathways to developing new medicines considerably. In the following sections we identify current practices, consider common challenges regarding drugs in the lungs and suggest opportunities to galvanize inhaled product development. 2. Dosimetry in inhaled product development Accurate dosimetry is essential in studies investigating drug safety and efficacy. At the Drugs in the Lungs Workshop the current opinion of the state-of-the-art concerning methods of quantifying the delivery of inhaled molecules was ascertained by considering: (i) How are non-clinical doses calculated or measured and is this consistent across the industry?, and (ii) Is there a scientific basis to support a more informed approach to guidelines on dosimetry in safety studies? 2.1. Dose calculations Two dose metrics are important for the design of non-clinical and clinical safety studies. The Delivered Dose (Eq. (1)) is the amount of drug inhaled by the animal or human subject and the Deposited Dose (Eq. (2)) is the actual amount of drug that is deposited in the lungs [11].   −1 Delivered Dose mg kg = ðC × RMV × D × IFÞ  BW

ð1Þ

  −1 = ðC × RMV × D × IF × DFÞ  BW Deposited Dose mg kg

ð2Þ

C RMV D BW IF

DF

concentration of substance in air (mg L− 1) respiratory minute volume or the volume of air inhaled in one minute (L min− 1) duration of exposure (min) body weight (kg) inhalable fraction, the proportion by weight of particles that is inhalable by the test species (IF is often assumed to be 100% if the test aerosol has a Mass Median Aerodynamic Diameter (MMAD) less than 3–4 μm), deposition fraction or the fraction of the Delivered Dose that is deposited in the lungs.

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Although both equations are used consistently across the industry, there are notable differences in the terminology and algorithms associated with both dose metrics. A number of different terms are in use to describe the Delivered Dose, with no particular term achieving greater acceptance than others; examples include total, inhaled, targeted and presented dose. Alternative terms for the Deposited Dose were the achieved or lung dose. This multiplicity of terms invites confusion and makes apparent the need for a harmonised terminology. For the purposes of this summary, we have adopted the terminology recommended by the AIT [11], where the Delivered Dose (Eq. (1)) is the amount of test substance inhaled by the test subject and Deposited Dose (Eq. (2)) is the amount of test substance calculated to be deposited in the lungs. In addition to variations in terminology, the Workshop identified that the algorithms used to determine RMV values for non-clinical species can also vary considerably [14–17]. However, the algorithm advocated by the AIT (Eq. (3); ref. [11]) appears to be gaining prominence based on the high number of Workshop attendees reporting its use. This algorithm was derived from RMV data from control animals collected in 18 datasets (comprising nearly 2000 individual observations) across four species in ten separate laboratories under Good Laboratory Practice (GLP) conditions. Since these conditions represent those used in current practice in regulatory inhalation toxicology studies, this algorithm is considered the most appropriate for use in the estimation of Delivered and Deposited Doses for non-clinical safety studies.   −1 0:852 RMV L min = 0:608 × BWð kgÞ

ð3Þ

In non-clinical studies for which the Delivered Dose is presented, the FDA's Division of Pulmonary, Allergy and Rheumatology Products applies default Deposition Fractions (DF) to calculate the Deposited Dose; this practice has been presented at numerous public meetings. For non-clinical safety studies and clinical trials, the FDA uses the following DF in their evaluations: 10% in rats, 25% in dogs and 100% in humans. These default values are based primarily on publications by Wolff and Dorato [18] and Snipes and co-workers [19]. Wolff and Dorato compiled published data on pulmonary deposition of particles across species commonly used in non-clinical pharmaceutical testing while Snipes and co-workers performed a meta-analysis of a number of different deposition studies (assuming particles with an MMAD of 2 μm) and calculated average depositions for each species. However, there are inherent limitations associated with the use of these standard FDA default values for calculating Deposited Doses, which was reflected in the lively and lengthy debate on the relevance and implications of lung deposition estimates at the Workshop. As a result, this issue is considered in greater depth in the following Sections 2.2 and 2.3. In contrast to non-clinical studies, current practice for dose estimation in clinical trials is to assume the delivery of the entire nominal dose (total amount of drug) rather than use Eqs. (1) and (2). Dose estimates in clinical studies would be more accurate if the amount of drug remaining in the device after administration were measured and subtracted from the nominal dose; this is especially relevant when nebulizers are used. 2.2. Estimating the deposited dose Although the FDA default DFs are used industry-wide, there is an overwhelming consensus that they do not necessarily reflect the actual Deposited Dose of pharmaceutical aerosols. Most researchers feel that the default DFs sometimes underestimate the deposition in non-clinical species and overestimate deposition in man. If this is true, it is possible that the use of more accurate measures of drug deposition would improve the analysis of non-clinical safety data

and make it easier to define realistic clinical dose ranges. To evaluate whether current estimates of Deposited Doses are reasonable requires an appreciation of: i) how the default DFs were established, ii) how these values are used in non-clinical safety studies, and iii) the implications of the above for study design and data interpretation. Snipes and co-workers [19] based their aerosol deposition analysis on a number of studies investigating the deposition of insoluble environmental particulates such as plutonium, europium, iron oxide and aluminium silicate in the lungs of a number of experimental species. Although these studies provide an excellent starting point for determining deposition profiles of pharmaceutical aerosols with similar aerodynamic properties, drug substances may behave quite differently in the respiratory tract compared with insoluble environmental particles. This is because drug compounds may be hygroscopic, possess variable dissolution rates or exhibit different transportation properties. The formulation and processes of aerosolisation, inhalation and deposition of pharmaceutical aerosols may also introduce deviations from predictions based on insoluble particles. Furthermore, differences in DF between mono- and polydisperse aerosols may be even greater than those between aerosols of different materials, yet similar size distributions. Therefore, it would be valuable and appropriate from a scientific standpoint to generate more accurate deposition data on actual pharmaceutical aerosols to develop a better understanding of their dosimetry and deposition in non-clinical species and in humans. The implications of using more realistic DFs for pharmaceutical aerosols in inhaled product development requires an understanding of how these values are used in the development process. Deposited Doses in non-clinical studies are used to determine acceptable dose ranges for human clinical trials. It is important to note that regulatory authorities generally require the application of a default DF value of 100% in man (rather than the average 40% described by Snipes and coworkers [19] for clinical trials). This represents a cautious approach where the actual deposition fraction is unknown. One consequence of this overestimation of deposition in man is that to achieve safety cover excessively high doses must be administered to non-clinical species, especially when the additional safety margins are applied; i.e. margins of 6-fold in dog and 10-fold in rat referenced to the No Observable Adverse Effect Level in these species. For compounds that are well-tolerated or require high clinical doses to achieve efficacy, it is often impractical to administer sufficiently high doses in nonclinical studies to achieve the required safety margins. One solution to the requirement for excessive doses in non-clinical studies would be to introduce a reasonable upper dose limit for welltolerated compounds. This strategy is currently covered by the guidelines [ICH Guidance for Industry M3(R2), 2010], albeit at dose levels that are only applicable for safety assessment of orally administered compounds which it is feasible to administer in larger quantities. Workshop participants discussed the feasibility of introducing reasonable upper limits for delivery by inhalation based, for example, upon the use of 1 mg/L as a maximum achievable concentration across a number of compounds with maximum dosing times set according to what is appropriate for different species. It would also help if clinical doses could be based on data-driven values of DFs. If deposition data for specific animal models and specific compounds were generated, the use of alternative DF could be justified on a case-by-case basis. This approach opens up the potential to apply higher Deposited Dose values in non-clinical studies than are possible using the current default DFs, which would in turn support higher dose levels in human clinical studies. It was intimated in Workshop discussions that European regulatory bodies are amenable to considering evidence for animal deposition values on a case-bycase basis. It was also believed that the FDA would be likely to consider alternative values to their default position on non-clinical dosimetry if appropriate supporting evidence were provided. In contrast, the regulatory assumption of 100% drug deposition in

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human clinical trials, like the applied safety margins, is based primarily on philosophical safety concerns rather than experimentally derived data; this makes it unlikely that data would be adequate to change this position. It was recognised that even if the regulatory authorities were to consider more realistic DF values for clinical studies, the overall impact on permitted dose levels would be limited. For example, the use of a 50% DF (as opposed to 100%) would only increase the permissible clinical dose by 2-fold, i.e. it would have limited impact on non-clinical safety study dosing requirements. However, if a change in this component of the overall safety evaluation were combined with changes in the evaluation of the non-clinical toxicology package (e.g. use of measured rather than estimated deposited doses, application of maximum doses, and reduction of safety margins) this would have the potential for a significant cumulative impact on the determination of acceptable clinical doses. It was noted that non-clinical administration methods typically involve sustained delivery by nebuliser and tidal breathing which does not reflect the clinical situation where exposure in humans is often by bolus with special inhalation manoeuvres. However, in mitigation, estimated dose and systemic exposure in non-clinical studies are used primarily to calculate safety cover for human studies in which clinical doses are based on a pragmatic, conservative philosophy. In summary, there was a consensus at the Workshop that it was both valuable and appropriate to generate deposition data relevant to pharmaceutical aerosols to improve understanding of real, compared to estimated, Deposited Dose in non-clinical species and man. In fact, although not required by the regulatory authorities, most companies represented at the meeting already often measure drug concentrations in the lung immediately after dosing or collect systemic PK data as proof of dosing in non-clinical studies (see Section 2.3).

pharmaceutical aerosols deposit and to compare real versus estimated Deposited Dose values. The two most prevalent methods are: i) measurement of systemic exposure, i.e. area under the plasma concentration curve (AUC) by inhalation compared to intravenous dose, and ii) excision of lung tissue immediately after administration and measurement of drug directly in lung tissue homogenate. The strengths and inherent limitations of each approach were discussed in the Workshop (Table 1). There was consensus that the AUC provides evidence of dosing following inhalation, especially when the drug compound exhibits both high solubility and permeability. Despite the issues listed in Table 1 and the caveats that must be applied, measuring systemic PK in the nonclinical phase provides proof of dosing, evidence of systemic safety assessment and a useful indication of clinical performance. In comparison to the relatively simple, but indirect measurement of the deposited dose using the AUC, determination of drug concentrations in lung homogenate provides a more direct measure of local deposited doses. This approach is preferred for the measurement of deposited doses of poorly soluble compounds and would also be applicable for poorly permeable compounds or inhaled pharmaceuticals where the formulation retards drug absorption for a significant amount of time. Measuring drug concentrations in homogenates prepared from excised lungs was recommended for safety studies of poorly soluble compounds, when accumulation after chronic dosing is a concern. Currently, there is no agreed protocol across the industry for measuring drug accumulation following chronic dosing, despite the fact that many new chemical entities exhibit low solubility profiles. Therefore, although no guidelines were agreed at the Workshop, development of evidence-based protocols was identified as a priority area for pre-competitive collaboration with the aim of establishing how best to measure real deposited doses after administration of pharmaceutical aerosols and how best to assess lung accumulation of poorly soluble drug compounds.

2.3. Measuring the deposited dose

2.4. Modelling pharmaceutical aerosol deposition

Different methods are currently used to verify drug delivery to the lung in non-clinical studies in order to achieve a greater understanding of how

In silico models have the potential to improve current understanding and estimates of dosimetry and there was a high level of

Table 1 Advantages and limitations of using systemic exposure (area under plasma concentration curve; AUC) or lung tissue homogenates for measuring Deposited Dose. AUC

Lung homogenate

Compound/formulation suitability

Complete absorption, no first pass extraction by the lung.

Method strengths

Dose determination (by comparison to reference PK data) after measuring an easily accessible systemic compartment. The ability to assess drug concentrations at various time points. Using basic PK principles it is possible to estimate the accumulation of non dissolved material in the lung. Rapid sample analysis. In non-clinical species, inhaled compound bioavailability can be influenced by both oral and nasal absorption. While oral absorption can be charcoal-blocked, nasal absorption cannot be controlled at present. The ability to model or block nasal absorption would be useful to fully understand inhaled bioavailability in non-clinical species. In contrast, nasal absorption is limited in human inhalation studies. Therefore, clinically some information can be gained from charcoal block, as long as clearance following intravenous dosing in the same species/strain at a comparable dose level is known in order to relate AUC to Deposited Dose. Non-clinical doses are often achieved by modifying the duration of exposure and/or inhaled drug concentration and different combinations of these may lead to variable pharmacokinetics for the same Deposited Dose. Accumulation of dissolved drug in the lung by any potential uptake mechanisms (transporters, lysosomal trapping) cannot be assessed by plasma AUC. Analytical sensitivity for low dose and (potentially) high clearance compounds. Proof of dosing. As a safety cover for systemic findings. As a useful indication of clinical performance.

Low solubility, low permeability compounds. Slow release formulations. Direct measure of local delivered doses. Possible assessment of compound accumulation after chronic dosing.

Limitations/caveats

Recommended applications

Complicated sample preparation and more time-consuming. Location of the drug in the lung is unknown with a homogenate concentration (e.g. upper or lower airway deposition differences). Measured lung concentrations will vary with time both during and after dosing. As lungs are typically collected after dosing, shorter inhalation periods will provide more reliable estimates. However, non-clinical doses are often achieved by modifying the duration of exposure and/or inhaled drug concentration and different combinations of these. Measured concentrations also include compound within the blood in the lung and compound passively distributed into the tissue (compounds with high volume of distributions and/or high plasma protein binding will influence these concentrations the most). Data is not readily comparable to clinical dose estimates.

Proof of dosing. As a useful indication of compound accumulation.

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interest in the current state-of-the-art in this area. Bahman Asgharian facilitated discussion and illustrated various approaches to modelling pharmaceutical aerosol deposition in the lungs of humans and species used in non-clinical studies [20,21]. There are two main approaches to calculating the deposition of delivered aerosols in the lung depending on the resolution of lung airway geometry and desired level of detail. The first approach involves calculations of the airflow in the selected lung geometry using computational fluid dynamics (CFD) and using the flow field predictions to solve for exact locations of particles depositing in the lung. Feasibility and practicability of this approach hinges on the availability of accurate airway geometry typically obtained from scanned images of the lung, as well as knowledge of airflow and pressure distribution in the deep lung to serve as outlet boundary conditions for the computational domain. The resolution clarity of existing imaging systems does not allow reconstruction of smaller airways of the lung (i.e., bronchial and pulmonary airways) and reconstructed airway geometry comprises less than 5% of the lung volume. Unless the region of interest in the lung lies within the domain of current resolution, the use of CFD in deposition predictions does not offer improved accuracy over other approaches. Uncertainty regarding airway geometry and airflow boundary conditions limits CFD application in the deep lung for the purpose of deposition predictions. Hence, detailed CFD approach is most useful in the extrathoracic airways [22–26] and first few airway generations of the tracheobronchial airways [27–39] for which airway passages are reconstructed from scanned images of airway coronal sections and combined with measured breathing parameters to form anatomically and physiologically realistic deposition models. Since the full transport equations with complete account of dominant physical mechanisms are solved, detailed information regarding the airflow and particle deposition is obtained, however, at the expense of a heavy computational cost (Fig. 1). The second and traditional approach to predicting particle deposition in the lung uses simplifying assumption to allow deposition calculations of particles for the entire lung. The lung geometry is modelled – based on the available measurements of airway parameters [40,41] – as a dichotomous, symmetric or asymmetric branching structure with each airway shaped as a cylinder. The idealised geometry of lung airways is compatible with the area-averaged, 1-dimensional mechanistic model of particle transport and deposition throughout the conducting tree and alveolar ducts of the lung. Although detailed site-specific or per-generation experimental comparisons of deposition are not warranted in the simplified geometry, dose to the lung and to the tracheobronchial and alveolar regions has been compared extensively [42]. In addition, the influence of gravity on pleural pressure has been neglected to allow uniform airflow distribution in the lung. Consequently, airflow at any location in the lung is proportional to the distal volume to that location. Hence, the need for detailed airflow computations using CFD is alleviated. Using cross-sectional-area-averaged particle concentration of inhaled aerosols, a simplified transport equation is obtained and solved to predict local and regional deposition of particles in the lung. This approach is ideal when regional lung deposition predictions are needed while at the same time the CFD approach is handicapped by enormity of the lung airways and lack of information on ventilation distribution in the lung [43–48]. As a result of making simplifying assumptions, computations can be carried out in a relatively short time, however, detailed, site-specific deposition information are either unavailable or unreliable (Fig. 2). The use of in silico approaches to improve estimations of deposited dose is currently limited by the lack of data and validation for pharmaceutical aerosols and lack of precedence for regulatory acceptance (e.g., the models have undergone limited development relating to deposition in species used in non-clinical studies). It was noted that the in silico human deposition models have been accepted

Fig. 1. Deposition distribution of 1 μm particles in the first few airway generations of a reconstructed human tracheobronchial tree.

by the US Environmental Protection Agency for environmental safety assessments and it is possible that the applicability of the models for human drug safety studies may not yet be fully realised by the FDA. To develop in silico models into useful tools for dosimetry and pharmacokinetic analysis, deposition data is required to validate and confirm the models. As individual datasets are small, pre-competitive information could be pooled across the industry for this purpose. For example, particle size determined using cascade impactors may help to predict the DF for in silico models in cases where this has been validated against experimental data. For normally distributed particles, 10% deposition in the rat appears to be reasonable as previously mentioned. For skewed distributions, adjustments are required to the DF. However, it was noted that current in vitro methods for aerosol characterisation, such as cascade impaction, are primarily developed for aerosol characterisation and are not designed for in vitro-in vivo prediction of dose to the lung [49,50]. The opportunity to build a databank based on drug-like molecules with different physico-chemical properties may provide an opportunity to generate pharmaceutically relevant deposition data to support more realistic deposition values in animals; this would inform dosimetry calculations in non-clinical studies and facilitate their translation into clinical studies. This was identified as a positive opportunity for industry to collaborate, pool data and, in conjunction with greater use of imaging techniques and integration of appropriate techniques to characterize lung deposition in non-clinical species and human (qualitatively and quantitatively), to improve our understanding of deposition.

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present Good Laboratory Practice (GLP) toxicology studies supporting clinical trials are standard [51] with histopathology as the primary endpoint of such studies [52]. Within these studies, however, there are issues with regard to (i) the consistency with which pathologies are defined and classified, (ii) biomarkers of toxicity, (iii) the background range of normal biological variation and how to distinguish an adverse reaction from a normal response, and (iv) the means and extent to which toxicokinetics should be applied. 3.1. Regulatory requirements

Fig. 2. Idealised geometry of the tracheobronchial tree of humans based on measurements of Raabe et al. (2006). Each airway is represented by a cylindrical tube.

2.5. Current practice and opportunities Cross-industry sharing of deposition data in non-clinical animal species provides an opportunity to pool data upon which more informed estimates of deposition could be made and negotiated for regulatory acceptance. The MHRA are amenable to considering evidence-based deposition data whereas the FDA has default expectations regarding deposition factors, although the FDA would also be likely to consider evidence-based deposition data if this were presented. The deposition factors used by the FDA for non-clinical studies (10% in rat; 25% in dog) are not entirely unreasonable, but there is scope for using actual data for a given compound to improve dose estimates. At present, there is no regulatory or scientific guidance on protocols to evaluate deposition and accumulation of compounds following inhaled administration. With extensive validation, current in vitro methods for aerosol characterisation (e.g. cascade impaction) can be used for in vitro-in vivo prediction of dose to the lung although this is not necessarily a 1:1 correlation; this does not, however, extend to prediction of regional deposition. Systemic exposure, although a useful indicator of dosing and systemic safety cover, cannot be used to estimate lung deposition or accumulation in non-clinical species. There is considerable potential for cross industry sharing of protocols for measuring deposition and dosimetry data to improve estimates of safety risk, our fundamental understanding of the deposition of pharmaceutical aerosols and to validate and develop in silico models of particle deposition for inhaled pharmaceuticals. 3. Inhalation safety studies Current practices in inhalation toxicology, how these might be improved and the challenges that might benefit from a collaborative approach were considered by asking the question, “what measurements can be used consistently across the industry to establish safety or toxicity to allow regulatory bodies to assess new chemical entities (NCE)?” A rational approach to developing inhalation safety science is of importance to industry and regulators, both of whom have an interest in maximising safety assurance while complying with the 3Rs requirement for eliminating unnecessary animal experimentation. At

Safety studies are performed early in product development and are designed to meet well-defined regulatory package specifications to permit first-in-human dosing. In addition to standard tests for mutagenicity and safety pharmacology, non-clinical repeat dosing studies are required utilising delivery via the relevant clinical route of exposure. Although in vitro methods are in development and have the potential to reduce costs and animal experimentation as well as provide mechanistic insights [53], there are currently no validated assays with regulatory acceptance that may be substituted for the acute and chronic in vivo studies that are required to enable single dose human clinical trials (ICH M3(R2) guideline) (Table 2). In these studies the core end-points measured are histopathology, hematology, and clinical chemistry. Although the histopathology procedures are standard, the terminology used to describe the nature and severity of end-points may be a source of inter-study variation which precludes meta-analyses and hinders the assessment of study outcomes. This is a recognised weakness which is being addressed by toxicology and pathology bodies, e.g. the International Harmonisation of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice “INHAND” [54]. This is a welcome initiative which aims to address the need for standard definitions. Ideally, the new nomenclature should be recognisable by specialist and non-specialist pathologists and regulators. However, by their nature histopathological measurements and severity ratings are semi-quantitative. Drug administration in these non-clinical studies is by aerosolisation with the attendant issues regarding dosimetry that are described in the previous section. The form, in which the test article is presented, for example powder or liquid droplet solution, may have a profound effect on the local lung effects. Ideally the same form should be used throughout the development of an inhaled product and certainly the definitive studies should be performed with the same or a very similar formulation to that which will be used for phase 3 studies and launch. However, practical limitations such as the early stage of pharmaceutical development and limited availability of the material may prevent this ideal approach. Acute studies are mandatory and establish doses for use in longer duration chronic administration studies. Although acute studies have limited ability to predict chronic outcomes, they are regarded as useful for toxicity screening, i.e. detection of readily identifiable toxicities and maximum

Table 2 Non-clinical toxicology required before commencing single dose human clinical trials of inhaled medicines.⁎ Inhalation toxicology

Description

Single dose

2 species (rodent and non-rodent, typically rat and dog or rat and monkey), ascending dose to maximum tolerated single dose 2 species, (rodent and non-rodent, typically rat and dog or rat and monkey), repeat dosing of 2 weeks–1 month duration In vitro and in vivo genotoxicity tests In vitro and in vivo studies related to cardiovascular, central nervous, and respiratory systems

Repeat dose Mutagenicity Safety pharmacology

⁎ This a brief outline of the studies required. More detail, particularly for longer than single dose clinical trials, is available in the references ([51,52]; ICH Guideline M3(R2)).

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doses to be used in repeat dose studies. In general, however, acute studies by their short-term nature have limited ability to predict subtle effects over the long term (ICH guidelines, M3 Section 4). 3.2. Biomarkers of toxicity For both acute and chronic toxicity testing, better biomarkers in bronchoalveolar lavage fluid (BALF) or blood and other supplementary end-points would be useful. It is necessary to consider these on a compound by compound basis and if pharmacodynamic end-points are available these should also be used. Endpoints that can be measured in early, acute and repeat dose studies include cell infiltrates and cytokines in BALF. These measurements can be particularly useful in early studies to assess toxicity relative to other candidate compounds or known toxicants. Pulmonary function is a safety pharmacology end-point and can also be useful in early studies to help assess whether there is any respiratory tract irritation, effect on lung surfactant or serious lung damage which are manifest by changes in breathing pattern or lung mechanics. Over the years, a variety of enzymatic markers have been monitored [55], without any particular marker gaining precedence as indicative of respiratory toxicity. More recently genomic approaches such as gene profiling have been utilised, for example to identify mechanisms of toxicity of excipients for use in inhaled formulations [56,57] and metabonomic studies are being undertaken increasingly to elucidate metabolite biomarkers for improved diagnosis of lung diseases such as asthma [58], chronic obstructive pulmonary disease (COPD) [59] and cystic fibrosis [60]. The study of metabolite signatures in lung tissue of COPD murine models and the acute effects of cigarette smoke on these signatures [59] is the first time that NMR-based metabonomic analysis has been reported for intact lung tissue; this has the potential to generate a reliable metabolic fingerprint to uniquely characterise the animal model of COPD and begin to identify strong biomarkers for the disease. There is the prospect that in the future such methods will be developed and applied to the identification of biomarkers of inhaled drug or excipient toxicity. 3.3. Interpretation of adverse effects Difficulties in interpreting toxicology findings include reconciling test sensitivity, background biological variation, normal responses to inhaled materials and drug or medicine-specific adverse effects. Identification of adverse end-points is an area where better control data sets might help discern true adverse effects from normal physiological lung response. The lung responds acutely to inhalation of irritant materials by hypersecretion of mucus, chemokine release, inflammatory cell recruitment and cough [61]. Collectively these may be characterised as non-specific irritancy. Chronic changes that could lead to pathology may be observed morphologically as epithelial damage, granuloma and phospholipidosis — including the appearance of “foamy” macrophages. The development of foamy macrophages is a poorly understood phenomenon that provides a conundrum regarding the implications of their observation in non-clinical testing. The foamy macrophage phenotype is a term used to describe the appearance of macrophages that have taken on a granular or vacuolated appearance under the light microscope (Fig. 3). The phenotype can be manifested as a result of a number of different processes including intracellular lipid accumulation due to excess surfactant in the lung tissue [62,63], phospholipidosis [64,65] or the uptake of insoluble particles [66,67]. The appearance of the phenotype throughout the lung post-dose is often heterologous and the foamy phenotype varies between compounds and dosing regimens. While the potential for induction of foamy macrophages should be investigated early in the drug development process, the implications

of inducing a foamy macrophage phenotype are relatively poorly understood. In some circumstances the appearance of foamy macrophages may simply be an adaptive and reversible response to the inhalation of particles [68]. However, the induction of foamy macrophages may also form part of the progression of a number of diseases including emphysema, pulmonary alveolar proteinosis [63] and certain infectious diseases [69] and can be considered an early marker of the progressive toxicity of inhaled drug particles [70]. The production of the foamy macrophage phenotype and activation of macrophages in vitro have been reported by a number of authors investigating the effects of insoluble environmental particulates [71– 73]. However, there is no definitive evidence regarding the relevance and implications of the formation of foamy macrophages in response to inhaled drug particles within in vitro systems, or with respect to the observation of foamy macrophages during chronic inhalation safety studies, which are generally carried out at very high particle doses. Better understanding of the implications and reversibility of any changes is therefore critical for developing new inhaled drugs. The measurement and interpretation of these phenomena are complicated by uncertainty regarding the specific or non-specific nature of the response to inhaled particles [74,75]. Developing a data set that clearly establishes normal and adaptive physiological responses is needed. This might be helped by pooling of industry-wide control data that includes sham, vehicle and particle controls. There is also a question regarding whether certain biological responses in nonclinical inhalation studies are acceptable and expected non-specific changes, i.e. normal adaptive and non-toxic. Benchmarking of the effects of known toxicants (as a positive control data set) and approved marketed inhaled pharmaceuticals (as a negative control data set) might be instructive in discerning what constitutes adverse versus non-adverse effects. This approach would also provide the necessary validation to make biomarker profiles decision-making in inhalation development projects. Where non-clinical data lead to uncertainty regarding toxicity, methods for monitoring safety in the clinic (i.e. measurable biomarkers) would enable progression and dose escalation in human clinical trials. Unfortunately, measures of respiratory toxicity in man are limited, but it would be extremely useful if some of the markers that can be measured in BALF in non-clinical studies could be measured readily in blood in clinical studies. 3.4. Toxicokinetics Toxicokinetics is the determination of the relationship between systemic (blood or plasma) levels of a compound and its toxicity and is increasingly being applied in toxicity testing. The extent to which lung kinetics are measured in inhalation safety studies across the industry varies. Measurement of systemic drug concentrations for pharmacokinetic studies is a standard procedure (Section 5) and this data is of interest for investigating the relationship between systemic toxicity and systemic exposure. However, drug concentration in lung has not traditionally been measured. As far as current industry practices are concerned, it transpires that drug levels in the lung are measured either (i) not at all, (ii) immediately after dosing or at the end of study, or (iii) at various time points. Data obtained immediately after dosing is used principally to verify that dosing has been successful. Samples collected at the end of the study can be compared to earlier equivalent samples to allow accumulation in the lung to be assessed. For more detailed lung toxicokinetic analysis, a greater number of time points are required and this data is not generally collected. The main barrier to wider use of measuring drug levels in the lungs in study protocols is the difficulty in interpreting lung concentrations or accumulation if it occurs. In addition, there is concern that if measurements that are not specified in regulatory guidelines are taken they may raise unwarranted concerns by the regulators. In

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Fig. 3. Left: A cytologic preparation of bronchoalveolar-lavage fluid from a patient showing “foamy” alveolar macrophages (buffered eosin and azure B, ×480). Comparison with the brown-staining cells also visible in these preparations shows that the macrophages are two to three times their normal size [63]. Right: Transmission Electron Microscopy image of a foamy macrophage containing numerous complex phospholipid inclusions (right; Bar represents 5 μm) [62].

contrast, the advantages of the toxicokinetic approach are that drug quantification in the lung can be useful for determining dose proportionality and interpreting histopathology, particularly if there is evidence of drug accumulation in lung. 3.5. Developments in safety science A number of developments in inhalation safety are alluded to above. The need for the harmonisation of terminology used to identify, describe and quantify histopathology is widely recognised and is being addressed. Measurement of systemic (blood/plasma) levels of drug and the determination of toxicokinetic parameters is now a routine in inhalation studies. However, there is not yet any standard in industry with respect to lung drug level measurements, which increase study costs, require additional resource, may be difficult to interpret and can raise questions from regulatory authorities. An example of the application of toxicokinetics in industry is the approach developed at Pfizer by Rhys Jones and Natasha Neef (presented at the APSGB Workshop on 10 June [1]); this described a method for modelling macrophage loading with poorly soluble particles in the lung. The aim was to examine the concept of overload doses in the lung, proposed to be N1 mg particles/g lung tissue [76], beyond which macrophage abnormalities are observed. A toxicokinetic model was developed to address the questions: (i) how do proposed lung thresholds for insoluble particles relate to studies in rodents with inhaled molecules? and (ii) do observed adverse changes in the lung correspond to accumulated doses above 1 mg/ g? Briefly, mass delivered was estimated from pulmonary deposition based on multiple path modelling (~ 10% in rat; [20]). This input was compared to clearance via mass absorbed following dissolution, which was calculated using the systemic exposure data, and macrophage uptake rates, which were based on data for removal of undissolved particulates by rat alveolar macrophages with a clearance of 0.007 day− 1 [77]. Using these parameters the thresholds confirmed that 0.1–1 mg particles/g lung tissue generated non-adverse adaptive changes, e.g. an increase in macrophage numbers, whereas lung accumulation at N1 mg particles/g lung tissue was associated with adverse changes such as inflammation and tissue degeneration [Note: the model assumes an absence of chemical toxicity and does not at present account for factors such as particle size or surface area]. This approach allowed lung burdens to be estimated successfully and used to interpret the histopathology findings. The outcomes were broadly consistent with published data on pathology associated with lung burdens of biologically inert material. Since the extent of accumulation can be estimated and extrapolated to levels at steadystate, this has the potential to inform dose selection for longer term

toxicology studies. The measurement of lung doses in non-clinical safety studies would help to validate this approach. Furthermore, the same approach can be applied to humans using the anticipated clinical dose using an estimated pulmonary absorption rate constant and an adjusted value for macrophage clearance in man 0.0035 day− 1 [78] compared to the value in rodents. This can provide confidence that lung burdens in human will remain well below threshold for adverse findings on chronic dosing. Industry-wide data sharing and collaborative research provide opportunities to improve on the current state-of-the-art. A possibility for data sharing is to pool control data from inhalation safety studies (i.e. share control data across the industry) to establish normal background biological variation. This would be useful for the regulators and was of interest to industry in order to help to assess which changes are adverse and which changes can be considered nonadverse. It was recognised that the logistics of such an exercise together with inter-laboratory differences in measurement and interpretation would provide considerable challenges. The value of sharing control data would be in generating larger data sets to aid evaluation of study data against a background of normal biological variation. A major research challenge for inhalation science is to develop better biomarkers, especially those that would enable dose escalation in the clinic when there are concerns regarding drug safety. Most widely applicable would be the identification of generic markers for different forms of lung damage. This could be complemented by specific markers for different inhaled drugs or drug classes. New endpoints also suffer from a lack of validation. A valuable means of addressing this would be benchmarking using known toxicants and approved inhaled pharmaceuticals to define markers of adverse effects. 4. Pulmonary drug disposition Key questions considered at the Workshop concerning pulmonary drug disposition were what is the relative importance of intrinsic and formulation-driven PK? What methods are available to determine PK and how are these being applied? What is known about the presence and influence of transporters on the fate of drugs in the lung? The major advantage of using inhaled medicines to target the lungs is the elevated drug concentration that can be achieved locally in the target tissues after administration via this route [79]. In studying pulmonary PK, the goal is to understand the temporal relationship between the delivered dose, the drug concentration at the sites of action within or outside the lungs, and the drug concentration in easily accessible reference fluids such as samples from the systemic circulation. However, the respiratory tract consists of several anatomically different regions that may, or may not

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constitute target regions for inhaled medications. These regions are difficult to target selectively with inhalation therapies, so most often nontarget tissues are exposed to drug in parallel with target tissues. The link between PK and therapeutic activity is considered in Section 5. Measuring pulmonary PK is complicated by the lack of easily accessible fluids for direct or surrogate measurement of the concentration of active substance in target tissues of the lung (see Section 5.2). If drug concentration is sampled in systemic blood, this is a compartment that is downstream of both target and non-target respiratory tissue. In addition, blood samples are generally taken from the venous side of the circulation, which means that drug has been exposed to interactions in other organs and the peripheral capillary bed since leaving the lungs. 4.1. Intrinsic and formulation-driven pharmacokinetics (PK) For inhalation therapies, both the intrinsic PK and formulationdriven PK are of interest. Intrinsic PK is the temporal disposition of the active compound following inhalation to the lung as determined by the inherent molecular characteristics of the drug. For example, the relationships between the absorption of drugs from the lung and their molecular weight, log P or polar surface area [80,81]. Formulation-driven PK is the influence of the formulation in modifying the intrinsic PK; often through controlling the liberation of the active compounds from a carrier (e.g., a solid particle or other drug reservoir). The latter implies the ability of the formulation to alter drug pharmacokinetic parameters, such as time to maximum drug concentration (Tmax), the maximum drug concentration, (Cmax), and the half life (t½). Formulation effects generally have a greater influence on over-all pulmonary kinetics for lower solubility drugs than for higher solubility drugs [82,83]. The dependency of PK on formulation might be seen, for example, as differences resulting from inhalation of a nebulised solution versus a dry powder formulation or between amorphous versus crystalline powders [93]. In addition, with all aerosol drug delivery procedures the formulation has a critical indirect influence on PK by delivering the carrier vehicles to different regions of the lungs according to their aerodynamic properties. This may alter PK as absorption rate, clearance mechanisms and metabolic capacity vary between different regions of the lung [84]. 4.2. Experimental models for PK studies There are a number of different models for studying pulmonary kinetics non-clinically including cell cultures, ex vivo organs and in vivo models (these have recently been reviewed [85,86]. Cell culture methods have a number of advantages for studying drug permeability and metabolism, particularly for mechanistic investigation [87]. However, the application of cell culture systems to study PK parameters in routine drug discovery and development is currently limited. Cell models have been used to rank different inhaled drugs according to their absorption properties [88], and they are beginning to be adapted to investigate particle-cell interactions and the influence of formulation on PK [89–91]. In the future a primary use of in vitro methods may be to provide rate constants for use in physiologically based PK models which are beginning to be developed to predict the in vivo local fate of soluble inhalants in the lungs [92]. In vivo, pulmonary PK would ideally be studied separately in each anatomical region and preferably with the dissolution and absorption phase studied separately from distribution and elimination. In most studies this cannot be achieved; in reality the anatomical regions are exposed in parallel, and most often absorption to the systemic circulation must be measured on the venous side. Rodents are the smallest animals used for in vivo studies; a PK profile may be obtained from a single rat, although their lungs are too small to permit regional exposures. In addition, most often rodents are exposed via nose-only inhalation, which gives a substantial deposition of drug in the non-

target region of the nasal airways. It is possible to use intubation to bypass the nasal airways and increase substantially the pulmonary targeting, however, most often these exposures are done by solution or suspension instillation instead of inhalation. Instillation gives a more patchy and central deposition of materials than aerosolisation and inhalation [94]. Regional exposures of the respiratory tract require larger lungs such as those of the pig, the dog or humans. In larger lungs the major regions can be targeted either by using differently sized aerosols of narrow size distribution [94], or by using a so called bolus technique to direct certain volume elements of air containing aerosol to controlled depths in the lungs [95]. With larger animals compared to rodents, more and larger blood samples are easily obtained and it is possible, although rare, to use protocols for direct sampling of blood on the arterial side of the circulation, mirroring the direct efflux of blood from the lungs downstream of the pulmonary circulation. In the ideal case where both sides of the systemic circulation are simultaneously sampled, the kinetics of absorption of inhaled solutes from the peripheral lung can be quantified by measuring the net increase in blood concentration upon passage of the lungs [96–98]. In human studies, for the most part repeated blood samples from the venous side with calculation of the AUC, Cmax and Tmax must be used as surrogate indices for pulmonary absorption [99]. The isolated, ventilated, and perfused lung (IPL) is a model that is particularly suitable for studying the lung-specific absorption of soluble substances following luminal administration. The lungs of small animals, most commonly the rat, are attached and ventilated via a tracheal catheter. The pulmonary circulation is perfused with an albuminphosphate buffer in either single-pass or recirculation mode [100]. The kinetics of pulmonary absorption of both small molecules and macromolecules can be studied in detail by repeatedly sampling perfusate following the intratracheal administration of substance, and is particularly relevant if techniques are used to administer test substance as fully respirable aerosols under normal ventilation [101,102]. Furthermore, ease of access to the lungs after a perfusion period allows accurate mass balance (i.e. recovery) of the originally administered dose. It is evident, therefore, that the IPL can be used to study the relationship between absorption rates and the physicochemical properties of inhaled drugs and solutes [6,102,103]. Fewer studies are available on vehicle- or formulation effects of drugs on absorption. However, a detailed study on a low-solubility organic toxicant indicated a profound effect of substance load on carrier particles on the overall pulmonary absorption rate and metabolism [104]. The uses of the IPL extend beyond drug disposition to the study of pharmacodynamic effects, such as bronchoconstriction [105] and vasoconstriction [106,107]. When used for these purposes the IPL provides an opportunity to study the relationship between PK and pharmacodynamics (PD; see Section 5), albeit with the caveat that the duration of experiments is limited and the isolation of the lung will affect certain functions. 4.3. The influence of drug transporters By influencing the distribution of drugs into cells and tissues, transporters expressed in cell membranes may be important determinants of drug disposition, efficacy and toxicity [108–110]. Transporters of the solute carrier family (SLC) facilitate the transfer of compounds across the plasma membrane. Commonly, these transporters mediate the uptake of drugs into cells but, depending on the driving force for the transport, some SLCs will also transport compounds in the other direction. Common drug transporting SLCs are organic cation transporters (OCT and OCTN/SLC22A family) and organic anion transporters (OAT/SLC22A family and OATP/SLCO family). Drug transporters of the ATP binding cassette family (ABC) transport compounds out of cells in an energy-dependent process driven by ATP hydrolysis. Examples of ABC transporters are the multidrug resistance proteins (MDR/ABCB), multidrug resistance

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associated proteins (MRP/ABCC) and breast cancer resistance protein (BCRP/ABCG2). Together with passive transmembrane diffusion, transporters of the SLC/SLCO and ABC families work in concert to regulate drug distribution and are key determinants of drug absorption and clearance [111]. The impact of drug transporters is most significant for compounds with low passive permeability as such molecules will be more dependent on an active transport process for significant passage across cellular membranes. Recent progress in the transporter area has resulted in an enhanced assessment of drug transporters in drug discovery and development. A review of clinically important drug transporters and recommendations of experimental studies and data interpretation has recently been published [112]. Current activity in the pharmaceutical industry is focused on transporters in the intestinal epithelium, hepatocytes, kidney proximal tubule cells and blood-brain barrier and their role in absorption, elimination and distribution of drugs as well as drug–drug interactions. Despite the rapidly expanding literature on drug transporters, relatively limited data is available on the functional role of transporters in the lung and their relevance for pulmonary PK is currently unclear [113,114]. Potentially, transporters in the lung epithelium may influence the absorption of drugs from airway to blood circulation, residence times of drugs in the lung, the intracellular concentration of drugs in pulmonary cells and consequently pharmacological efficacy as well as toxicity. In common with other organs in the body, many transporters of the SLC/SLCO and ABC families are expressed in the lung and comprehensive reviews in this area have recently been published [113,114]. Among the SLC/SLCO carriers, the organic cation transporters OCT1-3 and OCTN1-2 are expressed and immunohistochemistry has indicated a localisation on the luminal side of pulmonary epithelial cells [115–117]. Expression of OATPs has been observed on the mRNA level [115], but data on protein expression and cellular distribution in the lung is still lacking. In terms of ABC transporters, Pglycoprotein (MDR1), BCRP and a range of MRPs are expressed in the lung (reviewed by [113,114,118]). P-glycoprotein and BCRP are localised to the apical side of airway epithelium, whereas MRP1 has been found on the basolateral side. In general, P-glycoprotein is expressed to a lesser degree in lung compared to other barriers, e.g. the intestine [119]. However, since the lung is a highly heterogenous organ with a variety of different cell types, the expression may still be of functional importance in specific parts of the lung. There is a need to identify transporter distribution in different regions of the lung, between different cell types and at the subcellular level. There have been very few studies published to date on the functional impact of drug transporters on lung PK (reviewed in: [113,114]). Data from cell models and isolated perfused lung are emerging, but there is yet very little in vivo evidence. Any inhaled drugs with a relatively low passive permeability are potentially susceptible to an influence of drug transporters on their absorption and distribution into sub-compartments of the lung. Studies by Horvath and co-workers have indicated that the organic cation transporters are of importance for the transport of β-agonists in airway smooth muscle cells [120] and for a variety of substrates in lung epithelial cells [117]. Similarly, Nakamura and coworkers [121] demonstrated that uptake of ipratropium, an anticholinergic drug, by human bronchial epithelial cells was largely mediated by OCTNs. Several studies exploring the functional role of P-glycoprotein in lung have been published, although these report conflicting data. The absorption of Rhodamine, a fluorescent P-gp substrate, was inhibited by P-gp inhibitors in the isolated perfused lung [122] whereas another P-gp substrate, digoxin, was not affected in vitro [123] or in vivo [124]. The absorption of the p-glycoprotein substrates has also been reported in vivo [6]. In summary, the functional impact of transporters on the PK of small molecular weight drugs is still largely unknown. There is a clear need to extend our knowledge before consideration of transporters can be factored into respiratory drug discovery and development

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programmes. The functional activity of drug transporters may be studied in cellular systems but the impact on PK has to be assessed in a more complex system. Since specific transporter inhibitors are still lacking, it is a challenge to study the functional role in vivo in the presence of confounding factors. Transgenic animals have become more accessible and may be one useful tool to increase our understanding in this area. The isolated perfused rat lung may be another useful model as it offers the possibility of controlling the administration and sampling of the drug. 4.4. Current research activity An important goal of current research is to gain a better understanding of the basis for bioequivalence of inhaled medications [49]. Compared with other routes of exposure there are more complex relationships between the delivered dose and the therapeutic effect. Because the exposed target tissue is located upstream of the systemic circulation, most often parallel to exposed non-target tissues, there is poor correlation between the easily accessible drug concentrations of the systemic circulation and target-tissue concentration and ultimately therapeutic effect. A better understanding of the functional role of drug transporters on drug distribution in the lung is required as there is only assorted and sometimes inconsistent functional data available. In the absence of a demonstrated functional impact of drug transporters on drug disposition in the lung, academia rather than industry is currently taking the lead in research in this area. In future an understanding of the presence and function of transporters in the lung may become essential knowledge if the PK of inhaled drugs is to be interpreted rationally. 5. Pharmacokinetic-pharmacodynamic relationships in the lung 5.1. Pharmacodynamics (PD) in the lungs The previous section considered inhaled medicines with regard to the factors affecting PK; i.e. the quantitative and temporal relationship between administered drug dose and drug concentration measured in a distinct biological matrix. Whereas PK measures the fate of an inhaled drug molecule, it is PD that defines the quantitative relationship between drug concentration and pharmacological response. Linking PK/ PD quantitatively affords an understanding of the relationship between drug dose and the time of onset, intensity and duration of response. PK/ PD methodology has had a significant impact in pharmaceutical drug discovery and development [125], benefiting experimental design and informing decisions on target validation, lead identification/optimisation and efficacy/safety. Most typically, PK/PD relationships are established with drug concentrations determined in blood or bloodrelated matrices (serum or plasma), with assumptions that equilibrium will be achieved between free drug in, for example, plasma and free drug in the biophase of the pharmacological receptor. As noted above (Section 4), however, the use of blood-related matrices may not be appropriate for local effects following inhaled drug administration. There is a paucity of quantitative PK/PD data for studies involving inhaled drugs and local pulmonary response. This may be attributed to the relatively small size of the inhaled pharmaceuticals sector with few first-in-class molecules and the questionable value of incorporating systemic drug concentrations, such as plasma data, into the PK/PD analysis for locally acting inhaled therapeutics. For progress in pulmonary PK/PD, a better understanding of the benefits and limitations of measuring drug concentrations in lung is required to guide developments in experimental methodology. 5.2. Measuring pulmonary drug concentrations Harvesting lung samples for the measurement of drug concentrations is technically challenging, particularly in the clinic where

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opportunities for such sampling are more limited and not widely utilised. In the clinic, measurement of drug concentration in a bloodrelated matrix predominates to provide surrogate concentration data for clinical pulmonary PK/PD. While pivotal non-clinical lung dosing studies also focus on the measurement of drug in a blood-related matrix (thereby providing translational information for the clinic) it is increasingly routine in non-clinical studies to also measure drug concentrations in the lung tissue itself. Typically such measurements have served to assess Deposited Dose or the retention of drug in the lung (Section 2.3) or in toxicokinetic studies (Section 3.4) to gain a measure of organ exposure to the drug to aid safety profiling. However, a variety of lung sampling methods exist and provide an opportunity to relate time profiles to lung and plasma drug concentration and begin to address some of challenges of pulmonary PK/PD analysis. The lung sampling methods are described in more detail below. 5.2.1. Lung tissue homogenates Whole lung tissue is readily collected in the non-clinical setting and when done so immediately at the end of an intratracheal or inhaled drug administration it can provide an estimate of the Deposited Dose (Table 1). A caveat with this technique is that rapid absorption during the dosing and sampling period results in loss of drug from the lung, reducing the accuracy of dose determination. By combining lung dose with sacrifice of animals at various time points after dosing, however, an assessment can be made of the drug dose (concentration) remaining within the lung as a function of time. Acquiring such pulmonary drug concentration-time data may be particularly judicious if the tissue is used simultaneously for PD evaluation, e.g. levels of inflammatory markers. This approach is limited, however, by the lack of precision of homogenate data regarding the localisation of drug in the lung as is described below. Processing of lung tissue for drug analysis involves homogenisation to complete destruction, i.e. the formation of a uniformly dispersed suspension of macerated or crushed tissue in an aqueous diluent. This often involves the indiscriminate use of the whole lung tissue or various components thereof with data expressed as averaged drug concentration for the entire lung. However, average drug concentrations across the tissue may be difficult to relate to levels at a particular effector site within the lung. Even at a gross level, variation can be expected in drug concentrations between the individual lung lobes, arising through variability in lung deposition and the recognition that lobular deposition is not strictly proportional to lobular mass. While measurements incorporating information on individual lobes can be useful, this may still not account for localised concentrations of drug critical for driving pharmacological response. In acknowledging that pharmacological activity is more closely related to free drug concentration than total concentration, lung tissue homogenates can be subjected to equilibrium dialysis or ultrafiltration techniques to assess free drug fraction. An example of this approach is that of Wu and co-workers [126] using equilibrium dialysis of rat plasma and lung tissue homogenates to study the effects of the different plasma and tissue binding profiles displayed by synthetic glucocorticoids upon their occupancy of tissue glucocorticoid receptors. Following intravenously infused des-ciclesonide and budesonide, total drug concentrations in lung and plasma were reported to be comparable whereas the free concentration of budesonide in both lung and plasma was seven-fold greater than the free concentrations of des-ciclesonide; the greater free concentration for budesonide correlated with greater lung glucocorticoid receptor occupancy. The above example emphasises the critical importance of measuring free drug concentrations. However, the use of the equilibrium dialysis or ultra-filtration is not standard practice in PK/ PD investigations and indeed the measurement of free drug fraction in tissue homogenates may actually lead to erroneous estimates of the

true free concentration within the intact lung. For example, mechanical disruption and aqueous dilution of the tissue can release drug from low affinity binding sites, it will also disrupt the normal compartmentalisation of cellular proteins and indeed compartmentalised drug (e.g. drug entrapped within endosomal compartments). Similarly, homogenisation of lung tissue will release drug into solution that in the intact lung may remain undissolved, e.g. drug in suspension or dry powder dosage formulations. Indeed, the establishment of pulmonary PK/PD relationships for low solubility drugs is all the more challenging. In summary, while lung tissue can be readily collected in the non-clinical setting there exists very little published data on the use of lung tissue homogenates in PK/PD experimentation and there remains uncertainty on how best to exploit this biological matrix. 5.2.2. Bronchoscopic tissue biopsy Bronchoscopy involves the insertion into the airways of either a rigid bronchoscope or more commonly a flexible fibreoptic bronchoscope. Beyond tumor diagnosis, bronchoscopic tissue biopsy (either endobronchial or transbronchial biopsy) can be indicated in the diagnosis of infectious or immunological lung disease which may also involve a diffuse infiltrative component. Beyond tissue biopsy the technique is also amenable to obtaining bronchial epithelial cell brushings. The nature of the procedure means that any sampling for drug concentration analysis (e.g. for antibiotics, steroids, and immunosuppressants) is secondary to pathology needs. While a number of intra-patient samples can be taken at any given time, the technique will generally yield individual patient data for a single time point only. Many of the issues raised above for tissue homogenates will also be relevant to tissue biopsy samples. Bronchoscopic tissue biopsy has been applied to the study of antibiotic penetration and drug concentrations in the lungs of patients undergoing a diagnostic procedure [127,128]. 5.2.3. Microdialysis Microdialysis involves the insertion of a probe directly into a tissue for the continuous sampling of an unbound analyte such as a drug. The method depends upon a microdialysis probe tip consisting of a tubelike or capillary-like semi-permeable synthetic membrane, the core of which is continuously perfused with physiological solution. The semipermeable membrane restricts the passage of proteins and hence protein-bound drug but affords the movement of free low molecular weight drug. Once inserted into the tissue the unbound drug will move across the semi-permeable membrane by passive diffusion and the continually flowing dialysate in the probe will be recycled for bioanalysis. The method is applicable to both animal and human experimentation [129]. Microdialysis is not without its limitations. It is an invasive technique than can disrupt normal tissue architecture at the site of probe placement. Such tissue damage may lead to vascular leakage and an inflammatory response both of which may require a recovery period after probe implantation prior to experimental sampling. Due to the low dialysate flow rates in the probe the collection times to obtain a single sample for bioanalysis can be quite protracted (15–30 min). Such a relatively long collection interval may impact on studies where free drug concentrations in the tissue are changing rapidly. The technique may also suffer from variable and low recovery of the free drug into the probe which appears to be especially the case when the probe is inserted into an intact tissue, i.e. the free drug concentration in the tissue maybe much higher than in the microdialysis probe. This variable recovery may also be a feature of the physico-chemical nature of the analyte itself, with concerns that a number of lipophilic drugs are particularly poorly recovered [130] while others are not [131].

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An example of the use of microdialysis in the non-clinical setting is the examination of the penetration of the antibiotic, cefclor, into skeletal muscle and into lung tissue following intravenous administration in the rat [132]. Steady-state or equilibrium free concentrations of cefclor in skeletal muscle and lung while similar were only 26% of the free drug concentrations in plasma. Such findings have implications for PK/PD modelling of antibiotic treatment in interstitial lung infections. The same laboratory reported similar findings for cefpodoxime prior to conducting a clinical microdialysis study using skeletal muscle (where probe insertion is less hazardous) as a tissue surrogate for lung [133]. Indeed clinical microdialysis appears to be particularly used for drug studies of anti-infective agents [134], including human lung infections [135]. 5.2.4. Epithelial lining fluid Epithelial lining fluid (ELF) lies between the lung epithelial surface and the gas in the lung lumen. The volume and composition of the ELF will depend upon ion and water transport across the epithelium, passive liquid flow, evaporative water loss, secretions of glandular and goblet cells, and mucociliary transport. Albumin and other proteins are present in ELF but at much lower levels than in serum, e.g. despite the varied literature data all estimates of ELF albumin levels conclude it is ≤ 10% that of albumin concentration in serum [136,137]. However, the volume and composition of ELF can change with pulmonary disease. Compared to blood levels, drug concentrations in ELF may be more reflective of drug concentrations (total/free) in lung interstitium and are more likely to relate directly to PD targets within the ELF itself such as bacterial lung infection. Clinically, broncho-alveolar lavage (BAL) is used as a diagnostic technique to sample cells, proteins, and other constituents of the ELF. It usually involves consecutive instillations (three to six) of sterile saline (each 20 to 50 ml in volume) through a fibre-optic bronchoscope into a sub-segmental bronchus followed by immediate fluid retrieval by aspiration. While a standard inter-laboratory method for the non-clinical collection of ELF has not been formalised, general consensus at the Workshop indicated that the BAL procedure in the rat typically involves the tracheal instillation and subsequent recovery of three consecutive 5 ml volumes of aqueous solvent. The use of a 5 ml volume for each instillation implies that the significant majority of rat lung airways and alveolar sacs should be exposed to lavage fluid. Among non-clinical scientists there was some debate as to the nature of the solvent that should be used in rodent BAL, i.e. normal saline or phosphate-buffered saline, and while some anecdotal information (unpublished) may suggest that this choice could affect the recovery of pulmonary macrophages this is not broadly acknowledged. At the non-clinical level, BAL appears to be used increasingly as a routine component of pulmonary PK and PD investigations. It provides for the gathering of PD endpoints, e.g. immune cells infiltrates into the lung lumen and products of inflammation, while simultaneously affording the opportunity to estimate drug concentration in LF for PK/PD purposes. One of the problems in undertaking BAL, whether non-clinically or clinically, is that it represents a dilution of what is essentially an unknown volume of ELF; it does not allow for the direct measurement of drug concentration in ELF alone. This is compounded by the technical limitations in not being able to recover the full volume of instilled lavage fluid, and as a corollary only partial recovery of ELF. Therefore for meaningful estimates of drug concentration in ELF there is a need to determine by how much the ELF has been diluted by the BAL procedure. This is most commonly addressed by the simultaneous measurement of endogenous levels of urea in the retrieved BAL fluid and in serum. While not the only dilution indicator to be used, endogenous urea relies conveniently on its similar concentrations in both ELF and in serum; a urea concentration ratio (BAL fluid:serum) of less than unity indicates the dilution factor that should be applied to interpret ELF drug concentrations. Nevertheless, such methodology is

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not universally applied in non-clinical BAL studies even when they seek to address drug concentrations in ELF. Uncertainties do exist in the interpretation of BAL solute concentration data [137], which from a pharmaceutical perspective may limit the value of the technique in decision-making. For example, a study into the fluid dynamics during clinical bronchoalveolar lavage, using tritiated water, estimated that the ELF normally resident in the lung segment contributed no more than 2% to the total aspirated BAL volume and that approximately 40% of BAL fluid volume had come from the systemic circulation or surrounding interstitium [138]. The implication of the latter is that significant drug transfer into the lung from serum or tissue interstitium may lead to overestimation of ELF drug concentration. The impact of the BAL procedure upon diffusion of serum urea into the aspirated BAL fluid has been reported [139,140]. Variations in the BAL fluid volume instilled, the number of instillations and the contact “dwell” time of the BAL fluid with lung epithelium can all affect the diffusion of serum urea into the lung with the potential for overestimation of the volume of ELF recovered and inappropriate dilution calculations. While levels of serum proteins are significantly lower in ELF, their impact upon pulmonary PD through influencing free drug fraction still needs to be considered particularly for high protein bound drugs. The cellular component of ELF is often forgotten in PK studies and lysis of, for example, pulmonary macrophages during the BAL procedure may artificially elevate apparent free drug concentrations in ELF. From a drug delivery perspective, BAL fluid may solubilise drug from suspension or dry powder formulations that in the ELF was undissolved. BAL measurements bring technical challenges and the data are difficult to interpret. However, BAL fluid is obtainable both non-clinically and clinically and should prove to be a useful translational methodology for PK and PK/PD investigations. BAL does not allow for the direct measurement of drug concentration in ELF. Bronchoscopic microsampling (BMS) is a technique for sampling of ELF directly and repeatedly on the surface of a bronchus by using a polyester fibre probe. The technique has been exploited most frequently in measuring bronchial ELF concentrationtime profiles for systemically administered antibiotics [141–145] and determining the duration that ELF drug concentrations N minimum inhibitory drug concentration. 5.2.5. Induced sputum Although BAL represents a minimal risk procedure for patients it is invasive. Induced sputum has been widely used to investigate airway inflammatory disease and assess interstitial lung disease [146–149]. Sputum is induced by inhalation of hypertonic (4 to 5% w/v) saline solution delivered by ultrasonic nebuliser over variable periods of time. Expectoration of sputum is encouraged by cough and is generally attempted after a cumulative period of approximately 20 min saline inhalation. It can be repeated to collect more than one time-point. Induced sputum has been best utilised when looking at the cellular inflitrates of the more proximal airway ELF. While its use for PD endpoints is established, the suitability of induced sputum for assessing ELF drug concentrations in PK/PD studies has not been tested. 5.2.6. Imaging Imaging techniques can be used to evaluate dose deposition in the lung following instilled or inhaled pulmonary delivery. The imaging modalities available include gamma scintigraphy, a two-dimensional (2D) technique, and single photon emission computed tomography (SPECT) and positron emission tomography (PET), both threedimensional (3D) techniques. These techniques provide information on the amount of drug delivered to the whole lungs and the distribution of the drug within the airways [150–155]. In the case of gamma scintigraphy the main, but not only, gamma radiation-emitting isotope that is used is 99mTechnetium (99mTc) which can be incorporated into the pulmonary formulation, e.g.

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liposomes [156] or added to the formulation such that it forms a close physical (non-covalent) association with the drug particles allowing it to act as a marker for the drug. The latter approach is technically more demanding and requires careful validation [152]. Gamma scintigraphy has proved a valuable comparative technique for investigating pulmonary delivery, affording assessment of the extent and pattern (e.g. central versus peripheral) of lung deposition. However, the two-dimensional planar images obtained with gamma scintigraphy do not account for the three-dimensional nature of the lung tissue and the drug deposition profile in the two-dimensional images includes an overlay of structures of interest (alveoli, small and large airways). Precise and quantitative measurements of these individual regions are more difficult [157]. Gamma emitting isotopes are also used in SPECT, a technique in which the gamma camera is rotated through 360° and affords the reconstruction of a three-dimensional tomographic image. This allows a more complete profile of penetration through the lung compared to planar techniques [158,159] and is able to differentiate better between small and large airways. Regional deposition data may be expressed either as sections through the lungs in transverse, coronal, and sagittal planes, as a series of lung shaped concentric shells, or as the amount of drug in different airway generations. However, SPECT is more technically and computationally demanding than gamma scintigraphy [160]. Both planar gamma scintigraphy and SPECT are more widely used in clinical studies but both have also shown nonclinical utility, including use in models of lung disease [161]. PET produces a three-dimensional image from radiation emitted by short-half-life positron-emitting radionuclides, e.g. carbon-11, fluorine-18. Drugs can be radiolabelled and biodistribution scans obtained, including clinical examples of the use of PET in pulmonary deposition studies of inhaled drugs [162–164]. PET can also be used for non-clinical investigations [165,166] using small animal imaging scanners. PET imaging has even been used as a non-invasive biomarker to assess response in tuberculosis-infected lungs to treatment with systemically administered antibiotics [167]. 5.3. Pulmonary PD endpoints There is a general consensus that appropriate PD endpoints that have relevance to the progression of clinical disease, and which provide useful decision-making data, can be explored in non-clinical studies. The non-clinical PD endpoints of drug action that have found most utility include those that quantify pulmonary inflammation and airway hyper-reactivity/broncho-constrictiveness. BAL provides the opportunity to sample for inflammatory cytokines and leucocyte infiltration and can be incorporated within studies quantifying realtime broncho-constrictive responsiveness to challenge [168]. The potential also exists to use imaging approaches [169] for some of the cell-based inflammation PD endpoints. It is clear, however, that an improved understanding is needed of the disease pathology in acute and chronic non-clinical models and how this pathology relates to the clinical condition. This should lead to improved biomarkers that will enhance the translation of non-clinical data to the human disease, an objective which will also be facilitated by quantitative PK/PD modelling for inhaled drugs that act locally to bring about pulmonary responses. 5.4. PK/PD for locally acting inhaled drugs For drugs that access their respective pharmacological target following systemic exposure, the use of plasma concentration data can generally provide for appropriate and predictive PK/PD relationships. However, the value of incorporating systemic drug concentrations into the PK/PD analysis for locally acting inhaled therapeutics is more questionable [169]. Plasma is ‘downstream’ relative to the lung (following inhaled delivery) and inference of local ‘effect-site’

concentration from systemic drug concentration data is complicated by a number of factors — including multiphasic absorption processes (due to depot effects or differences in absorption rate from different regions of the lung), the presence of non-absorptive lung clearance processes and the fact that the swallowed component of the orally inhaled dose may contribute to systemic exposure (if the drug is orally bioavailable). In short, for inhaled drug delivery there is a clear deficiency in understanding of the relationship between drug concentrations in the lung and drug concentrations in plasma, a position further compounded by the complexity of the lung architecture, variable drug deposition patterns following inhaled delivery (Section 2) and the difficulty in sampling and interpreting drug concentrations in the lung. The problem is compounded by an almost complete lack of data examining local drug concentrations in the lung following pulmonary drug delivery and seeking to relate such concentrations to a measured pulmonary response. In contrast there is a body of work for systemically administered anti-infectives studying the relationship between blood levels of drug or area under the blood drug concentration time curve (AUC) or drug dose administered upon the clearance of lung infection; this includes non-clinical [170–172] and clinical examples [173]. Similarly there are examples of the clinical measurement of drug concentrations in ELF (see Section 5.2.4) following the oral or intravenous administration of anti-infectives; these related ELF drug concentrations to minimum inhibitory concentrations (MIC) for bacteriocidal activity [174–176] or to improved FEV in cystic fibrosis patients [177]. The pulmonary delivery of liposomal encapsulated ciprofloxacin in a rat model of pneumonia has recently been reported with drug levels determined in ELF, alveolar macrophages and in serum [178,179]. These levels were related to MIC necessary to kill a variety of bacterium, including intracellular organisms such M. tuberculosis. One aspect of local lung delivery which has received attention in review articles is that of the PK and PD considerations relating to inhaled corticosteroids, with notable contributions from the groups of Derendorf and Hochaus [180–185]. While the qualitative consideration of these issues is important there is still lacking, however, substantive quantitative relationships between the PK and PD of glucocorticoids although a useful technique for glucocorticoid PK/PD is the surrogate endpoint of ex-vivo pharmacological receptor occupancy (see also Section 5.2.1.). Measurement of ex-vivo glucocorticoid receptor binding has been used to assess the selectivity of lung targeting after pulmonary delivery [186] and the relationship between free drug concentrations in the lung and pulmonary glucocorticoid receptor binding [126]. This technique when applied to other receptors, e.g. muscarinic, in the lung could be expected to have greater utility in the development of quantitative PK/PD relationships. For example, such data may help develop cross-species prediction of the relationship between pulmonary dose and receptor occupancy. The prediction of systemic and pulmonary PK across species would enable input of forecasts into PD measures and will contribute to the further understanding and development of quantitative pulmonary PK/PD relationships; it is an important approach which can parallel laboratory or clinical experimentation. PK scaling between species by either allometric or physiological approaches is a mature discipline. There is confidence among non-clinical scientists that inter-species modelling of systemic blood levels following pulmonary drug administration can be achieved with a reasonable degree of confidence. Predicting human lung drug concentrations from nonclinical data may be more challenging but examples of the successful prediction of human drug concentrations in lung tissue from nonclinical species, albeit unpublished, are reported to exist in industry. With non-clinical PK/PD relationships established then predictions of human effect- versus time relationships can be tested. There are two recent published examples of predicting elements of the pulmonary

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PK/PD response [187,188]. Goutelle and co-workers [187] undertook population pharmacokinetic modelling and Monte Carlo simulation to explore the pulmonary PK and PD of rifampin. They found their modelling to describe well the actual measured rifampin concentrations in the plasma and ELF in a population in human volunteers. Based upon Cmax:MIC and AUC24 h:MIC ratios their work supported the need to evaluate higher doses of rifampin for the treatment of patients with tuberculosis. Agoram and co-workers [188] proposed a solution to interpreting in-vivo PD for a series of six inhaled longacting β2-agonists (LABAs) in the absence of PK data. Using a nonclinical model they established that the relationship between observed maximum effect and area under the effect-time curve for different doses of the LABAs could be described by a common sigmoidal relationship. They concluded for that particular series of molecules the in-vivo duration of action was dependent upon potency and not pulmonary PK differences; a modelling approach that aided the allocation of resources for lead candidate selection. Clearly such a method, or variants of it, if more generally applicable would rely upon precedented mechanisms of action within a class of drug molecule. In summary, quantitative PK/PD approaches involving inhaled drug and local pulmonary response are very much under-evidenced and a general uncertainty still exists regarding how best to use lung tissue data for PK/PD modelling. This current level of understanding and application needs to be addressed if the pharmaceutical industry is to avoid high attrition in the development of inhaled drugs that target novel mechanisms of actions. 6. Challenges and opportunities The factors needed to produce oral compounds with more druglike properties are defined by Lipinski's ‘rule of five’ [189,190]. While the lung may be a more complex organ, is it possible to pool data on the physical and chemical properties for inhaled delivery and generate a model that similarly reduces the attrition in development for inhaled compounds? Selecting the right molecule is critical and, although formulation plays a part, the chemical structure ultimately determines the fate of the molecule, so as much information should be gathered as early as possible prior to engaging in long and costly

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animal studies. Pharmaceutical companies generate large amounts of data, much of which is archived or may be forgotten when molecules fail during the discovery or development phases. This information may be of limited value within any one company, but combined between companies (while avoiding potential IP pitfalls) such data could constitute a large reservoir of shared information to aid drug design, PK, dosimetry paradigms, toxicology outcomes and appropriate methodologies. In fact, it can be argued that common value could far outweigh the risks of pooling such information and would benefit all by reducing the need to generate additional internal data. There is a paucity of published data on the dosimetry of inhaled drugs compared with the wealth of data available for environmental particulates. If the industry wishes to demonstrate clearly the true nature of novel inhaled medicines, then current practices will need to advance considerably in order to understand how the drug behaves once it has been administered to the lung. Here resides yet another opportunity to establish the facts collectively to build a better understanding of how drug concentrations are attained in the lungs and how this affects drug action. In toxicology studies, drug attrition remains high across the industry for many different reasons including, inter alia: molecular characteristics, unexpected toxicologies and pulmonary accumulation. A collective understanding of the factors that preclude successful “developability” would benefit the entire industry and avoid the escalating costs of drug development. Again, opportunities to share common cross-species control data in inhalation toxicology studies, to understand common biological responses to inhaled material (e.g. the development of foamy macrophages in the alveoli of non-clinical species) and adoption of common approaches to recording toxicological findings (with a common terminology) would all aid progression. This may also apply to the development of in vitro systems for early screening of common phenomena such as phospholipidosis — this would eliminate those molecules with a limited chance of successfully transitioning the development cycle. Developing new respiratory medicines remains challenging at many levels. Estimating the duration of action is an area that requires further investment. Understanding cellular trafficking during inflammatory episodes in patients and how such phenomena can be monitored early

Table 3 Opportunities for collaborative working to enhance inhaled product development through strategic advances in inhalation science. The objectives are pre-competitive topics of common interest to innovator companies, contract research organisations, academia and regulators. Collaboration

Objective

Activity

Network

Harmonise/inform practice Develop expertise Develop an in silico model of drug deposition Improve dosimetry methods 1) Protocol development and validation for drug administration to non-clinical species 2) Protocol development and validation for measuring drug deposition/accumulation in the lung Benchmark normal variation for toxicology studies

Scientific meetings and dialogue with regulators Collaborative research projects Pool data for the development of an in silico model of drug deposition Compare drug distribution in the lungs of non-clinical species (e.g. fluorescent beads, etc.) after different administration techniques. Develop evidence-based protocols for how best to measure real deposited doses after administration of pharmaceutical aerosols and how best to assess lung accumulation of poorly soluble drug compounds. Sharing of toxicological control data to establish normal biological variation and normal responses to inhalation exposure Pooling of data to link physicochemical properties and inhaled pharmacokinetic profiles Use of imaging to assess dosimetry, impact of delivery devices on dosimetry, in vitro-in vivo correlation. The aim will be to harmonise dose estimations. Characterisation of lung fluids, development of simulated fluids and assays to establish solubility and dissolution parameters for modelling formulation effects on pharmacokinetics Mechanism-based research to understand the phenomena of foamy macrophage formation and the implications for safety The use of known irritants and marketed medicines to discriminate adverse from non-adverse effects Studies into mechanisms and impacts related to active drug transport (for details see the accompanying article: Gumbleton et al., 2010) Use of imaging to measure precedented molecule receptor occupancy and help understand the relevant biophase for pharmacokinetic modelling

Data sharing

Identify the physicochemical determinants of pulmonary PK Research

Profiling of lung deposition

Develop methods for drug solubility and dissolution

Understand the pathophysiology of phospholipidosis and foamy macrophages Develop and validate biomarkers Understand the impact and role of transporters in lung disposition Extend PK/PD modelling and identify target localisation

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in non-clinical models and during therapy also remains unresolved. In addition, there are difficulties in relating effects in animal models to human disease. A greater understanding of drug deposition post inhalation remains an area poorly satisfied as the imaging techniques currently available do not provide sufficient detail to allow such assessments with confidence. Clinical data suggests that particle size can influence the PD of inhaled medicines [94], yet how such findings can be exploited for more novel medicines remains unanswered. Surely all these areas cannot remain untouched and a coordinated effort across academia and industry is what is required to bring some of these challenges forward. Cost efficiencies continue to be sought internally within companies in order to reduce costs and raise productivity, but this will have a limited impact upon global success. Pooling information and sharing challenges common to the industry are more likely to be successful in the long term than the prevailing silo mentality. A shift in approach is required to bring together expertise and allow common challenges to be tackled and the benefits shared. It is hoped that the Workshop at GlaxoSmithKline in Stevenage on 10th July 2010 marked the beginning of a common understanding that a progressive approach is required to aid development of new inhaled medicines. The immediate challenge is to generate the impetus to take advantage of the opportunities for collaborative working discussed in this article (Table 3). This approach aims to work to the benefit of all; not least in expediting the development of new inhaled medicines for the patients who need them.

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