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

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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, UKb Applied Research Associates, 8537 Six Forks Road, Raleigh NC 27615, USAc AstraZeneca R & D, Bakewell Road, Loughborough LE11 5RH, UKd Karolinska Institutet, Stockholm SE-171 77, Swedene Inhalation Sciences Sweden AB, Stockholm SE-171 77, Swedenf University of Cardiff, Welsh School of Pharmacy, King Edward VII Avenue, Cardiff CF10 3NB, UKg AstraZeneca R & D, Lund SE-221-87, Swedenh Huntingdon Life Sciences, Woolley Road, Huntingdon PE28 4HS, UKi GlaxoSmithKline R & D, Gunnels Wood Road, Stevenage SG1 2NY, UKj Pfizer R & D, Sandwich, Kent CT13 9NJ, UKk Novartis Horsham Research Centre, Wimblehurst Road, Horsham, RH12 5AB, UKl SciLucent LLC, 585 Grove Street, Herndon, VA 20170, USA

☆ This article is based upon an international workshopthe Drugs in the Lungs Network. The meeting aimed to ipresentations, discussions and the consensus achieved amore detailed perspective on the topics discussed and c⁎ Corresponding author.

E-mail address: [email protected] (B. Forbes).

0169-409X/$ – see front matter © 2010 Elsevier B.V. Adoi:10.1016/j.addr.2010.11.004

a b s t r a c t
a r t i c l e i n f o
Article history:Received 5 October 2010Accepted 25 November 2010Available online 6 December 2010

Keywords:Aerosol dosimetryDepositionInhalation toxicologyADMEIsolated perfused lungTransporterPharmacokineticsPharmacodynamics

Dosimetry, safety and the efficacy of drugs in the lungs are critical factors in the development of inhaledmedicines. This article considers the challenges in each of these areas with reference to current industrypractices for developing inhaled products, and suggests collaborative scientific approaches to address thesechallenges. The portfolio of molecules requiring delivery by inhalation has expanded rapidly to include noveldrugs for lung disease, combination therapies, biopharmaceuticals and candidates for systemic delivery viathe lung. For these drugs to be developed as inhaled medicines, a better understanding of their fate in thelungs and how this might be modified is required. Harmonised approaches based on ‘best practice’ areadvocated for dosimetry and safety studies; this would provide coherent data to help product developers andregulatory agencies differentiate new inhaled drug products. To date, there are limited reports describing fulltemporal relationships between pharmacokinetic (PK) and pharmacodynamic (PD) measurements. A betterunderstanding of pulmonary PK and PK/PD relationships would help mitigate the risk of not engagingsuccessfully or persistently with the drug target as well as identifying the potential for drug accumulation inthe lung or excessive systemic exposure. Recommendations are made for (i) better industry-academia-regulatory co-operation, (ii) sharing of pre-competitive data, and (iii) open innovation through collaborativeresearch in key topics such as lung deposition, drug solubility and dissolution in lung fluid, adaptive responsesin safety studies, biomarker development and validation, the role of transporters in pulmonary drugdisposition, target localisation within the lung and the determinants of local efficacy following inhaled drugadministration.

held by the Academy of Pharmaceutical Sciences Great Brdentify common challenges facing those undertaking inhre freely available on the APSGB website [1]. This articleonclusions reached at the meeting.

ll rights reserved.

© 2010 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 701.1. Drugs in the lungs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 701.2. Inhaled product development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

itain on 10 June 2010 at GlaxoSmithKline, Stevenage, UK, to launchaled product development. Details of the Workshop participants,by the meeting organisers and expert speakers aims to deliver a

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70 B. Forbes et al. / Advanced Drug Delivery Reviews 63 (2011) 69–87

1.3. Common challenges in developing inhaled medicines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 711.4. Open innovation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

2. Dosimetry in inhaled product development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 712.1. Dose calculations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 712.2. Estimating the deposited dose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 722.3. Measuring the deposited dose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 732.4. Modelling pharmaceutical aerosol deposition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 732.5. Current practice and opportunities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

3. Inhalation safety studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 753.1. Regulatory requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 753.2. Biomarkers of toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 763.3. Interpretation of adverse effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 763.4. Toxicokinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 763.5. Developments in safety science . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

4. Pulmonary drug disposition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 774.1. Intrinsic and formulation-driven pharmacokinetics (PK) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 784.2. Experimental models for PK studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 784.3. The influence of drug transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 784.4. Current research activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

5. Pharmacokinetic-pharmacodynamic relationships in the lung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 795.1. Pharmacodynamics (PD) in the lungs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 795.2. Measuring pulmonary drug concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

5.2.1. Lung tissue homogenates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 805.2.2. Bronchoscopic tissue biopsy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 805.2.3. Microdialysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 805.2.4. Epithelial lining fluid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 815.2.5. Induced sputum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 815.2.6. Imaging. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

5.3. Pulmonary PD endpoints . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 825.4. PK/PD for locally acting inhaled drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82

6. Challenges and opportunities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

1. Introduction

This article considers inhaled product development with anemphasis on dosimetry, safety and efficacy. A commentary on currentindustry practices in these areas is provided based on the experienceof the authors together with the consensus views of the APSGB Drugsin the Lungs Workshop on 10 June 2010 where these topics werediscussed in a series of structured debates [1]. Furthermore,consideration is given to the scientific developments and collabora-tive approaches required for industry to move towards a moreefficient paradigm for developing inhaled medicines.

1.1. Drugs in the lungs

The successful integration of novel drugs with devices capable ofdelivering defined doses to the respiratory tract has resulted in aproven track record for inhalation as a route of administration thatlimits systemic exposure and provides localised topical delivery. Thus,a number of orally inhaled products have been developed successfullyover the last 50 years, providing symptomatic relief to millions ofpatients with asthma and chronic obstructive pulmonary disease(COPD) [2]. Inhalation is also a proven means of systemic delivery fordrugs that have limited bioavailability by other routes or wouldbenefit from rapid onset of action and a variety of products are indevelopment for this purpose [3,4].

In recent decades, advances in device design and formulationscience have addressed the need for more efficient inhalers that arecapable of delivering larger doses to the lung with low extra-thoracic deposition [2,5]. Once deposited in the lungs, drugdisposition (dissolution, absorption, distribution, metabolism andelimination) and the influence of pulmonary pharmacokinetics (PK)on drug efficacy and safety are the critical determinants of clinicaloutcomes. Pulmonary disposition remains poorly understood de-spite modern capabilities in imaging, analytical and biological

science which make measurement of drug disposition and mode ofaction more accessible. This raises the question, how can the fate ofdrugs in the lungs be understood better to allow improvements incurrent therapy and expedite the development of new inhaledmedicines?

1.2. Inhaled product development

In the early stages of drug discovery a sound scientific case is builtto rationalise and validate a potential biological target. As a whole, theindustry is well versed and able to undertake these tasks in anefficient manner. Once a drug target has been accepted as part of awider portfolio of mechanisms, the intellectual property aroundknow-how and tractability grows. However, the development of newmedicines depends not only upon understanding the disease andtarget, but also how amenable the drug molecule is to pharmaceuticaldevelopment. Regulatory guidelines dictate well-defined non-clinicaland clinical phases of medicine development, but escalating costs,high attrition (failure to reach market) for novel therapies, poorproduct differentiation for reimbursement and generic competitionare increasingly severe challenges to bringing novel medicines tomarket.

The success stories in inhaled therapy of lung diseases arerestricted to a small number of target classes, notably β2 receptoragonists, antimuscarinic drugs and corticosteroids [6] which for β2

receptor agonists and antimuscarinic drugs are associated with themuscular bronchioles and the airways of the proximal lung. Inaddition, most inhaled therapies do not modify to any great extent theunderlying diseases, although steroids may impart some beneficialeffects. In this regard, there is considerable scope to develop novelnew medicines which seek to modify respiratory disease processesdirectly and to embrace respiratory disease areas for which therapy isinadequate or non-existent.

71B. Forbes et al. / Advanced Drug Delivery Reviews 63 (2011) 69–87

1.3. Common challenges in developing inhaled medicines

The inhaled route of delivery has always been associated with aconsiderable challenge in getting the drug to its target. The lungs are ahighly complex organ designed to filter inspired air with manydifferent cell types contributing to their function. Furthermore, thelungs may change dramatically when afflicted by disease resulting inan internal environment that works against the drug reaching andinteracting successfully with the target. For targets in the upperairways this may have lesser significance, but drug delivery to thedeep lung may be impeded by changes such as mucus hypersecretionor thickening, airway narrowing or collapse, fibrosis and poor bloodcirculation. To mitigate the risk of failing to deliver an inhaledmolecule to its site of action, a far greater understanding of the impactof disease on lung pathophysiology is required.

The health and economic burden of respiratory disease [7] not onlyprovides a hugemarket for inhaled therapy, but also invokes a need toevaluate current practices and identify ways to develop new andbetter inhaled medicines. In contrast to precedented mechanisms,future drug targets in the lung are likely to be novel and necessitatenew classes of molecules, engaging unprecedented mechanisms forwhich limited biological information is available [7–9]. For example,new approaches to disease modification will require pulmonarydelivery of biopharmaceuticals, genes and small interfering RNA. Thepharmaceutical portfolio for delivery by inhalation will be increasedfurther by the emergence of drugs for systemic delivery via inhalation[3,4] and combination therapies (9). This expansion in the numberand classes of drugs for delivery to the lungs will bring new challengesin establishing their safety and efficacy. This is likely to require newmethods and collaborative approaches to the way that industrycurrently develops inhaledmedicines, as the achievements of the pastwill be no guarantee of success in the future.

Development of best practice may require new ways of crosscompany collaboration that break current conventions. Pertinentquestions include:

• How consistent is the industry approach to developing inhaledmedicines?

• What are the molecular characteristics that make a good respiratorydrug?

• Are standardised validated methods used for drug administration innon-clinical settings?

• Do pharmaceutical companies measure similar parameters and atwhat stage in the discovery and development cycle are safety andefficacy studies conducted?

• Are toxicological data obtained and reported similarly betweencompanies and is inconsistent reporting of pathology creating amore complex picture for the industry and regulators than isnecessary?

• Do the endpoints of clinical trials show sufficient commonalityacross companies to demonstrate the true value of a newmedicine?

1.4. Open innovation

Given the considerable challenges outlined above, can pharmaceu-tical companies continue to work in isolation to develop inhaledmedicines or is it possible for cross company collaborations in a pre-competitive environment to increase the future chances of success forall? The latter will depend upon what information can aid not only theprogression of targets through the drug development pipeline, but alsoguide regulatory authorities, payers and medical practitioners to abetter understanding of the potential of a new drug. If common barriersand bottlenecks to progression are identified it may be possible to findbetter ways to develop understanding of inhaled medicines throughwider collaborations between industry, academia and contract researchorganisations. This will need to encompass areas of mutual benefit,

while maintaining intellectual property rights which so often tend tostifle innovative approaches. While intellectual property is of greatimportance to each pharmaceutical company, this need not preventwider collaboration between companies to develop such assets wherethe nature of the challenges facing the industry is common.

Pre-competitive collaborations in the pharmaceutical sciences areemerging and being advocated, for example, in the bioinformatics field[10]. In inhalation science, initiatives include the recent publication bythe Association of Inhalation Toxicologists (AIT) encouraging aharmonised data-driven approach to calculating delivered dose innon-clinical toxicology studies [11] and the recent U-BIOPRED consor-tium (unbiased markers for the prediction of respiratory diseaseoutcomes; part of the European Innovative Medicines Initiative),which is seeking to improve the diagnosis of asthma to aid bettertreatment [12,13]. In addition, the Cross Company AnimalModels group(CCAMS; incorporating pharmaceutical companies, academia andCROs) is seeking to unify models for chronic respiratory diseases andmethods used during drug discovery by gaining consensus on thosetechniques best able to predict a drug effect in vivo. Adoption of bestpracticewould allow commonmodels to be used, reduce the number ofanimals used overall and aid regulatory authorities by providingcomparable data across licensing submissions.

At a timewhen development costs are rising and payers are seekinggreater differentiation as well as value for money for new drugs, thetime of stand-alone pharmaceutical companies may be coming to anend. Sharing best practice and undertaking pre-competitive approachesmay help to reduce the pathways to developing new medicinesconsiderably. In the following sections we identify current practices,consider common challenges regarding drugs in the lungs and suggestopportunities to galvanize inhaled product development.

2. Dosimetry in inhaled product development

Accurate dosimetry is essential in studies investigating drug safetyand efficacy. At the Drugs in the Lungs Workshop the current opinionof the state-of-the-art concerningmethods of quantifying the deliveryof inhaled molecules was ascertained by considering: (i) How arenon-clinical doses calculated or measured and is this consistent acrossthe industry?, and (ii) Is there a scientific basis to support a moreinformed approach to guidelines on dosimetry in safety studies?

2.1. Dose calculations

Two dose metrics are important for the design of non-clinical andclinical safety studies. TheDeliveredDose (Eq. (1)) is theamount of druginhaled 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].

DeliveredDose mg kg−1� �

= C × RMV × D × IFð Þ � BW ð1Þ

DepositedDose mg kg−1� �

= C × RMV × D × IF × DFð Þ � BW ð2Þ

C concentration of substance in air (mg L−1)RMV respiratory minute volume or the volume of air inhaled in

one minute (L min−1)D duration of exposure (min)BW body weight (kg)IF inhalable fraction, the proportion by weight of particles that

is inhalable by the test species (IF is often assumed to be100% if the test aerosol has a Mass Median AerodynamicDiameter (MMAD) less than 3–4 μm),

DF deposition fraction or the fraction of the Delivered Dose thatis deposited in the lungs.


Although both equations are used consistently across the industry,there are notable differences in the terminology and algorithmsassociated with both dose metrics. A number of different terms are inuse to describe the Delivered Dose, with no particular term achievinggreater acceptance than others; examples include total, inhaled,targeted and presented dose. Alternative terms for the Deposited Dosewere the achieved or lung dose. This multiplicity of terms invitesconfusion andmakes apparent the need for a harmonised terminology.For the purposes of this summary, we have adopted the terminologyrecommended by the AIT [11], where the Delivered Dose (Eq. (1)) isthe amount of test substance inhaled by the test subject and DepositedDose (Eq. (2)) is the amount of test substance calculated to bedeposited in the lungs.

In addition to variations in terminology, the Workshop identifiedthat the algorithms used to determine RMV values for non-clinicalspecies can also vary considerably [14–17]. However, the algorithmadvocated by the AIT (Eq. (3); ref. [11]) appears to be gainingprominence based on the high number of Workshop attendeesreporting its use. This algorithm was derived from RMV data fromcontrol animals collected in 18 datasets (comprising nearly 2000individual observations) across four species in ten separate laboratoriesunder Good Laboratory Practice (GLP) conditions. Since these condi-tions represent those used in current practice in regulatory inhalationtoxicology studies, this algorithm is considered themost appropriate foruse in the estimation of Delivered and Deposited Doses for non-clinicalsafety studies.

RMV L min−1� �

= 0:608 × BW kgð Þ0:852 ð3Þ

In non-clinical studies for which the Delivered Dose is presented,the FDA's Division of Pulmonary, Allergy and Rheumatology Productsapplies default Deposition Fractions (DF) to calculate the DepositedDose; this practice has been presented at numerous public meetings.For non-clinical safety studies and clinical trials, the FDA uses thefollowing DF in their evaluations: 10% in rats, 25% in dogs and 100% inhumans. These default values are based primarily on publications byWolff and Dorato [18] and Snipes and co-workers [19]. Wolff andDorato compiled published data on pulmonary deposition of particlesacross species commonly used in non-clinical pharmaceutical testingwhile Snipes and co-workers performed a meta-analysis of a numberof different deposition studies (assuming particles with an MMAD of2 μm) and calculated average depositions for each species. However,there are inherent limitations associated with the use of thesestandard FDA default values for calculating Deposited Doses, whichwas reflected in the lively and lengthy debate on the relevance andimplications of lung deposition estimates at theWorkshop. As a result,this issue is considered in greater depth in the following Sections 2.2and 2.3.

In contrast to non-clinical studies, current practice for doseestimation in clinical trials is to assume the delivery of the entirenominal dose (total amount of drug) rather than use Eqs. (1) and (2).Dose estimates in clinical studies would be more accurate if theamount of drug remaining in the device after administration weremeasured and subtracted from the nominal dose; this is especiallyrelevant when nebulizers are used.

2.2. Estimating the deposited dose

Although the FDA default DFs are used industry-wide, there is anoverwhelming consensus that they do not necessarily reflect theactual Deposited Dose of pharmaceutical aerosols. Most researchersfeel that the default DFs sometimes underestimate the deposition innon-clinical species and overestimate deposition inman. If this is true,it is possible that the use of more accurate measures of drugdeposition would improve the analysis of non-clinical safety data

and make it easier to define realistic clinical dose ranges. To evaluatewhether current estimates of Deposited Doses are reasonable requiresan appreciation of: i) how the default DFs were established, ii) howthese values are used in non-clinical safety studies, and iii) theimplications of the above for study design and data interpretation.

Snipes and co-workers [19] based their aerosol deposition analysison a number of studies investigating the deposition of insolubleenvironmental particulates such as plutonium, europium, iron oxideand aluminium silicate in the lungs of a number of experimentalspecies. Although these studies provide an excellent starting point fordetermining deposition profiles of pharmaceutical aerosols withsimilar aerodynamic properties, drug substances may behave quitedifferently in the respiratory tract compared with insoluble environ-mental particles. This is because drug compounds may be hygroscop-ic, possess variable dissolution rates or exhibit differenttransportation properties. The formulation and processes of aero-solisation, inhalation and deposition of pharmaceutical aerosols mayalso introduce deviations from predictions based on insolubleparticles. Furthermore, differences in DF between mono- andpolydisperse aerosols may be even greater than those betweenaerosols of different materials, yet similar size distributions. There-fore, it would be valuable and appropriate from a scientific standpointto generate more accurate deposition data on actual pharmaceuticalaerosols to develop a better understanding of their dosimetry anddeposition in non-clinical species and in humans.

The implications of using more realistic DFs for pharmaceuticalaerosols in inhaled product development requires an understandingof how these values are used in the development process. DepositedDoses in non-clinical studies are used to determine acceptable doseranges for human clinical trials. It is important to note that regulatoryauthorities generally require the application of a default DF value of100% inman (rather than the average 40% described by Snipes and co-workers [19] for clinical trials). This represents a cautious approachwhere the actual deposition fraction is unknown. One consequence ofthis overestimation of deposition in man is that to achieve safetycover excessively high doses must be administered to non-clinicalspecies, especially when the additional safety margins are applied; i.e.margins of 6-fold in dog and 10-fold in rat referenced to the NoObservable Adverse Effect Level in these species. For compounds thatare well-tolerated or require high clinical doses to achieve efficacy, itis often impractical to administer sufficiently high doses in non-clinical studies to achieve the required safety margins.

One solution to the requirement for excessive doses in non-clinicalstudies would be to introduce a reasonable upper dose limit for well-tolerated compounds. This strategy is currently covered by theguidelines [ICH Guidance for Industry M3(R2), 2010], albeit at doselevels that are only applicable for safety assessment of orallyadministered compounds which it is feasible to administer in largerquantities. Workshop participants discussed the feasibility of intro-ducing reasonable upper limits for delivery by inhalation based, forexample, upon the use of 1 mg/L as a maximum achievableconcentration across a number of compounds with maximum dosingtimes set according to what is appropriate for different species. Itwould also help if clinical doses could be based on data-driven valuesof DFs. If deposition data for specific animal models and specificcompounds were generated, the use of alternative DF could bejustified on a case-by-case basis. This approach opens up the potentialto apply higher Deposited Dose values in non-clinical studies than arepossible using the current default DFs, which would in turn supporthigher dose levels in human clinical studies. It was intimated inWorkshop discussions that European regulatory bodies are amenableto considering evidence for animal deposition values on a case-by-case basis. It was also believed that the FDA would be likely toconsider alternative values to their default position on non-clinicaldosimetry if appropriate supporting evidence were provided. Incontrast, the regulatory assumption of 100% drug deposition in


human clinical trials, like the applied safety margins, is basedprimarily on philosophical safety concerns rather than experimentallyderived data; this makes it unlikely that data would be adequate tochange this position.

It was recognised that even if the regulatory authorities were toconsider more realistic DF values for clinical studies, the overallimpact on permitted dose levels would be limited. For example, theuse of a 50% DF (as opposed to 100%) would only increase thepermissible clinical dose by 2-fold, i.e. it would have limited impact onnon-clinical safety study dosing requirements. However, if a change inthis component of the overall safety evaluation were combined withchanges in the evaluation of the non-clinical toxicology package (e.g.use of measured rather than estimated deposited doses, application ofmaximum doses, and reduction of safety margins) this would havethe potential for a significant cumulative impact on the determinationof acceptable clinical doses.

It was noted that non-clinical administration methods typicallyinvolve sustained delivery by nebuliser and tidal breathing which doesnot reflect the clinical situation where exposure in humans is often bybolus with special inhalation manoeuvres. However, in mitigation,estimated dose and systemic exposure in non-clinical studies are usedprimarily to calculate safety cover for human studies in which clinicaldoses are based on a pragmatic, conservative philosophy. In summary,there was a consensus at the Workshop that it was both valuable andappropriate to generate deposition data relevant to pharmaceuticalaerosols to improve understanding of real, compared to estimated,Deposited Dose in non-clinical species and man. In fact, although notrequired by the regulatory authorities, most companies represented atthe meeting already often measure drug concentrations in the lungimmediately afterdosingor collect systemic PKdata asproof of dosing innon-clinical studies (see Section 2.3).

2.3. Measuring the deposited dose

Differentmethodsare currentlyused toverifydrugdelivery to the lungin non-clinical studies in order to achieve a greater understanding of how

Table 1Advantages and limitations of using systemic exposure (area under plasma concentration c

AUC

Compound/formulation suitability Complete absorption, no first pass extraction by the

Method strengths Dose determination (by comparison to reference PKmeasuring an easily accessible systemic compartmenThe ability to assess drug concentrations at various tUsing basic PK principles it is possible to estimate theof non dissolved material in the lung.Rapid sample analysis.

Limitations/caveats In non-clinical species, inhaled compound bioavailabinfluenced by both oral and nasal absorption. While ocan be charcoal-blocked, nasal absorption cannot bepresent. The ability to model or block nasal absorptiouseful to fully understand inhaled bioavailability in nspecies. In contrast, nasal absorption is limited in humstudies. Therefore, clinically some information can bcharcoal block, as long as clearance following intraventhe same species/strain at a comparable dose level isorder to relate AUC to Deposited Dose.Non-clinical doses are often achieved by modifying texposure and/or inhaled drug concentration and diffcombinations of these may lead to variable pharmacthe same Deposited Dose.Accumulation of dissolved drug in the lung by any pomechanisms (transporters, lysosomal trapping) cannby plasma AUC.Analytical sensitivity for low dose and (potentially) hcompounds.

Recommended applications Proof of dosing.As a safety cover for systemic findings.As a useful indication of clinical performance.

pharmaceutical aerosols deposit and to compare real versus estimatedDeposited Dose values. The two most prevalent methods are: i)measurement of systemic exposure, i.e. area under the plasma concen-tration curve (AUC) by inhalation compared to intravenous dose, and ii)excision of lung tissue immediately after administration and measure-ment of drug directly in lung tissue homogenate. The strengths andinherent limitations of each approach were discussed in the Workshop(Table 1). There was consensus that the AUC provides evidence of dosingfollowing inhalation, especially when the drug compound exhibits bothhigh solubility and permeability. Despite the issues listed in Table 1 andthe caveats that must be applied, measuring systemic PK in the non-clinical phase provides proof of dosing, evidence of systemic safetyassessment and a useful indication of clinical performance.

In comparison to the relatively simple, but indirect measurement ofthe deposited dose using the AUC, determination of drug concentrationsin lung homogenate provides a more direct measure of local depositeddoses. This approach is preferred for the measurement of depositeddoses of poorly soluble compounds and would also be applicable forpoorly permeable compounds or inhaled pharmaceuticals where theformulation retards drug absorption for a significant amount of time.Measuring drug concentrations in homogenates prepared from excisedlungswas recommended for safety studies of poorly soluble compounds,when accumulation after chronic dosing is a concern. Currently, there isno agreed protocol across the industry formeasuring drug accumulationfollowing chronic dosing, despite the fact that many new chemicalentities exhibit low solubility profiles. Therefore, although no guidelineswere agreed at theWorkshop, development of evidence-basedprotocolswas identified as a priority area for pre-competitive collaboration withthe aim of establishing how best to measure real deposited doses afteradministration of pharmaceutical aerosols and how best to assess lungaccumulation of poorly soluble drug compounds.

2.4. Modelling pharmaceutical aerosol deposition

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

urve; AUC) or lung tissue homogenates for measuring Deposited Dose.

Lung homogenate

lung. Low solubility, low permeability compounds.Slow release formulations.

data) aftert.

Direct measure of local delivered doses.Possible assessment of compound accumulation after chronic dosing.

ime points.accumulation

ility can beral absorptioncontrolled atn would beon-clinicalan inhalatione gained fromous dosing inknown in

he duration oferentokinetics for

tential uptakeot be assessed

igh clearance

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

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

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


interest in the current state-of-the-art in this area. Bahman Asgharianfacilitated discussion and illustrated various approaches to modellingpharmaceutical aerosol deposition in the lungs of humans and speciesused in non-clinical studies [20,21].

There are two main approaches to calculating the deposition ofdelivered aerosols in the lung depending on the resolution of lungairway geometry and desired level of detail. The first approachinvolves calculations of the airflow in the selected lung geometryusing computational fluid dynamics (CFD) and using the flow fieldpredictions to solve for exact locations of particles depositing in thelung. Feasibility and practicability of this approach hinges on theavailability of accurate airway geometry typically obtained fromscanned images of the lung, as well as knowledge of airflow andpressure distribution in the deep lung to serve as outlet boundaryconditions for the computational domain. The resolution clarity ofexisting imaging systems does not allow reconstruction of smallerairways of the lung (i.e., bronchial and pulmonary airways) andreconstructed airway geometry comprises less than 5% of the lungvolume. Unless the region of interest in the lung lies within thedomain of current resolution, the use of CFD in deposition predictionsdoes not offer improved accuracy over other approaches. Uncertaintyregarding airway geometry and airflow boundary conditions limitsCFD application in the deep lung for the purpose of depositionpredictions. Hence, detailed CFD approach is most useful in theextrathoracic airways [22–26] and first few airway generations of thetracheobronchial airways [27–39] for which airway passages arereconstructed from scanned images of airway coronal sections andcombined with measured breathing parameters to form anatomicallyand physiologically realistic deposition models. Since the fulltransport equations with complete account of dominant physicalmechanisms are solved, detailed information regarding the airflowand particle deposition is obtained, however, at the expense of aheavy computational cost (Fig. 1).

The second and traditional approach to predicting particledeposition in the lung uses simplifying assumption to allowdeposition calculations of particles for the entire lung. The lunggeometry is modelled – based on the available measurements ofairway parameters [40,41] – as a dichotomous, symmetric orasymmetric branching structure with each airway shaped as acylinder. The idealised geometry of lung airways is compatible withthe area-averaged, 1-dimensional mechanistic model of particletransport and deposition throughout the conducting tree and alveolarducts of the lung. Although detailed site-specific or per-generationexperimental comparisons of deposition are not warranted in thesimplified geometry, dose to the lung and to the tracheobronchial andalveolar regions has been compared extensively [42]. In addition, theinfluence of gravity on pleural pressure has been neglected to allowuniform airflow distribution in the lung. Consequently, airflow at anylocation in the lung is proportional to the distal volume to thatlocation. Hence, the need for detailed airflow computations using CFDis alleviated. Using cross-sectional-area-averaged particle concentra-tion of inhaled aerosols, a simplified transport equation is obtainedand solved to predict local and regional deposition of particles in thelung. This approach is ideal when regional lung deposition predictionsare needed while at the same time the CFD approach is handicappedby enormity of the lung airways and lack of information on ventilationdistribution in the lung [43–48]. As a result of making simplifyingassumptions, computations can be carried out in a relatively shorttime, however, detailed, site-specific deposition information areeither unavailable or unreliable (Fig. 2).

The use of in silico approaches to improve estimations of depositeddose is currently limited by the lack of data and validation forpharmaceutical aerosols and lack of precedence for regulatoryacceptance (e.g., the models have undergone limited developmentrelating to deposition in species used in non-clinical studies). It wasnoted that the in silico human deposition models have been accepted

by the US Environmental Protection Agency for environmental safetyassessments and it is possible that the applicability of the models forhuman drug safety studies may not yet be fully realised by the FDA.

To develop in silico models into useful tools for dosimetry andpharmacokinetic analysis, deposition data is required to validate andconfirm the models. As individual datasets are small, pre-competitiveinformation could be pooled across the industry for this purpose. Forexample, particle size determined using cascade impactors may help topredict the DF for in silicomodels in cases where this has been validatedagainst 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 aerosolcharacterisation and are not designed for in vitro-in vivo prediction ofdose to the lung [49,50]. The opportunity to build a databank based ondrug-like molecules with different physico-chemical properties mayprovide an opportunity to generate pharmaceutically relevant deposi-tion data to support more realistic deposition values in animals; thiswould inform dosimetry calculations in non-clinical studies andfacilitate their translation into clinical studies. This was identified as apositive opportunity for industry to collaborate, pool data and, inconjunction with greater use of imaging techniques and integration ofappropriate techniques to characterize lung deposition in non-clinicalspecies and human (qualitatively and quantitatively), to improve ourunderstanding of deposition.

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

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

Inhalationtoxicology

Description

Single dose 2 species (rodent andnon-rodent, typically rat anddogor rat andmonkey), ascending dose to maximum tolerated single dose

Repeat dose 2species, (rodentandnon-rodent, typically rat anddogor rat andmonkey), repeat dosing of 2 weeks–1month duration

Mutagenicity In vitro and in vivo genotoxicity testsSafety pharmacology In vitro and in vivo studies related to cardiovascular, central

nervous, and respiratory systems

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


2.5. Current practice and opportunities

Cross-industry sharing of deposition data in non-clinical animalspecies provides an opportunity to pool data upon which moreinformed estimates of deposition could be made and negotiated forregulatory acceptance. The MHRA are amenable to consideringevidence-based deposition data whereas the FDA has defaultexpectations regarding deposition factors, although the FDA wouldalso be likely to consider evidence-based deposition data if this werepresented. The deposition factors used by the FDA for non-clinicalstudies (10% in rat; 25% in dog) are not entirely unreasonable, butthere is scope for using actual data for a given compound to improvedose estimates. At present, there is no regulatory or scientificguidance on protocols to evaluate deposition and accumulation ofcompounds following inhaled administration. With extensive valida-tion, current in vitromethods for aerosol characterisation (e.g. cascadeimpaction) can be used for in vitro-in vivo prediction of dose to thelung although this is not necessarily a 1:1 correlation; this does not,however, extend to prediction of regional deposition. Systemicexposure, although a useful indicator of dosing and systemic safetycover, cannot be used to estimate lung deposition or accumulation innon-clinical species. There is considerable potential for cross industrysharing of protocols for measuring deposition and dosimetry data toimprove estimates of safety risk, our fundamental understanding ofthe deposition of pharmaceutical aerosols and to validate and developin silico models of particle deposition for inhaled pharmaceuticals.

3. Inhalation safety studies

Current practices in inhalation toxicology, how these might beimproved and the challenges that might benefit from a collaborativeapproach were considered by asking the question, “what measure-ments can be used consistently across the industry to establish safetyor toxicity to allow regulatory bodies to assess new chemical entities(NCE)?” A rational approach to developing inhalation safety science isof importance to industry and regulators, both of whom have aninterest in maximising safety assurance while complying with the 3Rsrequirement for eliminating unnecessary animal experimentation. At

present Good Laboratory Practice (GLP) toxicology studies supportingclinical trials are standard [51] with histopathology as the primaryendpoint of such studies [52]. Within these studies, however, thereare issues with regard to (i) the consistency with which pathologiesare defined and classified, (ii) biomarkers of toxicity, (iii) thebackground range of normal biological variation and how todistinguish an adverse reaction from a normal response, and (iv)the means and extent to which toxicokinetics should be applied.

3.1. Regulatory requirements

Safety studies are performed early in product development and aredesigned to meet well-defined regulatory package specifications topermit first-in-human dosing. In addition to standard tests formutagenicity and safety pharmacology, non-clinical repeat dosingstudies are required utilising delivery via the relevant clinical route ofexposure. Although in vitromethods are in development and have thepotential to reduce costs and animal experimentation as well asprovide mechanistic insights [53], there are currently no validatedassays with regulatory acceptance that may be substituted for theacute and chronic in vivo studies that are required to enable singledose 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 histopathologyprocedures are standard, the terminology used to describe the natureand severity of end-points may be a source of inter-study variationwhich precludes meta-analyses and hinders the assessment of studyoutcomes. This is a recognised weakness which is being addressed bytoxicology and pathology bodies, e.g. the International Harmonisationof Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice“INHAND” [54]. This is a welcome initiative which aims to address theneed for standard definitions. Ideally, the new nomenclature shouldbe recognisable by specialist and non-specialist pathologists andregulators. However, by their nature histopathological measurementsand severity ratings are semi-quantitative.

Drug administration in these non-clinical studies is by aerosolisa-tion with the attendant issues regarding dosimetry that are describedin the previous section. The form, in which the test article ispresented, for example powder or liquid droplet solution, may havea profound effect on the local lung effects. Ideally the same formshould be used throughout the development of an inhaled productand certainly the definitive studies should be performed with thesame or a very similar formulation to that whichwill be used for phase3 studies and launch. However, practical limitations such as the earlystage of pharmaceutical development and limited availability of thematerial may prevent this ideal approach. Acute studies aremandatory and establish doses for use in longer duration chronicadministration studies. Although acute studies have limited ability topredict chronic outcomes, they are regarded as useful for toxicityscreening, i.e. detection of readily identifiable toxicities andmaximum

image of Fig.�2


doses to be used in repeat dose studies. In general, however, acutestudies by their short-term nature have limited ability to predictsubtle effects over the long term (ICH guidelines, M3 Section 4).

3.2. Biomarkers of toxicity

For both acute and chronic toxicity testing, better biomarkers inbronchoalveolar lavage fluid (BALF) or blood and other supplemen-tary end-points would be useful. It is necessary to consider these on acompound by compound basis and if pharmacodynamic end-pointsare available these should also be used. Endpoints that can bemeasured in early, acute and repeat dose studies include cellinfiltrates and cytokines in BALF. These measurements can beparticularly useful in early studies to assess toxicity relative to othercandidate compounds or known toxicants. Pulmonary function is asafety pharmacology end-point and can also be useful in early studiesto help assess whether there is any respiratory tract irritation, effecton lung surfactant or serious lung damage which are manifest bychanges in breathing pattern or lung mechanics.

Over the years, a variety of enzymatic markers have beenmonitored [55], without any particular marker gaining precedenceas indicative of respiratory toxicity. More recently genomicapproaches such as gene profiling have been utilised, for example toidentify mechanisms of toxicity of excipients for use in inhaledformulations [56,57] and metabonomic studies are being undertakenincreasingly to elucidate metabolite biomarkers for improved diag-nosis of lung diseases such as asthma [58], chronic obstructivepulmonary disease (COPD) [59] and cystic fibrosis [60]. The study ofmetabolite signatures in lung tissue of COPD murine models and theacute effects of cigarette smoke on these signatures [59] is the firsttime that NMR-based metabonomic analysis has been reported forintact lung tissue; this has the potential to generate a reliablemetabolic fingerprint to uniquely characterise the animal model ofCOPD and begin to identify strong biomarkers for the disease. There isthe prospect that in the future such methods will be developed andapplied to the identification of biomarkers of inhaled drug or excipienttoxicity.

3.3. Interpretation of adverse effects

Difficulties in interpreting toxicology findings include reconcilingtest sensitivity, background biological variation, normal responses toinhaled materials and drug or medicine-specific adverse effects.Identification of adverse end-points is an area where better controldata sets might help discern true adverse effects from normalphysiological lung response. The lung responds acutely to inhalationof irritant materials by hypersecretion of mucus, chemokine release,inflammatory cell recruitment and cough [61]. Collectively these maybe characterised as non-specific irritancy. Chronic changes that couldlead to pathology may be observed morphologically as epithelialdamage, granuloma and phospholipidosis— including the appearanceof “foamy” macrophages.

The development of foamy macrophages is a poorly understoodphenomenon that provides a conundrum regarding the implicationsof their observation in non-clinical testing. The foamy macrophagephenotype is a term used to describe the appearance of macrophagesthat have taken on a granular or vacuolated appearance under thelight microscope (Fig. 3). The phenotype can be manifested as a resultof a number of different processes including intracellular lipidaccumulation 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 isoften heterologous and the foamy phenotype varies betweencompounds and dosing regimens.

While the potential for induction of foamymacrophages should beinvestigated early in the drug development process, the implications

of inducing a foamy macrophage phenotype are relatively poorlyunderstood. In some circumstances the appearance of foamy macro-phages may simply be an adaptive and reversible response to theinhalation of particles [68]. However, the induction of foamymacrophages may also form part of the progression of a number ofdiseases including emphysema, pulmonary alveolar proteinosis [63]and certain infectious diseases [69] and can be considered an earlymarker of the progressive toxicity of inhaled drug particles [70]. Theproduction of the foamy macrophage phenotype and activation ofmacrophages in vitro have been reported by a number of authorsinvestigating the effects of insoluble environmental particulates [71–73]. However, there is no definitive evidence regarding the relevanceand implications of the formation of foamy macrophages in responseto inhaled drug particles within in vitro systems, or with respect to theobservation of foamy macrophages during chronic inhalation safetystudies, which are generally carried out at very high particle doses.

Better understanding of the implications and reversibility of anychanges is therefore critical for developing new inhaled drugs. Themeasurement and interpretation of these phenomena are complicat-ed by uncertainty regarding the specific or non-specific nature of theresponse to inhaled particles [74,75]. Developing a data set thatclearly establishes normal and adaptive physiological responses isneeded. This might be helped by pooling of industry-wide controldata that includes sham, vehicle and particle controls. There is also aquestion regarding whether certain biological responses in non-clinical inhalation studies are acceptable and expected non-specificchanges, i.e. normal adaptive and non-toxic. Benchmarking of theeffects of known toxicants (as a positive control data set) andapproved marketed inhaled pharmaceuticals (as a negative controldata set) might be instructive in discerning what constitutes adverseversus non-adverse effects. This approach would also provide thenecessary validation to make biomarker profiles decision-making ininhalation development projects.

Where non-clinical data lead to uncertainty regarding toxicity,methods for monitoring safety in the clinic (i.e. measurablebiomarkers) would enable progression and dose escalation inhuman clinical trials. Unfortunately, measures of respiratory toxicityin man are limited, but it would be extremely useful if some of themarkers that can be measured in BALF in non-clinical studies could bemeasured readily in blood in clinical studies.

3.4. Toxicokinetics

Toxicokinetics is the determination of the relationship betweensystemic (blood or plasma) levels of a compound and its toxicity andis increasingly being applied in toxicity testing. The extent to whichlung kinetics are measured in inhalation safety studies across theindustry varies. Measurement of systemic drug concentrations forpharmacokinetic studies is a standard procedure (Section 5) and thisdata is of interest for investigating the relationship between systemictoxicity and systemic exposure. However, drug concentration in lunghas not traditionally been measured. As far as current industrypractices are concerned, it transpires that drug levels in the lung aremeasured either (i) not at all, (ii) immediately after dosing or at theend of study, or (iii) at various time points. Data obtained immediatelyafter dosing is used principally to verify that dosing has beensuccessful. Samples collected at the end of the study can be comparedto earlier equivalent samples to allow accumulation in the lung to beassessed. For more detailed lung toxicokinetic analysis, a greaternumber of time points are required and this data is not generallycollected.

Themain barrier to wider use of measuring drug levels in the lungsin study protocols is the difficulty in interpreting lung concentrationsor accumulation if it occurs. In addition, there is concern that ifmeasurements that are not specified in regulatory guidelines aretaken they may raise unwarranted concerns by the regulators. In

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 thebrown-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 afoamy macrophage containing numerous complex phospholipid inclusions (right; Bar represents 5 μm) [62].


contrast, the advantages of the toxicokinetic approach are that drugquantification in the lung can be useful for determining doseproportionality and interpreting histopathology, particularly if thereis evidence of drug accumulation in lung.

3.5. Developments in safety science

A number of developments in inhalation safety are alluded toabove. The need for the harmonisation of terminology used toidentify, describe and quantify histopathology is widely recognisedand is being addressed. Measurement of systemic (blood/plasma)levels of drug and the determination of toxicokinetic parameters isnow a routine in inhalation studies. However, there is not yet anystandard in industry with respect to lung drug level measurements,which increase study costs, require additional resource, may bedifficult to interpret and can raise questions from regulatoryauthorities.

An example of the application of toxicokinetics in industry is theapproach developed at Pfizer by Rhys Jones and Natasha Neef(presented at the APSGB Workshop on 10 June [1]); this described amethod for modelling macrophage loading with poorly solubleparticles in the lung. The aim was to examine the concept of overloaddoses in the lung, proposed to be N1 mg particles/g lung tissue [76],beyond which macrophage abnormalities are observed. A toxicoki-netic model was developed to address the questions: (i) how doproposed lung thresholds for insoluble particles relate to studies inrodents with inhaled molecules? and (ii) do observed adversechanges in the lung correspond to accumulated doses above 1 mg/g? Briefly, mass delivered was estimated from pulmonary depositionbased on multiple path modelling (~10% in rat; [20]). This input wascompared to clearance via mass absorbed following dissolution,which was calculated using the systemic exposure data, andmacrophage uptake rates, which were based on data for removal ofundissolved particulates by rat alveolar macrophages with a clearanceof 0.007 day−1 [77]. Using these parameters the thresholds confirmedthat 0.1–1 mg particles/g lung tissue generated non-adverse adaptivechanges, e.g. an increase in macrophage numbers, whereas lungaccumulation at N1 mg particles/g lung tissue was associated withadverse changes such as inflammation and tissue degeneration [Note:the model assumes an absence of chemical toxicity and does not atpresent account for factors such as particle size or surface area].

This approach allowed lung burdens to be estimated successfullyand used to interpret the histopathology findings. The outcomes werebroadly consistent with published data on pathology associated withlung burdens of biologically inert material. Since the extent ofaccumulation can be estimated and extrapolated to levels at steady-state, this has the potential to inform dose selection for longer term

toxicology studies. The measurement of lung doses in non-clinicalsafety studies would help to validate this approach. Furthermore, thesame approach can be applied to humans using the anticipatedclinical dose using an estimated pulmonary absorption rate constantand an adjusted value for macrophage clearance in man 0.0035 day−1

[78] compared to the value in rodents. This can provide confidencethat lung burdens in human will remain well below threshold foradverse findings on chronic dosing.

Industry-wide data sharing and collaborative research provideopportunities to improve on the current state-of-the-art. A possibilityfor data sharing is to pool control data from inhalation safety studies(i.e. share control data across the industry) to establish normalbackground biological variation. This would be useful for theregulators and was of interest to industry in order to help to assesswhich changes are adverse andwhich changes can be considered non-adverse. It was recognised that the logistics of such an exercisetogether with inter-laboratory differences in measurement andinterpretation would provide considerable challenges. The value ofsharing control data would be in generating larger data sets to aidevaluation of study data against a background of normal biologicalvariation.

A major research challenge for inhalation science is to developbetter biomarkers, especially those that would enable dose escalationin the clinic when there are concerns regarding drug safety. Mostwidely applicable would be the identification of generic markers fordifferent forms of lung damage. This could be complemented byspecific markers for different inhaled drugs or drug classes. New end-points also suffer from a lack of validation. A valuable means ofaddressing this would be benchmarking using known toxicants andapproved inhaled pharmaceuticals to define markers of adverseeffects.

4. Pulmonary drug disposition

Key questions considered at the Workshop concerning pulmonarydrug disposition were what is the relative importance of intrinsic andformulation-driven PK? What methods are available to determine PKand how are these being applied? What is known about the presenceand influence of transporters on the fate of drugs in the lung?

Themajor advantage of using inhaledmedicines to target the lungs isthe elevated drug concentration that can be achieved locally in the targettissues after administration via this route [79]. In studying pulmonary PK,the goal is to understand the temporal relationshipbetween the delivereddose, the drug concentration at the sites of action within or outside thelungs, and thedrug concentration in easily accessible referencefluids suchas samples from the systemic circulation. However, the respiratory tractconsists of several anatomically different regions that may, or may not

image of Fig.�3


constitute target regions for inhaled medications. These regions aredifficult to target selectivelywith inhalation therapies, somost often non-target tissues are exposed to drug in parallel with target tissues. The linkbetween PK and therapeutic activity is considered in Section 5.

Measuring pulmonary PK is complicated by the lack of easilyaccessible fluids for direct or surrogate measurement of theconcentration of active substance in target tissues of the lung (seeSection 5.2). If drug concentration is sampled in systemic blood, this isa compartment that is downstream of both target and non-targetrespiratory tissue. In addition, blood samples are generally taken fromthe venous side of the circulation, which means that drug has beenexposed to interactions in other organs and the peripheral capillarybed since leaving the lungs.

4.1. Intrinsic and formulation-driven pharmacokinetics (PK)

For inhalation therapies, both the intrinsic PK and formulation-driven PK are of interest. Intrinsic PK is the temporal disposition of theactive compound following inhalation to the lung as determined bythe inherent molecular characteristics of the drug. For example, therelationships between the absorption of drugs from the lung and theirmolecular weight, log P or polar surface area [80,81].

Formulation-driven PK is the influence of the formulation inmodifying the intrinsic PK; often through controlling the liberation ofthe active compounds from a carrier (e.g., a solid particle or other drugreservoir). The latter implies the ability of the formulation to alterdrug pharmacokinetic parameters, such as time to maximum drugconcentration (Tmax), the maximum drug concentration, (Cmax), andthe half life (t½). Formulation effects generally have a greaterinfluence on over-all pulmonary kinetics for lower solubility drugsthan for higher solubility drugs [82,83]. The dependency of PK onformulation might be seen, for example, as differences resulting frominhalation of a nebulised solution versus a dry powder formulation orbetween amorphous versus crystalline powders [93].

In addition, with all aerosol drug delivery procedures the formula-tion has a critical indirect influence on PK by delivering the carriervehicles to different regions of the lungs according to their aerodynamicproperties. This may alter PK as absorption rate, clearance mechanismsand 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 pulmonarykinetics non-clinically including cell cultures, ex vivo organs and in vivomodels (these have recently been reviewed [85,86]. Cell culturemethodshave a number of advantages for studying drug permeability andmetabolism, particularly for mechanistic investigation [87]. However,the application of cell culture systems to study PK parameters in routinedrug discovery and development is currently limited. Cell models havebeen used to rank different inhaled drugs according to their absorptionproperties [88], and they are beginning to be adapted to investigateparticle-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 rateconstants for use in physiologically based PKmodelswhich are beginningto be developed to predict the in vivo local fate of soluble inhalants in thelungs [92].

In vivo, pulmonary PK would ideally be studied separately in eachanatomical region and preferably with the dissolution and absorptionphase studied separately from distribution and elimination. In moststudies this cannot be achieved; in reality the anatomical regions areexposed in parallel, and most often absorption to the systemiccirculation must be measured on the venous side. Rodents are thesmallest animals used for in vivo studies; a PK profile may be obtainedfrom a single rat, although their lungs are too small to permit regionalexposures. In addition, most often rodents are exposed via nose-onlyinhalation, which gives a substantial deposition of drug in the non-

target region of the nasal airways. It is possible to use intubation tobypass the nasal airways and increase substantially the pulmonarytargeting, however, most often these exposures are done by solutionor suspension instillation instead of inhalation. Instillation gives amore patchy and central deposition of materials than aerosolisationand inhalation [94].

Regional exposures of the respiratory tract require larger lungssuch as those of the pig, the dog or humans. In larger lungs the majorregions can be targeted either by using differently sized aerosols ofnarrow size distribution [94], or by using a so called bolus techniqueto direct certain volume elements of air containing aerosol tocontrolled depths in the lungs [95]. With larger animals comparedto rodents, more and larger blood samples are easily obtained and it ispossible, although rare, to use protocols for direct sampling of bloodon the arterial side of the circulation, mirroring the direct efflux ofblood from the lungs downstream of the pulmonary circulation. In theideal case where both sides of the systemic circulation aresimultaneously sampled, the kinetics of absorption of inhaled solutesfrom the peripheral lung can be quantified by measuring the netincrease in blood concentration upon passage of the lungs [96–98]. Inhuman studies, for the most part repeated blood samples from thevenous side with calculation of the AUC, Cmax and Tmax must be usedas surrogate indices for pulmonary absorption [99].

The isolated, ventilated, and perfused lung (IPL) is a model that isparticularly suitable for studying the lung-specific absorption of solublesubstances following luminal administration. The lungs of small animals,most commonly the rat, are attached and ventilated via a trachealcatheter. The pulmonary circulation is perfused with an albumin-phosphate buffer in either single-pass or recirculation mode [100]. Thekinetics of pulmonary absorption of both small molecules and macro-molecules can be studied in detail by repeatedly sampling perfusatefollowing the intratracheal administrationof substance, and isparticularlyrelevant if techniques are used to administer test substance as fullyrespirable aerosols under normal ventilation [101,102]. Furthermore, easeof access to the lungs after a perfusion period allows accurate massbalance (i.e. recovery) of the originally administered dose.

It is evident, therefore, that the IPL can be used to study therelationship between absorption rates and the physicochemical proper-ties of inhaled drugs and solutes [6,102,103]. Fewer studies are availableon vehicle- or formulation effects of drugs on absorption. However, adetailed study on a low-solubility organic toxicant indicated a profoundeffect of substance load on carrier particles on the overall pulmonaryabsorption rate andmetabolism [104]. The uses of the IPL extend beyonddrug disposition to the study of pharmacodynamic effects, such asbronchoconstriction [105] and vasoconstriction [106,107].When used forthese purposes the IPL provides an opportunity to study the relationshipbetween PK and pharmacodynamics (PD; see Section 5), albeit with thecaveat that the duration of experiments is limited and the isolation of thelung 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 importantdeterminants of drug disposition, efficacy and toxicity [108–110].Transporters of the solute carrier family (SLC) facilitate the transfer ofcompounds across the plasma membrane. Commonly, these trans-porters mediate the uptake of drugs into cells but, depending on thedriving force for the transport, some SLCs will also transportcompounds in the other direction. Common drug transporting SLCsare organic cation transporters (OCT and OCTN/SLC22A family) andorganic anion transporters (OAT/SLC22A family and OATP/SLCOfamily). Drug transporters of the ATP binding cassette family (ABC)transport compounds out of cells in an energy-dependent processdriven by ATP hydrolysis. Examples of ABC transporters are themultidrug resistance proteins (MDR/ABCB), multidrug resistance


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 toregulate drug distribution and are key determinants of drugabsorption and clearance [111]. The impact of drug transporters ismost significant for compounds with low passive permeability as suchmolecules will be more dependent on an active transport process forsignificant passage across cellular membranes.

Recent progress in the transporter area has resulted in anenhanced assessment of drug transporters in drug discovery anddevelopment. A review of clinically important drug transporters andrecommendations of experimental studies and data interpretation hasrecently been published [112]. Current activity in the pharmaceuticalindustry is focused on transporters in the intestinal epithelium,hepatocytes, kidney proximal tubule cells and blood-brain barrier andtheir role in absorption, elimination and distribution of drugs as wellas drug–drug interactions. Despite the rapidly expanding literature ondrug transporters, relatively limited data is available on the functionalrole of transporters in the lung and their relevance for pulmonary PKis currently unclear [113,114]. Potentially, transporters in the lungepithelium may influence the absorption of drugs from airway toblood circulation, residence times of drugs in the lung, theintracellular concentration of drugs in pulmonary cells and conse-quently pharmacological efficacy as well as toxicity.

In common with other organs in the body, many transporters ofthe SLC/SLCO and ABC families are expressed in the lung andcomprehensive reviews in this area have recently been published[113,114]. Among the SLC/SLCO carriers, the organic cation transpor-ters OCT1-3 and OCTN1-2 are expressed and immunohistochemistryhas indicated a localisation on the luminal side of pulmonaryepithelial cells [115–117]. Expression of OATPs has been observedon the mRNA level [115], but data on protein expression and cellulardistribution in the lung is still lacking. In terms of ABC transporters, P-glycoprotein (MDR1), BCRP and a range of MRPs are expressed in thelung (reviewed by [113,114,118]). P-glycoprotein and BCRP arelocalised to the apical side of airway epithelium, whereas MRP1 hasbeen found on the basolateral side. In general, P-glycoprotein isexpressed to a lesser degree in lung compared to other barriers, e.g.the intestine [119]. However, since the lung is a highly heterogenousorgan with a variety of different cell types, the expression may still beof functional importance in specific parts of the lung. There is a needto identify transporter distribution in different regions of the lung,between different cell types and at the subcellular level.

There have beenvery few studies published to date on the functionalimpact of drug transporters on lung PK (reviewed in: [113,114]). Datafrom cell models and isolated perfused lung are emerging, but there isyet very little in vivo evidence. Any inhaled drugs with a relatively lowpassive permeability are potentially susceptible to an influence of drugtransporters on their absorption and distribution into sub-compart-ments of the lung. Studies by Horvath and co-workers have indicatedthat the organic cation transporters are of importance for the transportof β-agonists in airway smooth muscle cells [120] and for a variety ofsubstrates in lung epithelial cells [117]. Similarly, Nakamura and co-workers [121] demonstrated that uptake of ipratropium, an anticholin-ergic drug, by human bronchial epithelial cells was largely mediated byOCTNs. Several studies exploring the functional role of P-glycoprotein inlung have been published, although these report conflicting data. Theabsorption of Rhodamine, a fluorescent P-gp substrate, was inhibited byP-gp inhibitors in the isolatedperfused lung [122]whereas another P-gpsubstrate, digoxin, was not affected in vitro [123] or in vivo [124]. Theabsorption of the p-glycoprotein substrates has also been reported invivo [6].

In summary, the functional impact of transporters on the PK ofsmall molecular weight drugs is still largely unknown. There is a clearneed to extend our knowledge before consideration of transporterscan be factored into respiratory drug discovery and development

programmes. The functional activity of drug transporters may bestudied in cellular systems but the impact on PK has to be assessed in amore complex system. Since specific transporter inhibitors are stilllacking, it is a challenge to study the functional role in vivo in thepresence of confounding factors. Transgenic animals have becomemore accessible and may be one useful tool to increase ourunderstanding in this area. The isolated perfused rat lung may beanother useful model as it offers the possibility of controlling theadministration and sampling of the drug.

4.4. Current research activity

An important goal of current research is to gain a betterunderstanding of the basis for bioequivalence of inhaled medications[49]. Comparedwith other routes of exposure there are more complexrelationships between the delivered dose and the therapeutic effect.Because the exposed target tissue is located upstream of the systemiccirculation, most often parallel to exposed non-target tissues, there ispoor correlation between the easily accessible drug concentrations ofthe systemic circulation and target-tissue concentration and ulti-mately therapeutic effect. A better understanding of the functionalrole of drug transporters on drug distribution in the lung is required asthere is only assorted and sometimes inconsistent functional dataavailable. In the absence of a demonstrated functional impact of drugtransporters on drug disposition in the lung, academia rather thanindustry is currently taking the lead in research in this area. In futurean understanding of the presence and function of transporters in thelung may become essential knowledge if the PK of inhaled drugs is tobe interpreted rationally.

5. Pharmacokinetic-pharmacodynamic relationships in the lung

5.1. Pharmacodynamics (PD) in the lungs

The previous section considered inhaled medicines with regard tothe factors affecting PK; i.e. the quantitative and temporal relationshipbetween administered drug dose and drug concentrationmeasured in adistinct biological matrix. Whereas PK measures the fate of an inhaleddrug molecule, it is PD that defines the quantitative relationshipbetweendrug concentration and pharmacological response. Linking PK/PD quantitatively affords an understanding of the relationship betweendrug dose and the time of onset, intensity and duration of response. PK/PD methodology has had a significant impact in pharmaceutical drugdiscovery and development [125], benefiting experimental design andinforming decisions on target validation, lead identification/optimisa-tion and efficacy/safety. Most typically, PK/PD relationships areestablished with drug concentrations determined in blood or blood-relatedmatrices (serum or plasma), with assumptions that equilibriumwill be achievedbetween freedrug in, for example, plasmaand freedrugin the biophase of the pharmacological receptor. As noted above(Section 4), however, the use of blood-related matrices may not beappropriate for local effects following inhaled drug administration.There is a paucity of quantitative PK/PD data for studies involvinginhaled drugs and local pulmonary response. This may be attributed tothe relatively small size of the inhaled pharmaceuticals sector with fewfirst-in-class molecules and the questionable value of incorporatingsystemic drug concentrations, such as plasma data, into the PK/PDanalysis for locally acting inhaled therapeutics. For progress inpulmonary PK/PD, a better understandingof the benefits and limitationsof measuring drug concentrations in lung is required to guidedevelopments in experimental methodology.

5.2. Measuring pulmonary drug concentrations

Harvesting lung samples for the measurement of drug concentra-tions is technically challenging, particularly in the clinic where


opportunities for such sampling are more limited and not widelyutilised. In the clinic, measurement of drug concentration in a blood-related matrix predominates to provide surrogate concentration datafor clinical pulmonary PK/PD. While pivotal non-clinical lung dosingstudies also focus on the measurement of drug in a blood-relatedmatrix (thereby providing translational information for the clinic) itis increasingly routine in non-clinical studies to also measure drugconcentrations in the lung tissue itself. Typically such measurementshave served to assess Deposited Dose or the retention of drug inthe lung (Section 2.3) or in toxicokinetic studies (Section 3.4) to gain ameasure of organ exposure to the drug to aid safety profiling.However, a variety of lung sampling methods exist and provide anopportunity to relate time profiles to lung and plasma drugconcentration and begin to address some of challenges of pulmonaryPK/PD analysis. The lung sampling methods are described in moredetail below.

5.2.1. Lung tissue homogenatesWhole lung tissue is readily collected in the non-clinical setting

and when done so immediately at the end of an intratracheal orinhaled drug administration it can provide an estimate of theDeposited Dose (Table 1). A caveat with this technique is that rapidabsorption during the dosing and sampling period results in loss ofdrug from the lung, reducing the accuracy of dose determination. Bycombining lung dose with sacrifice of animals at various time pointsafter 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 beparticularly judicious if the tissue is used simultaneously for PDevaluation, e.g. levels of inflammatory markers. This approach islimited, however, by the lack of precision of homogenate dataregarding the localisation of drug in the lung as is described below.

Processing of lung tissue for drug analysis involves homogenisationto complete destruction, i.e. the formation of a uniformly dispersedsuspension of macerated or crushed tissue in an aqueous diluent. Thisoften involves the indiscriminate use of the whole lung tissue orvarious components thereof with data expressed as averaged drugconcentration for the entire lung. However, average drug concentra-tions across the tissue may be difficult to relate to levels at a particulareffector site within the lung. Even at a gross level, variation can beexpected in drug concentrations between the individual lung lobes,arising through variability in lung deposition and the recognition thatlobular deposition is not strictly proportional to lobular mass. Whilemeasurements incorporating information on individual lobes can beuseful, this may still not account for localised concentrations of drugcritical for driving pharmacological response.

In acknowledging that pharmacological activity is more closelyrelated to free drug concentration than total concentration, lungtissue homogenates can be subjected to equilibrium dialysis or ultra-filtration techniques to assess free drug fraction. An example of thisapproach is that of Wu and co-workers [126] using equilibriumdialysis of rat plasma and lung tissue homogenates to study the effectsof the different plasma and tissue binding profiles displayed bysynthetic glucocorticoids upon their occupancy of tissue glucocorti-coid receptors. Following intravenously infused des-ciclesonide andbudesonide, total drug concentrations in lung and plasma werereported to be comparable whereas the free concentration ofbudesonide in both lung and plasma was seven-fold greater thanthe free concentrations of des-ciclesonide; the greater free concen-tration for budesonide correlated with greater lung glucocorticoidreceptor occupancy.

The above example emphasises the critical importance ofmeasuring free drug concentrations. However, the use of theequilibrium dialysis or ultra-filtration is not standard practice in PK/PD investigations and indeed themeasurement of free drug fraction intissue 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 releasedrug from low affinity binding sites, it will also disrupt the normalcompartmentalisation of cellular proteins and indeed compartmen-talised drug (e.g. drug entrapped within endosomal compartments).Similarly, homogenisation of lung tissue will release drug intosolution that in the intact lung may remain undissolved, e.g. drug insuspension or dry powder dosage formulations. Indeed, the estab-lishment of pulmonary PK/PD relationships for low solubility drugs isall the more challenging. In summary, while lung tissue can be readilycollected in the non-clinical setting there exists very little publisheddata on the use of lung tissue homogenates in PK/PD experimentationand there remains uncertainty on how best to exploit this biologicalmatrix.

5.2.2. Bronchoscopic tissue biopsyBronchoscopy involves the insertion into the airways of either a

rigid bronchoscope or more commonly a flexible fibreoptic broncho-scope. Beyond tumor diagnosis, bronchoscopic tissue biopsy (eitherendobronchial or transbronchial biopsy) can be indicated in thediagnosis of infectious or immunological lung disease which may alsoinvolve a diffuse infiltrative component. Beyond tissue biopsy thetechnique is also amenable to obtaining bronchial epithelial cellbrushings.

The nature of the procedure means that any sampling for drugconcentration analysis (e.g. for antibiotics, steroids, and immuno-suppressants) is secondary to pathology needs. While a number ofintra-patient samples can be taken at any given time, the techniquewill generally yield individual patient data for a single time point only.Many of the issues raised above for tissue homogenates will also berelevant to tissue biopsy samples. Bronchoscopic tissue biopsy hasbeen applied to the study of antibiotic penetration and drugconcentrations in the lungs of patients undergoing a diagnosticprocedure [127,128].

5.2.3. MicrodialysisMicrodialysis involves the insertion of a probe directly into a tissue

for the continuous sampling of an unbound analyte such as a drug. Themethod depends upon a microdialysis probe tip consisting of a tube-like or capillary-like semi-permeable synthetic membrane, the core ofwhich is continuously perfused with physiological solution. The semi-permeable membrane restricts the passage of proteins and henceprotein-bound drug but affords the movement of free low molecularweight drug. Once inserted into the tissue the unbound drug willmove across the semi-permeable membrane by passive diffusion andthe continually flowing dialysate in the probe will be recycled forbioanalysis. The method is applicable to both animal and humanexperimentation [129].

Microdialysis is not without its limitations. It is an invasivetechnique than can disrupt normal tissue architecture at the site ofprobe placement. Such tissue damage may lead to vascular leakageand an inflammatory response both of which may require a recoveryperiod after probe implantation prior to experimental sampling. Dueto the low dialysate flow rates in the probe the collection timesto obtain a single sample for bioanalysis can be quite protracted(15–30 min). Such a relatively long collection interval may impact onstudies where free drug concentrations in the tissue are changingrapidly. The technique may also suffer from variable and low recoveryof the free drug into the probe which appears to be especially the casewhen the probe is inserted into an intact tissue, i.e. the free drugconcentration in the tissue maybe much higher than in themicrodialysis probe. This variable recovery may also be a feature ofthe physico-chemical nature of the analyte itself, with concerns that anumber of lipophilic drugs are particularly poorly recovered [130]while others are not [131].


An example of the use of microdialysis in the non-clinical setting isthe examination of the penetration of the antibiotic, cefclor, intoskeletal muscle and into lung tissue following intravenous adminis-tration in the rat [132]. Steady-state or equilibrium free concentra-tions of cefclor in skeletal muscle and lung while similar were only26% of the free drug concentrations in plasma. Such findings haveimplications for PK/PDmodelling of antibiotic treatment in interstitiallung infections. The same laboratory reported similar findings forcefpodoxime prior to conducting a clinical microdialysis study usingskeletal muscle (where probe insertion is less hazardous) as a tissuesurrogate for lung [133]. Indeed clinical microdialysis appears to beparticularly used for drug studies of anti-infective agents [134],including human lung infections [135].

5.2.4. Epithelial lining fluidEpithelial lining fluid (ELF) lies between the lung epithelial surface

and the gas in the lung lumen. The volume and composition of the ELFwill depend upon ion and water transport across the epithelium,passive liquid flow, evaporativewater loss, secretions of glandular andgoblet cells, and mucociliary transport. Albumin and other proteinsare present in ELF but at much lower levels than in serum, e.g. despitethe varied literature data all estimates of ELF albumin levels concludeit is ≤10% that of albumin concentration in serum [136,137].However, the volume and composition of ELF can change withpulmonary disease. Compared to blood levels, drug concentrations inELF may be more reflective of drug concentrations (total/free) in lunginterstitium and are more likely to relate directly to PD targets withinthe ELF itself such as bacterial lung infection.

Clinically, broncho-alveolar lavage (BAL) is used as a diagnostictechnique to sample cells, proteins, and other constituents of the ELF.It usually involves consecutive instillations (three to six) of sterilesaline (each 20 to 50 ml in volume) through a fibre-optic broncho-scope into a sub-segmental bronchus followed by immediate fluidretrieval by aspiration. While a standard inter-laboratory method forthe non-clinical collection of ELF has not been formalised, generalconsensus at the Workshop indicated that the BAL procedure in therat typically involves the tracheal instillation and subsequent recoveryof three consecutive 5 ml volumes of aqueous solvent. The use of a5 ml volume for each instillation implies that the significant majorityof rat lung airways and alveolar sacs should be exposed to lavage fluid.Among non-clinical scientists there was some debate as to the natureof the solvent that should be used in rodent BAL, i.e. normal saline orphosphate-buffered saline, and while some anecdotal information(unpublished) may suggest that this choice could affect the recoveryof pulmonary macrophages this is not broadly acknowledged. At thenon-clinical level, BAL appears to be used increasingly as a routinecomponent of pulmonary PK and PD investigations. It provides forthe gathering of PD endpoints, e.g. immune cells infiltrates into thelung lumen and products of inflammation, while simultaneouslyaffording the opportunity to estimate drug concentration in LF forPK/PD purposes.

One of the problems in undertaking BAL, whether non-clinically orclinically, is that it represents a dilution of what is essentially anunknown volume of ELF; it does not allow for the direct measurementof drug concentration in ELF alone. This is compounded by thetechnical limitations in not being able to recover the full volume ofinstilled lavage fluid, and as a corollary only partial recovery of ELF.Therefore for meaningful estimates of drug concentration in ELF thereis a need to determine by how much the ELF has been diluted by theBAL procedure. This is most commonly addressed by the simultaneousmeasurement of endogenous levels of urea in the retrieved BAL fluidand in serum. While not the only dilution indicator to be used,endogenous urea relies conveniently on its similar concentrations inboth ELF and in serum; a urea concentration ratio (BAL fluid:serum) ofless than unity indicates the dilution factor that should be applied tointerpret ELF drug concentrations. Nevertheless, such methodology is

not universally applied in non-clinical BAL studies even when theyseek to address drug concentrations in ELF.

Uncertainties do exist in the interpretation of BAL soluteconcentration data [137], which from a pharmaceutical perspectivemay 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 thelung segment contributed no more than 2% to the total aspirated BALvolume and that approximately 40% of BAL fluid volume had comefrom the systemic circulation or surrounding interstitium [138]. Theimplication of the latter is that significant drug transfer into the lungfrom serum or tissue interstitium may lead to overestimation of ELFdrug concentration. The impact of the BAL procedure upon diffusion ofserum urea into the aspirated BAL fluid has been reported [139,140].Variations in the BAL fluid volume instilled, the number of instillationsand the contact “dwell” time of the BAL fluid with lung epithelium canall affect the diffusion of serum urea into the lung with the potentialfor overestimation of the volume of ELF recovered and inappropriatedilution calculations. While levels of serum proteins are significantlylower in ELF, their impact upon pulmonary PD through influencingfree drug fraction still needs to be considered particularly for highprotein bound drugs. The cellular component of ELF is often forgottenin PK studies and lysis of, for example, pulmonary macrophagesduring the BAL procedure may artificially elevate apparent free drugconcentrations in ELF. From a drug delivery perspective, BAL fluidmaysolubilise drug from suspension or dry powder formulations that inthe ELF was undissolved. BAL measurements bring technical chal-lenges and the data are difficult to interpret. However, BAL fluid isobtainable both non-clinically and clinically and should prove to be auseful translational methodology for PK and PK/PD investigations.

BAL does not allow for the direct measurement of drug con-centration in ELF. Bronchoscopic microsampling (BMS) is a techniquefor sampling of ELF directly and repeatedly on the surface of abronchus by using a polyester fibre probe. The technique has beenexploited most frequently in measuring bronchial ELF concentration-time profiles for systemically administered antibiotics [141–145] anddetermining the duration that ELF drug concentrationsNminimuminhibitory drug concentration.

5.2.5. Induced sputumAlthough BAL represents a minimal risk procedure for patients it is

invasive. Induced sputum has been widely used to investigate airwayinflammatory disease and assess interstitial lung disease [146–149].Sputum is induced by inhalation of hypertonic (4 to 5% w/v) salinesolution delivered by ultrasonic nebuliser over variable periods oftime. Expectoration of sputum is encouraged by cough and isgenerally attempted after a cumulative period of approximately20 min saline inhalation. It can be repeated to collect more than onetime-point. Induced sputum has been best utilised when looking atthe cellular inflitrates of the more proximal airway ELF. While its usefor PD endpoints is established, the suitability of induced sputum forassessing ELF drug concentrations in PK/PD studies has not beentested.

5.2.6. ImagingImaging techniques can be used to evaluate dose deposition in the

lung following instilled or inhaled pulmonary delivery. The imagingmodalities available include gamma scintigraphy, a two-dimensional(2D) technique, and single photon emission computed tomography(SPECT) and positron emission tomography (PET), both three-dimensional (3D) techniques. These techniques provide informationon the amount of drug delivered to the whole lungs and thedistribution of the drug within the airways [150–155].

In the case of gamma scintigraphy the main, but not only, gammaradiation-emitting isotope that is used is 99mTechnetium (99mTc)which can be incorporated into the pulmonary formulation, e.g.


liposomes [156] or added to the formulation such that it forms aclose physical (non-covalent) association with the drug particlesallowing it to act as a marker for the drug. The latter approach istechnically more demanding and requires careful validation [152].Gamma scintigraphy has proved a valuable comparative technique forinvestigating pulmonary delivery, affording assessment of the extentand pattern (e.g. central versus peripheral) of lung deposition.However, the two-dimensional planar images obtained with gammascintigraphy do not account for the three-dimensional nature of thelung tissue and the drug deposition profile in the two-dimensionalimages includes an overlay of structures of interest (alveoli, small andlarge airways). Precise and quantitative measurements of theseindividual regions are more difficult [157].

Gamma emitting isotopes are also used in SPECT, a technique inwhich the gamma camera is rotated through 360° and affords thereconstruction of a three-dimensional tomographic image. This allowsa more complete profile of penetration through the lung compared toplanar techniques [158,159] and is able to differentiate betterbetween small and large airways. Regional deposition data may beexpressed either as sections through the lungs in transverse, coronal,and sagittal planes, as a series of lung shaped concentric shells, or asthe amount of drug in different airway generations. However, SPECT ismore technically and computationally demanding than gammascintigraphy [160]. Both planar gamma scintigraphy and SPECT aremore widely used in clinical studies but both have also shown non-clinical utility, including use in models of lung disease [161].

PET produces a three-dimensional image from radiation emittedby short-half-life positron-emitting radionuclides, e.g. carbon-11,fluorine-18. Drugs can be radiolabelled and biodistribution scansobtained, including clinical examples of the use of PET in pulmonarydeposition studies of inhaled drugs [162–164]. PET can also be usedfor non-clinical investigations [165,166] using small animal imagingscanners. PET imaging has even been used as a non-invasivebiomarker to assess response in tuberculosis-infected lungs totreatment with systemically administered antibiotics [167].

5.3. Pulmonary PD endpoints

There is a general consensus that appropriate PD endpoints thathave relevance to the progression of clinical disease, and whichprovide useful decision-making data, can be explored in non-clinicalstudies. The non-clinical PD endpoints of drug action that have foundmost utility include those that quantify pulmonary inflammation andairway hyper-reactivity/broncho-constrictiveness. BAL provides theopportunity to sample for inflammatory cytokines and leucocyteinfiltration and can be incorporated within studies quantifying real-time broncho-constrictive responsiveness to challenge [168]. Thepotential also exists to use imaging approaches [169] for some of thecell-based inflammation PD endpoints.

It is clear, however, that an improved understanding is needed ofthe disease pathology in acute and chronic non-clinical models andhow this pathology relates to the clinical condition. This should lead toimproved biomarkers that will enhance the translation of non-clinicaldata to the human disease, an objective which will also be facilitatedby quantitative PK/PD modelling for inhaled drugs that act locally tobring about pulmonary responses.

5.4. PK/PD for locally acting inhaled drugs

For drugs that access their respective pharmacological targetfollowing systemic exposure, the use of plasma concentration datacan generally provide for appropriate and predictive PK/PD relation-ships. However, the value of incorporating systemic drug concentra-tions into the PK/PD analysis for locally acting inhaled therapeutics ismore 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 complicatedby a number of factors — including multiphasic absorption processes(due to depot effects or differences in absorption rate from differentregions of the lung), the presence of non-absorptive lung clearanceprocesses and the fact that the swallowed component of the orallyinhaled dosemay contribute to systemic exposure (if the drug is orallybioavailable). In short, for inhaled drug delivery there is a cleardeficiency in understanding of the relationship between drugconcentrations in the lung and drug concentrations in plasma, aposition further compounded by the complexity of the lungarchitecture, variable drug deposition patterns following inhaleddelivery (Section 2) and the difficulty in sampling and interpretingdrug concentrations in the lung.

The problem is compounded by an almost complete lack of dataexamining local drug concentrations in the lung following pulmonarydrug delivery and seeking to relate such concentrations to a measuredpulmonary response. In contrast there is a body of work forsystemically administered anti-infectives studying the relationshipbetween blood levels of drug or area under the blood drugconcentration time curve (AUC) or drug dose administered uponthe clearance of lung infection; this includes non-clinical [170–172]and clinical examples [173]. Similarly there are examples of theclinical 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 inhibitoryconcentrations (MIC) for bacteriocidal activity [174–176] or toimproved FEV in cystic fibrosis patients [177]. The pulmonary deliveryof liposomal encapsulated ciprofloxacin in a rat model of pneumoniahas recently been reported with drug levels determined in ELF,alveolar macrophages and in serum [178,179]. These levels wererelated to MIC necessary to kill a variety of bacterium, includingintracellular organisms such M. tuberculosis.

One aspect of local lung delivery which has received attention inreview articles is that of the PK and PD considerations relating toinhaled corticosteroids, with notable contributions from the groups ofDerendorf and Hochaus [180–185]. While the qualitative consider-ation of these issues is important there is still lacking, however,substantive quantitative relationships between the PK and PD ofglucocorticoids although a useful technique for glucocorticoid PK/PDis the surrogate endpoint of ex-vivo pharmacological receptoroccupancy (see also Section 5.2.1.). Measurement of ex-vivo gluco-corticoid receptor binding has been used to assess the selectivity oflung targeting after pulmonary delivery [186] and the relationshipbetween free drug concentrations in the lung and pulmonaryglucocorticoid receptor binding [126]. This technique when appliedto other receptors, e.g. muscarinic, in the lung could be expected tohave greater utility in the development of quantitative PK/PDrelationships. For example, such data may help develop cross-speciesprediction of the relationship between pulmonary dose and receptoroccupancy.

The prediction of systemic and pulmonary PK across species wouldenable input of forecasts into PD measures and will contribute to thefurther understanding and development of quantitative pulmonaryPK/PD relationships; it is an important approach which can parallellaboratory or clinical experimentation. PK scaling between species byeither allometric or physiological approaches is a mature discipline.There is confidence among non-clinical scientists that inter-speciesmodelling of systemic blood levels following pulmonary drugadministration can be achieved with a reasonable degree ofconfidence. Predicting human lung drug concentrations from non-clinical data may be more challenging but examples of the successfulprediction of human drug concentrations in lung tissue from non-clinical species, albeit unpublished, are reported to exist in industry.With non-clinical PK/PD relationships established then predictions ofhuman effect- versus time relationships can be tested. There are tworecent published examples of predicting elements of the pulmonary


PK/PD response [187,188]. Goutelle and co-workers [187] undertookpopulation pharmacokinetic modelling andMonte Carlo simulation toexplore the pulmonary PK and PD of rifampin. They found theirmodelling to describe well the actual measured rifampin concentra-tions in the plasma and ELF in a population in human volunteers.Based upon Cmax:MIC and AUC24 h:MIC ratios their work supportedthe need to evaluate higher doses of rifampin for the treatment ofpatients with tuberculosis. Agoram and co-workers [188] proposed asolution to interpreting in-vivo PD for a series of six inhaled long-acting β2-agonists (LABAs) in the absence of PK data. Using a non-clinical model they established that the relationship betweenobserved maximum effect and area under the effect-time curve fordifferent doses of the LABAs could be described by a commonsigmoidal relationship. They concluded for that particular series ofmolecules the in-vivo duration of action was dependent upon potencyand not pulmonary PK differences; a modelling approach that aidedthe allocation of resources for lead candidate selection. Clearly such amethod, or variants of it, if more generally applicable would rely uponprecedented mechanisms of action within a class of drug molecule.

In summary, quantitative PK/PD approaches involving inhaleddrug and local pulmonary response are very much under-evidencedand a general uncertainty still exists regarding how best to use lungtissue data for PK/PD modelling. This current level of understandingand application needs to be addressed if the pharmaceutical industryis to avoid high attrition in the development of inhaled drugs thattarget novel mechanisms of actions.

6. Challenges and opportunities

The factors needed to produce oral compounds with more drug-like properties are defined by Lipinski's ‘rule of five’ [189,190]. Whilethe lung may be a more complex organ, is it possible to pool data onthe physical and chemical properties for inhaled delivery andgenerate a model that similarly reduces the attrition in developmentfor inhaled compounds? Selecting the right molecule is critical and,although formulation plays a part, the chemical structure ultimatelydetermines the fate of themolecule, so asmuch information should begathered as early as possible prior to engaging in long and costly

Table 3Opportunities for collaborative working to enhance inhaled product development throughcommon interest to innovator companies, contract research organisations, academia and re

Collaboration Objective

Network Harmonise/inform practiceDevelop expertise

Data sharing Develop an in silico model of drug depositionImprove dosimetry methods1) Protocol development and validation for drug

administration to non-clinical species2) Protocol development and validation for measuring drug

deposition/accumulation in the lungBenchmark normal variation for toxicology studies

Identify the physicochemical determinants of pulmonary PK

Research Profiling of lung deposition

Develop methods for drug solubility and dissolution

Understand the pathophysiology of phospholipidosis andfoamy macrophagesDevelop and validate biomarkers

Understand the impact and role of transporters in lungdispositionExtend PK/PD modelling and identify target localisation

animal studies. Pharmaceutical companies generate large amounts ofdata, much of which is archived or may be forgotten when moleculesfail during the discovery or development phases. This informationmay be of limited value within any one company, but combinedbetween companies (while avoiding potential IP pitfalls) such datacould constitute a large reservoir of shared information to aid drugdesign, PK, dosimetry paradigms, toxicology outcomes and appropri-ate methodologies. In fact, it can be argued that common value couldfar outweigh the risks of pooling such information and would benefitall by reducing the need to generate additional internal data.

There is a paucity of published data on the dosimetry of inhaleddrugs compared with the wealth of data available for environmentalparticulates. If the industry wishes to demonstrate clearly the truenature of novel inhaled medicines, then current practices will need toadvance considerably in order to understand how the drug behavesonce it has been administered to the lung. Here resides yet anotheropportunity to establish the facts collectively to build a betterunderstanding of how drug concentrations are attained in the lungsand how this affects drug action.

In toxicology studies, drug attrition remains high across theindustry for many different reasons including, inter alia: molecularcharacteristics, unexpected toxicologies and pulmonary accumula-tion. A collective understanding of the factors that preclude successful“developability” would benefit the entire industry and avoid theescalating costs of drug development. Again, opportunities to sharecommon cross-species control data in inhalation toxicology studies, tounderstand common biological responses to inhaledmaterial (e.g. thedevelopment of foamy macrophages in the alveoli of non-clinicalspecies) and adoption of common approaches to recording toxico-logical findings (with a common terminology) would all aidprogression. This may also apply to the development of in vitrosystems for early screening of common phenomena such asphospholipidosis — this would eliminate those molecules with alimited chance of successfully transitioning the development cycle.

Developing new respiratorymedicines remains challenging atmanylevels. Estimating the duration of action is an area that requires furtherinvestment. Understanding cellular trafficking during inflammatoryepisodes in patients and how such phenomena can be monitored early

strategic advances in inhalation science. The objectives are pre-competitive topics ofgulators.

Activity

Scientific meetings and dialogue with regulatorsCollaborative research projectsPool data for the development of an in silico model of drug depositionCompare 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 depositeddoses after administration of pharmaceutical aerosols and how best toassess lung accumulation of poorly soluble drug compounds.Sharing of toxicological control data to establish normal biologicalvariation and normal responses to inhalation exposurePooling of data to link physicochemical properties and inhaledpharmacokinetic profilesUse of imaging to assess dosimetry, impact of delivery devices ondosimetry, in vitro-in vivo correlation. The aim will be to harmonise doseestimations.Characterisation of lung fluids, development of simulated fluids and assaysto establish solubility and dissolution parameters for modellingformulation effects on pharmacokineticsMechanism-based research to understand the phenomena of foamymacrophage formation and the implications for safetyThe use of known irritants and marketed medicines to discriminateadverse from non-adverse effectsStudies into mechanisms and impacts related to active drug transport (fordetails see the accompanying article: Gumbleton et al., 2010)Use of imaging to measure precedented molecule receptor occupancy andhelp understand the relevant biophase for pharmacokinetic modelling


in non-clinical models and during therapy also remains unresolved. Inaddition, there are difficulties in relating effects in animal models tohuman disease. A greater understanding of drug deposition postinhalation remains an area poorly satisfied as the imaging techniquescurrently available do not provide sufficient detail to allow suchassessmentswith confidence. Clinical data suggests thatparticle size caninfluence the PD of inhaledmedicines [94], yet how suchfindings can beexploited for more novel medicines remains unanswered. Surely allthese areas cannot remain untouched and a coordinated effort acrossacademia and industry is what is required to bring some of thesechallenges forward. Cost efficiencies continue to be sought internallywithin companies in order to reduce costs and raise productivity, butthis will have a limited impact upon global success. Pooling informationand sharing challenges common to the industry are more likely to besuccessful in the long term than the prevailing silo mentality. A shift inapproach is required to bring together expertise and allow commonchallenges to be tackled and the benefits shared.

It is hoped that the Workshop at GlaxoSmithKline in Stevenage on10th July 2010 marked the beginning of a common understandingthat a progressive approach is required to aid development of newinhaled medicines. The immediate challenge is to generate theimpetus to take advantage of the opportunities for collaborativeworking discussed in this article (Table 3). This approach aims toworkto the benefit of all; not least in expediting the development of newinhaled medicines for the patients who need them.

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Challenges in inhaled product development and opportunities for open innovation

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