Validation of a General In Vitro Approach for Predictionof Total Lung Deposition in Healthy Adults

for Pharmaceutical Inhalation Products

Bo Olsson, PhD, Lars Borgstrom, PhD, Hans Lundback, PhD, and Marten Svensson, PhD

Abstract

Background: A validated method to predict lung deposition for inhaled medication from in vitro data is lackingin spite of many attempts to correlate in vitro and in vivo outcomes. By using an in vivo-like in vitro setup andanalyzing inhalers from the same batches, both in vitro and in vivo, we wanted to create a situation whereinformation from the in vitro and in vivo outcomes could be analyzed at the same time.Method: Nine inhalation products containing either budesonide or AZD4818 were evaluated. These comprisedtwo pressurized metered dose inhalers (pMDIs), a pMDI plus a spacer, four dry powder inhalers, and twodosimetric nebulizers. In vitro, an in vivo-like setup consisting of anatomically correct inlet throats were linked toa flow system that could replay actual inhalation flow profiles through the throat to a filter or to an impactor.In vivo, total lung deposition was measured in healthy adults by pharmacokinetic methods.Results and Conclusion: We could show that the amount of drug escaping filtration in a realistic throat modelunder realistic delivery conditions predicts the typical total lung deposition in trained healthy adult subjects inthe absence of significant exhaled mass. We could further show that by using combinations of throat models andflow profiles that represent realistic deviations from the typical case, variations in ex-cast deposition reflectbetween-subject variation in lung deposition. Further, we have demonstrated that ex-cast deposition collectedeither by a simple filter or by a cascade impactor operated at a fixed flow rate using a mixing inlet, to accom-modate a variable flow profile through the inhaler, predicts equally well the lung deposited dose. Additionally,the ex-cast particle size distribution measured by this method may be relevant for predicting exhaled fractionand regional lung deposition by computational models.

Key words: in vitro, throat cast, lung deposition, prediction, validation, inhalation, dry powder inhaler,pressurized metered dose inhaler, nebulizer

Introduction

The deposition of drug in the lung that would be, or hasbeen, achieved with a particular inhalation product in a

certain population using a specified set of user instructions isan important piece of information that can enlighten the in-terpretation of clinical outcome parameters, assist dose deci-sions when switching device and/or formulation in earlyclinical development, or guide inhalation product develop-ment. A general in vivo predictive in vitro method for lungdeposited dose has been a highly desirable goal for decades.The chief reason, of course, is that the measurement of lung

deposition is a time-consuming and expensive task that, as istrue for all intervention studies, carries some elements of risk.Such studies are therefore carried out less often than merited.An overview of the present level of understanding was pub-lished by Newman and Chan in 2008,(1) and a recent article onin vitro/in vivo correlations was published by Delvadia et al.(2)

For a more general overview of the subject, see the report fromthe ‘‘Thousand Years of Pharmaceutical Aerosols’’ conferencein Iceland in 2009.(3)

From the start of development of inhaled pharmaceuticals,it was obvious to scientists that, for particles to be depositedin the lung, these had to be small. Particle size distribution

AstraZeneca R&D, S-43283 Molndal, Sweden.The present address of Dr. Svensson is: EMMACE Consulting AB, S-24733 Sodra Sandby, Sweden.

JOURNAL OF AEROSOL MEDICINE AND PULMONARY DRUG DELIVERYVolume 26, Number 0, 2013ª Mary Ann Liebert, Inc.Pp. 1–15DOI: 10.1089/jamp.2012.0986

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measurement methodology was adopted from varioushealth protection fields whose chief interest was samplingaerosols from the environment. Inertial classification using(multistage) impactors served both fields well, because thematerial collected could be subsequently analyzed for itsproperties (such as content of chemicals or viable microor-ganisms). When used for pharmaceutical analysis, the inletto the impactor, which normally was vertically arranged,had for practical reasons a 90� bend in a conduit to accom-modate the need for most inhalers to be used in a horizontalposition: hence, an inlet throat. Rival designs of inlet throatswere promoted by different groups, but at this stage focushad shifted from in vivo predictability to quality control ofpharmaceutical inhalation products. The British Pharmaco-poeia (BP) provided the first standards in 1988. Includedwere the ‘‘metal impinger’’ inlet throat, a right-angled mi-tered metal tube [the forerunner of the present inlet throatspecified by the United States Pharmacopeia and the Euro-pean Pharmacopoeia (EP)], and also the ‘‘twin impinger’’inlet throat: a 50-mL glass bulb with inlet and outlet posi-tioned at 90� to each other. At about the same time, and forthe same reason, there was a need to define a cutoff size limitunder which particles were assumed to deposit in the lung[variously called the ‘‘respirable dose’’ or, less presumptu-ously, the ‘‘fine particle dose’’ (FPD)], and above whichparticles were assumed to deposit in the oropharynx. Var-ious limits at around 5 lm were used, in each case practicallydefined by the cutoff size at the calibrated flow rate of asuitable stage of the selected impactor. Later, in 1998, the EPdefined FPD as the emitted mass of drug in particles that hada measured (interpolated) aerodynamic particle diameter of5 lm and below in conjunction with the standardization ofthe flow rate at which a dry powder inhaler (DPI) should beassessed (that which gives a pressure drop over the DPI of4 kPa). Needless to say, a practical quality control limit haslittle to do with in vivo predictability. The inlet throat to theimpactor was much debated in a rather confused mix ofquality control versus clinical relevance arguments. Discus-sions ranged between advocating very large inlets, 0.5 L andabove, to allow a maximum fraction of the delivered dose toenter the impactor for size measurement,(4) to investigatingcasts of human cadaver throats to mimic the in vivo situa-tion.(5) That the inlet throat markedly influenced the mea-surements, especially for pressurized metered dose inhalers(pMDIs), was clear,(6) begging the question of which onewas most useful. The answer could perhaps depend on thepurpose—quality control or in vivo relevance—although,arguably, quality control of important performance charac-teristics needs to be in vivo–relevant. Comparing the FPDobtained with one of Swift’s adult throat casts(5) and the BPglass throat for three DPIs and one pMDI containing salbu-tamol, we showed a much improved correlation betweenlung deposited dose and FPD for the throat cast.(7) The im-provement mostly depended on a much larger fraction of thedose delivered from the pMDI being deposited in the throatcast than in the glass inlet, whereas the effect on theDPI measurements was relatively minor. This could not beexplained by the measured size distribution and clearlydemonstrated an interaction between inlet throat and aerosolcloud characteristics, showing that numerical manipulationof the size distribution measured with an inappropriate inletthroat cannot remedy such a flawed measurement. Even

using the anatomically more correct cast of Swift, the in vitromeasure overpredicted lung deposition, perhaps because apostmortem cast does not represent the throat of a livingperson, because the particular person’s anatomy was notrepresentative of the adult population, or for some otherreason. There are reports in the literature of attempts to finda better match between FPD and lung deposited dose byinvestigating the definition of FPD in terms of different cutoffvalues.(1) Because the generally observed discrepancy wasthat FPD, defined as particles smaller than about 5 lm,overestimated lung deposition, this then resulted in a sug-gestion of lower cutoff limits than 5 lm, based on the ob-servation that a better adherence to the identity line was thenobtained. But, as by definition the FPD becomes smaller byapplying a smaller cutoff value, this approach can only ad-dress the mean statistical mismatch, not the root cause of themismatch. It is obvious from the extensive literature on thedegree of lung deposition as functions of various parametersthat no specific cutoff limit exists; the probability for a par-ticle to deposit in the lung depends on a multitude of factors,each of which (including size) gradually changes the prob-ability of deposition. However, a more profound reason toreject the concept that lung deposition can be predictedbased on particle size downstream of the inlet throat is that,at this point, much, if not all, of the cloud dynamics havedissipated. Two examples may illustrate this importantpoint: (1) For a pMDI, the aerosol is expelled in a dynamicprocess where initially large droplets at high velocities veryquickly evaporate and lose momentum, the specifics ofwhich are device- and formulation-dependent. Once driedout (the idea behind large-volume inlets; see above), virtuallyall droplets leave residues that are small, but all informationon their initial dynamics is then gone. No manipulation ofthe size distribution of the residual particles can recreate theinitial dynamics and predict the interaction between the ex-pelled cloud and a human throat. (2) For a DPI, the aerosol isdelivered through a mouthpiece that, for different designs,imparts different spatial velocity profiles on the emittedparticles. As velocity, particle size, and vicinity of obstaclesdetermine the probability of impaction, again, no manipu-lation of the size distribution can recreate the initial dy-namics. For both cases, the interaction between clouddynamics and inlet throat will depend on the geometry ofthe inlet throat, but the interaction cannot be deduced fromthe measured size distribution or be translatable to anotherthroat geometry, for example, that of a human. However, fordevices that deliver a gentle cloud, we would expect thatinlet throat geometry is of minor importance. So, to improvepredictability and not just correlation, an in vitro parameterthat is causally related to lung deposition is needed. We ar-gue that a necessary component of such a parameter is arealistic measure of throat deposition. However, an equallyimportant component is that the dose delivered from a de-vice in vitro is made to equal the dose delivered to humansinhaling from the device.

By definition, total lung deposition equals the amount ofdrug that penetrates the throat region and is not exhaled. Byusing realistic drug delivery conditions (most importantly,we believe, are realistic flow-time profiles and device han-dling, although environmental conditions may also be in-fluential), the amount and quality of a dose delivered froman inhaler in vitro could be made to equal that delivered

2 OLSSON ET AL.

in vivo. By additionally using realistic throat models, theinteraction between the delivered aerosol cloud and this re-gion in vivo could be mimicked in vitro such that the amountand quality of drug deposited in this region becomes equal.As experience shows that the exhaled fraction is negligiblefor most pharmaceutical aerosols (with some important ex-ceptions to be dealt with below), the surviving aerosolshould then equal the lung deposited dose in both cases. Inprinciple then, total lung deposition could be predictedin vitro using a simple filter to collect the amount of drugpenetrating a realistic throat model under realistic deliveryconditions.

For prediction of regional deposition within the lung, thesize distribution of drug in the surviving aerosol should beuseful, provided the relationship between aerodynamicparticle size and deposition site is known for the inhalationregime in question. The size distribution may be determinedby directing the aerosol into a cascade impactor, and it hasbeen shown that the size distribution of the surviving aerosolis affected by the degree of filtration imposed by the throatmodel,(8) as expected. Arguably, therefore, the size distribu-tion for prediction of regional lung deposition (and exhaledfraction) should be measured downstream of a realisticthroat model.

Although a single throat model would be useful for esti-mating typical deposition, a range of models could poten-tially extend beyond a point estimate and, in addition,capture the expected between-subject variation in a popula-tion. By an initiative of Patricia Burnell, a consortium com-prised of scientists from GlaxoSmithKline, AstraZeneca, andsanofi-aventis initiated a cooperation with the HammersmithHospitals, London, UK, in order to develop a range of real-istic models of the oropharyngeal region. These were de-veloped based on magnetic resonance images of adultvolunteers inhaling from experimental devices.(9) Aerosoldeposition in these throat models has been studied usingmonodisperse particles,(10) as well as typical inhalationproducts.(11) The essential conclusion from these studies wasthat throat model deposition depended on the total volumeof the model, given the constraints of the overall shape of theoropharynx. Throat models representing high, medium, andlow filtering efficiency were identified using a pMDI, twoDPIs, and a nebulizer,(11) but there were no data to indicatewhether the filtering efficiency of the selected models trulyrepresented the range of the adult population. We used thethree selected throat models together with three selectedinhalation flow profiles in a pilot study to characterizein vitro deposition for two different budesonide DPIs andcompared these with lung deposition in adult volunteers forthe same DPIs.(12) The flow profiles had been selected fromhistorical recordings on the principle that they should rep-resent low, medium, and high flow rates for adults inhalingthrough these DPIs. The results supported the hypothesisthat lung deposition could be directly predicted by theamount of drug that penetrated the in vitro throat, with nopostprocessing of the data.

In the present article, we report on an extension of thebudesonide pilot study(12) to include one further DPI, twopMDIs (one of which was used both with and withoutspacer), and one dosimetric nebulizer. For completeness, weinclude the results of the pilot study. In addition, we describein lesser detail a pharmacokinetic study of AZD4818 [a small

molecule C-C chemokine receptor 1 (CCR1) antagonist(13)] todetermine lung deposited dose from a DPI and a dosimetricnebulizer, in which the same in vitro methodology as in thebudesonide studies was used to predict lung deposition.

Materials and Methods

Investigational products

Most of the inhalation products used here as test objectswere noncommercial but with release specifications similarto commercial products. Below we provide high-level de-scriptions of their nature.

DPIs. DPI_A contained a lactose carrier–based formu-lation with 5.5% micronized budesonide. The device was asingle-dose prefilled inhaler with a flow resistance (R) of 66Pa0.5 sec L–1.

DPI_B contained only micronized budesonide. The devicewas a multidose reservoir inhaler with R = 73 Pa0.5 sec L–1.

DPI_C was similar to DPI_B, but with a lower delivereddose.

pMDIs. MDI_A contained micronized budesonide sus-pended in HFA134a.

MDI_B contained micronized budesonide suspended inHFA227.

MDI_BS is the same pMDI as MDI_B, but used with anAerochamber Plus Flow-Vu anti-static Valved HoldingChamber (Trudell Medical International, London, ON, Ca-nada) modified to prevent any air from entering through thewhistle. The volume of this spacer is 149 mL.

Nebulizer. NEB_A was a Spira� dosimetric nebulizer(Respiratory Care Center, Hameenlinna, Finland) set at 1-secnebulization time after the start of inhalation for each breathand contained an aqueous suspension of micronized bude-sonide 0.5 mg/g.

The same batch of each product was used for both thein vivo and in vitro part of the studies.

In vitro methods

Throat models. Three throat models representing high,medium, and low filtering efficiency were used in this study.In the work where these were selected based on their filter-ing efficiency, they were designated L, C, and D, respec-tively.(11) These will here be referred to as small, medium,and large with reference to the buccal volume (for this and 10other key dimensional variables of the models, see Burnellet al.(11)). The stereolithography (STL) files defining the air-spaces of these models are published on the homepage of theInternational Society of Aerosol Medicine (ISAM; www.isam.org) and have recently been updated with information onhow to position the inhaler mouthpiece in relation to the inletof the anatomical model cavities. Several copies of eachmodel were manufactured by the following steps: embodi-ment of the airspace by stereolithography using the STL files,construction of a primary mold around the stereolithograph,molding of a silicone body in the primary mold, and finallymolding of the final model as a single part using polyure-thane as material with the silicone body in the center, fol-lowed by removal of the silicone part when the polymer had

IN VITRO APPROACH FOR PREDICTION OF LUNG DEPOSITION 3

solidified. Equivalence testing of the copies versus the orig-inal throat models was performed by volume measurementand dose measurements (dose ex-model) using a DPI and apMDI. As an alternative to the above-mentioned method,rapid prototyping [e.g., selective laser sintering/stereo-lithography (SLS/SLA)] can also be used to produce copiesof anatomical models, which is the method used by thecommercial supplier (EMMACE Consulting, Sodra Sandby,Sweden; www.emmace.se).

Inhalation flow-time profile targets. Historic flow-timeprofiles (n = 296) for 74 healthy adults instructed to inhaledeeply and forcefully from a DPI with a flow resistance of 66Pa0.5 sec L–1 were examined. The peak inspiratory flow (PIF)and the flow increase rate (FIR) between 10 and 40 L/min(FIR10–40) were calculated from the profiles (Fig. 1), and foreach of the two parameters the 5, 50, and 95 percentiles weredetermined. The three actual profiles closest to both 5 per-centiles, both 50 percentiles, and both 95 percentiles wereselected, and denoted weak, medium, and strong (Figs. 1 and2). The strong profile, for example, thus combined rapid FIRand high peak flow, following the general correlation be-tween these two parameters. Inhaled volume was dis-regarded in the flow profile selection, because the lower tailof the distribution (1.6 L) was far above that needed tocompletely empty the DPIs (for other populations or devices,volume may be an important selection criterion). Because theflow resistance of the device used for the profile samplingwas the same as that of DPI_A, the selected profiles wereused as recorded for this device. The flow resistance ofDPI_B and DPI_C was higher and, before being used forDPI_B and DPI_C, the flow rate values were scaled with theratio of the device resistances raised to the power of 0.84,(14)

i.e., they were 8% lower, in order to adjust to the likely lowerflow rates obtainable through these devices.

Collection of flow profiles for the pMDI devices, both withand without spacer, was performed in-house and includedmeasurement of the actual flow profile, but also where on thetime scale the press-fire operation (triggering) of the pMDIwas done. For this purpose, an instrumental recording sys-tem was developed that shunted all air intake to the pMDIthrough a silicone cap (allowing pressing of the canister tofire a dose) connected to a flow meter (Fig. 3). Three fullflow-time profiles, including time of triggering, were re-corded by this apparatus for each of 12 healthy volunteers,trained and overseen by a study nurse, for both the pMDIalone and for the pMDI with spacer. Instructions were tofully exhale, then start to inhale slowly, press down the can,and continue to inhale slowly and deeply. The averagetriggering delay compared with the start of inhalation wasabout a quarter of a second with large within-subject varia-tion (Fig. 4). Reasoning that the flow profile before triggeringwas irrelevant, and that the inhaled volume after triggeringwas sufficiently large ( > 0.7 L), we disregarded these featureswhen selecting profiles for the study. Instead the profileswere characterized by two parameters related to flow: (1) theflow rate at triggering, and (2) the average flow rate during1 sec (pMDI alone) or 3 sec (pMDI with spacer) after trig-gering. The 5, 50, and 95 percentiles were determined forthese parameters, and the three actual profiles closest tothese values for both parameters were selected (Figs. 5 and6), i.e., low flow rate at triggering was combined with lowaverage flow rate after triggering, etc., following the generalcorrelation trend for these parameters. The added flow re-sistance due to the flow measurement apparatus amountedto 0.5 kPa extra underpressure at a flow rate of 40 L/mincompared with the device alone, almost all of which was dueto resistance in the silicone cap. The difference between in-haling through the device alone and connected to the flowmeasurement apparatus was barely perceptible, but may

FIG. 1. FIR10-40L/min versus PIF for healthy adults inhalingdeeply and forcefully through DPI with flow resistance of 66Pa0.5 sec L–1, with the three selected profiles highlighted bylarger filled symbols.

FIG. 2. Selected flow profiles for DPIs (shown for flow re-sistance of 66 Pa0.5 sec L–1) as recorded in vivo (thick) andreplayed in vitro (thin). The mismatch for the strong profileafter about 3 sec depends on the limited volume capacity ofthe in vitro system and is judged to be of no importance forthe tested DPIs, because the powder is delivered in the be-ginning of the ‘‘inhalation.’’

4 OLSSON ET AL.

have led to the recording of slightly lower flow rates thanwould have occurred without the apparatus. We judge thatthis error, which could have been avoided by a more care-fully designed silicone cap, is of no material importance atthe low flow rates applied in the study.

The instructions for use of the nebulizer were to inhaleslowly at a target flow of 18 L/min for a volume of at least 1L and then exhale freely, and to repeat this for a set numberof inhalations. During inhalation, the subjects were guidedby watching a flow and volume meter under the supervisionof a study nurse. Nominally, this produces a flow-timeprofile that is essentially flat during the inhalation phase. Forthe in vitro assessments, flat profiles at 13, 18, or 24 L/minwere used to capture the assumed variation of flow profilesproduced by the subjects. This span represents the lowestoperable flow rate and the limit where the supervising nursewould react.

Setup. The throat casts were connected to different flow-generating and particle-collecting systems, depending oncircumstances. For particle size determination of the ex-castaerosol, the cast was connected to a mixing inlet (NepheleEnterprises, St. Paul, MN)(15) situated at the entrance port ofa Next Generation Impactor (NGI; Model 170, MSP Corp.,

MN), the outlet of which was coupled to a critical flowcontrol valve and a vacuum drain. The side port of themixing inlet was connected to a pressurized air source in linewith a breath profile generator (BPG; Nephele Enterprises,St. Paul, MN) consisting of a computer-controlled servomotor-operated piston in a 5-L cylinder (Fig. 7). Alternatively, whenparticle size was not measured, the cast was connected to aRespirGard IITM 303 glass fiber filter (Vital Signs Inc.,Englewood, CO) directly connected to the BPG. To charac-terize the pMDIs and pMDI plus spacer with different flowprofiles and coordination times, a mechanical hand wasconstructed. This was a grip around the pMDI body with apneumatic press function, which emulated the press actionfrom human handling. The press action was triggered from acontrol box, which in turn was triggered by the start of theBPG servomotor controller. The actual coordination time wasadjusted via the control box, which enabled a profile to beused with its recorded coordination time. The mixing inletand BPG were not needed for the flat profiles used with thenebulizer, and for this device the cast-NGI and cast-filtersetups were connected to the vacuum drain via a critical flowcontrol valve and a timed on/off valve. Airtight fitting of theinhalers to the casts was achieved by in-house molded sili-cone adapters designed for each device such that the inhaler

FIG. 3. Schematic of the setup used to measure flow profileand triggering time. The air inlet into the pMDI was re-stricted to a tube connected to a specially designed siliconecap that enclosed the upper part of the pMDI. A differentialpressure sensor (MPL 501, Micro Pneumatic Logic, Ft. Lau-derdale, FL) and a laminar flow element (LFE type 1, SpecialInstruments, GmbH, Nordlingen, Germany) were mounted inline along the air inlet tube. An optical sensor (FUE200C1004,Baumer Electric, Frauenfeld, Switzerland) was fixed at thepMDI, which reacted on canister movement. The pressuresensor and the optical sensor were connected to a device thatmeasured the time between the start of flow profile andcanister movement. The LFE coupled to a differential pressuretransducer (Digima Premo, SI-special instruments, GmbH,Nordlingen, Germany) enabled recording of the volumetricflow profile at 100 Hz via the analog-to-digital converter of anin-house–developed computerized lab system.

FIG. 4. Delay of triggering from start of inhalation (nega-tive if triggering preceded inhalation) for pMDI (upper panel)and pMDI with spacer (lower panel).

IN VITRO APPROACH FOR PREDICTION OF LUNG DEPOSITION 5

was fixed in a horizontal position perpendicular to the castopening with the front end of the mouthpiece flush with thecast opening.

Generation of flow-time profiles. The flow profile of in-terest was programmed into the BPG controller, and a re-cording volumetric flow meter equipped with a downstreamrestrictor having the same air flow resistance as the inhaler tobe assessed was placed in the opening of the cast using anairtight seal. For particle size measurement (Fig. 7), the im-pactor flow was first set to a volumetric flow rate about5 L/min above the peak flow of the target profile, with thepressurized air supply shut off. The pressurized air was thenturned on and adjusted to give zero flow in the cast. Thepiston of the BPG was set in motion by the BPG controller, sothat air was withdrawn from the supply line according to theprogrammed inhalation profile. As the air flow drawn by thevacuum drain was controlled by a critical valve, in whichsonic flow conditions defined the volumetric flow and held itconstant,(16) the volumetric flow entering the impactor re-mained the same, resulting in a compensating air flowthrough the cast with a profile similar to the target. Due toair compressibility, however, the programmed and replayedprofiles were not the same, the difference depending onpressure drops and dead volumes throughout the system,and thus were affected by inhaler (restrictor) resistance andflow rates. For each combination of target profile and inhalerresistance, an iterative process was used where the loadedprofile was adjusted in each loop to make the replayed andtarget profiles successively more alike. The replayed profilewas accepted as sufficiently similar to the target when FIR,PIF, and inhaled volume each differed less than 5% from thetarget values, and when the shape of the profiles showedoverall similarity (Figs. 2 and 6). The finally accepted BPGprogrammed profile for a specific target profile and inhaler

was saved to a disk for reuse in the actual performance tests.For the filter measurements, the iterative adjustment of theprofile loaded into the BPG controller essentially followedthe above procedure, although the process was simpler be-cause the filter was coupled directly to the BPG with noimpactor, mixing inlet, pressurized air supply, or vacuumdrain.

For the nebulizer, the air drain was simply a vacuumpump that was switched on every 15 sec to produce one ofthe three selected flow rates at the mouthpiece of the nebu-lizer. The operating time for the vacuum pump was adjustedso that 2 and 4 L was drawn through the filter only and NGI,respectively.

Procedures. To reduce bounce, the interior surfaces ofthe throat models were coated with glycerol/Brij 35 as de-scribed previously,(11) but with a shorter drain time (15–20 min instead of 120 min), as this was found sufficient to

FIG. 5. Average flow rate post triggering versus flow rateat triggering for healthy adults inhaling slowly and deeplythrough pMDI (filled symbols) and pMDI plus spacer (opensymbols), with the three profiles selected for each kindhighlighted by larger symbols.

FIG. 6. Selected flow profiles for pMDI (upper panel) andpMDI plus spacer (lower panel) as recorded in vivo (thick) andreplayed in vitro (thin). The vertical bars indicate the momentat which the pMDI was triggered to fire.

6 OLSSON ET AL.

reach a state where no more fluid was drained. Impactorcups were similarly coated.

All in vitro tests were performed in controlled facilitieswith the following environment: 30% relative humidity (RH)and 21�C. Prior to each in vitro experiment, it was confirmedthat the target flow profile was replayed correctly throughthe inhaler. The mouthpiece of a primed DPI, pMDI, ornebulizer was connected to the inlet of one of the throat casts.For each determination, four doses were withdrawn from theDPIs and pMDIs. For the nebulizer, 100 ‘‘inhalations’’ wereused for a determination and the filter unit was replacedafter each set of 20.

For DPI_A and DPI_B, all determinations used the im-pactor setup. For the other devices, one round of determi-nations (i.e., the nine combinations of throat models and flowprofiles) used the impactor setup, and two to four roundsused the filter setup (see Table 3 for number of replications).

The amount of budesonide deposited in the filter unit andon the various parts of the NGI was determined by a vali-dated liquid chromatography method. Quantitative recovery(95–105%) of budesonide deposited in the impactor, filters,and throat models was demonstrated.

Characteristics of the mixing inlet–BPG system. Thecharacteristics of the mixing inlet–BPG system describedabove were evaluated by a series of validation tests. Theleakage at an underpressure of 4 kPa in the system wastypically around 100 Pa/sec, i.e., in accordance with thespecification for the NGI leakage test according to the man-ufacturer. Patient profiles with a maximum PIF of about 95L/min could be replayed with the NGI-based system, be-cause the maximum impactor flow was 100 L/min, and thismust be higher than the flow through the inhaler. Themaximum effective volume of the piston was 4 L, which gavea maximum volume inspired through the inlet cast of thesame. The stability of the pressurized and vacuum drivenflows was determined by a flow meter at the cast inlet afterthese had been set to balance each other out to zero flow inthe inlet. The average flow rate over 1-sec periods wasmeasured for 5 min at two flow rates through the impactor,28.3 and 80 L/min. At both impactor flows, the standarddeviation (n = 300) of these measurements was < 0.1 L/min,indicating both a short- and long-term stability judged to besufficient to preclude any release of powder from a DPI si-tuated in the inlet before a flow profile was initiated. Themaximum flow acceleration for a DPI with R = 78 Pa0.5 sec L–1

was determined at an impactor flow of 66 L/min and aloaded square wave profile (0–60 L/min in 10 msec). Theresulting flow profile through the DPI had a FIR10–30L/min of16 L/sec2. Repeatability was measured for a target profilewith FIR10–30L/min = 8 L/sec2, PIF = 70 L/min, volume = 0.61L, for R = 66 Pa0.5 sec L–1 and an impactor flow of 77 L/min.The coefficient of variation was 0.4, 1.8, and 1.1% for PIF,FIR, and volume, respectively. The accuracy of replayedversus target triggering delay for pMDIs was found to be– 20 msec. An important feature of the mixing inlet is thatthe entire dose released from the inhaler is to be collected inthe impactor. A prerequisite for this is that losses in themixing inlet are low. This has been evaluated in more than 30studies of a variety of pMDI and DPI inhalers. The deposi-tion in the mixing inlet (percentage of total recovered dose)ranged between 0.4 and 2.8%, averaging 1.1% in thesestudies. The performance is summarized in Table 1, and wasjudged fit for the purpose for all investigated parameters.

Statistical methods. All analyses were performed usinglog-transformed data, the results of which were back-transformed to the linear scale by exponentiation. Reportedaverages are thus all geometric means. Coefficient of varia-tion (CV) was calculated from 100exp(var-1), where var is thevariance of the log- transformed data, or the estimated factorvariance from an ANOVA on log-transformed data. p < 0.05was regarded as significant.

In vivo methods

Procedure. The two in vivo studies that formed part ofthis investigation were both open-label, randomized, single-dose, single-center, and crossover studies. The studies wereperformed to estimate pharmacokinetic parameters afterpulmonary and intravenous administration of budesonideto healthy volunteers. Both the pilot(12) and the main studywere approved by the local ethics committee before start.The primary objective of both studies was to estimate thepulmonary bioavailability of budesonide. It has previouslybeen shown that the oral administration of activated char-coal blocks the gastrointestinal uptake of swallowed-downbudesonide and creates a situation where all systemicallyavailable budesonide has reached the systemic circulationvia the lungs, and it can thus be used as a surrogate for thepulmonary bioavailability.(17) It is also well known thatlung deposition is linear with given dose and that

FIG. 7. Schematic of the mixing inlet/BPG/impactor setup. (Left panel) Balanced flow (no piston movement, no flowthrough inhaler). (Right panel) Movement of piston creates flow through inhaler according to BPG program, but holdsvolumetric flow rate in impactor constant. The flow values cited are arbitrary examples to illustrate the principle.

IN VITRO APPROACH FOR PREDICTION OF LUNG DEPOSITION 7

budesonide does not undergo metabolic degradation in thehuman lung. Pulmonary bioavailability therefore can beequated to lung deposition. To create this situation, subjectswere administered an aqueous slurry of 10 g of activatedcharcoal (Carbomix, Solvay Pharma, Brussels, Belgium)before inhalation and 5 min, 1 hr, and 2 hr after inhala-tion.(18) The slurry was swirled around in the oral cavitybefore being swallowed.

Before the actual study days, the subjects trained on in-halation from the different devices to be used in the study,according to the respective instructions for use (see Inhalationflow-time profile targets above). On the actual study day, thesubjects trained on inhalation from the particular device tobe used on that day. The inhalation of study drug was per-formed in a room separate from the room where bloodsamples were taken. Inhalations were performed at ambientindoor conditions at the clinic. Subjects and clinical person-nel wore protective clothing and vinyl gloves to avoid sub-sequent contamination of blood samples. All inhalers wereprimed by the study nurse before use. Subjects were in anupright standing position while inhaling from the handhelddevices, and sitting while inhaling from the nebulizer. Theconsecutive inhalations from the handheld devices weredone without any unnecessary pauses. For the pMDI plusspacer inhalations, each actuation into the spacer was fol-lowed by one inhalation. For the nebulizer, the subjects wereallowed a short pause after each set of 25 inhalations. On thelast treatment day, the subjects received a 10-min intrave-nous infusion of budesonide (200 lg) 4 hr after the inhala-tion.(19) Treatments were spaced with at least 6 days inbetween, which is known to result in complete washout ofbudesonide in the plasma.(18) Plasma samples were collectedup until 8 hr after the last administration (inhaled or intra-venous). The pilot study comprised 12 healthy, nonsmokingvolunteers (8 males, 4 females, average age 25.3 years) whoinhaled two doses of budesonide from either DPI_A orDPI_B in each of the two study arms. The main studycomprised 16 healthy, nonsmoking volunteers (14 males, 2females, average age 28.4 years) who inhaled four doses ofbudesonide from either DPI_C, MDI_A, MDI_B, or MDI_BS,or 100 breaths from NEB_A, in each of the five study arms.

Results

In vitro results

The mean amount of budesonide delivered from eachdevice type was determined by taking the geometric meanover all experimental conditions (Table 2). For the DPIs andthe nebulizer, this nominal delivered dose (NDD) was de-rived by adding the amount deposited in the throat model tothat exiting the throat model and recovered from the filter orimpactor. There was no significant effect on NDD due to theselection of either throat model or flow profile (ANOVA,p > 0.05) except for DPI_A, where the delivered dose wasaffected by flow profile, but not throat model (about 10%,and statistically significantly lower for the weak comparedwith the strong flow profile; Fig. 2). For the pMDIs, NDDwas determined from separate measurements of the dosedelivered to a filter at 30 L/min (without cast) interspersedwith cast experiments (as dose delivery from the pMDIs isflow-independent in the flow range used, this simplificationis adequate and avoids the need to analyze cast depositions).As the spacer was used with all three flow profiles beforebeing rinsed, the spacer deposition is an average over theflow profiles. The geometric mean spacer deposition was123 lg (CV 17.1%, n = 9) with no effect of throat model(ANOVA, p > 0.05).

The amounts of budesonide exiting the casts for the dif-ferent combinations of throat model and flow profile aregiven in Table 3 as percentages of the respective NDD, to-gether with ANOVA results. The effect of flow profile wassignificant for all devices except DPI_A and NEB, and withlarger CVs for the two pMDIs without spacer. Where sig-nificant, the amount of budesonide that penetrated the throatmodel tended to be lower with the weaker flow profiles.Note that for the pMDIs, the coordination time is part of theflow profile. The effect of throat model was significant for alldevices except MDI_BS, with very large CVs for the twopMDIs without spacer. Where significant, the amount ofbudesonide penetrating the model was lower with thesmaller throat models. For all devices except MDI_BS, theeffect of throat model was stronger (higher CVs) than theeffect of flow profile. However, this observation is condi-tional on the fact that the flow profiles are from healthyvolunteers in a controlled setting and show a rather smallvariation compared with patient studies (as is probably truealso for the in vivo part of present study). The interactionterm between throat model and flow profile was statisticallyinsignificant for all devices, indicating that the ANOVA

Table 1. Summary of the Performance of the Breath

Profile Generator, Mixing Inlet, and pMDI Trigger

Validation parameter Results

Leak test of the whole system 100 Pa/sec @ - 4 kPaMaximum flow entering cast 95 L/minMaximum volume of inspiration 4 LStability of no flow baseline Standard deviation

< 0.1 L/min for 1-secperiods over 5 min

Maximum flow acceleration(FIR10–30L/min)

16 L sec - 2 @ R = 78Pa0.5 sec L - 1

Repeatability of DPI flowprofile; PIF = 70 L/min,FIR10-30L/min = 8 L sec–2,volume = 0.61 L

< 2% CV for PIF,FIR, and volume

Deposition losses inmixing inlet (DPIs, pMDIs)

1–3% of total recovereddose

Triggering delay time for pMDI – 20 msec from target

Table 2. Nominal Delivered Dose In Vitro

(NDD) Scaled for the Same Number

of Actuations Used During In Vivo Testing

Device Geometric mean (lg) CV (%) n

DPI_A 505 6.3 27DPI_B 714 7.7 27DPI_C 663 6.6 27MDI_A 756 7.3 24MDI_Ba 601 4.1 48NEB_A 434 8.0 27

aIncluding results for MDI_B during spacer experiments(MDI_BS).

8 OLSSON ET AL.

model was appropriate. There were no significant differencesbetween the ex-cast amount recovered from the impactorand filter setup (paired t test) except for NEB_A, which hadabout 10% lower amounts in the impactor compared withthe filters, due to about a 10% difference in the delivereddose measured between these ex-cast setups. The reason forthis is not known.

In vivo results

All subjects received all treatments in the two studies,apart from one subject who did not receive the DPI_Ctreatment. Data from the two studies were pooled andevaluated with a drug disposition model, based on a two-compartment model of the systemic fate of budesonide. Inthe model, drug is absorbed from a depot compartment intothe central compartment (inhaled drug) or directly infusedinto the central compartment (intravenous drug) from whichit is eliminated or transiently distributed to a peripheralcompartment. The model was fitted to all data simulta-neously by nonlinear mixed effect modeling (NONMEM�

ver. 7.2, ICON Development Solutions, Ellicott City, MD).The 95% confidence interval for the typical pulmonary bio-availability (lung deposition) was estimated by Log-likeli-hood Profiling using PsN software (Perl-speaks-NONMEMver. 3.4.2, Uppsala University, Uppsala, Sweden).

Actual plasma curves with typical-value fit are given inFigure 8. Estimated lung deposition of budesonide for thedifferent devices is given in Table 4 together with between-subject variability and precision estimates of these parame-ters, as well as the measured exhaled fraction. The estimatedvalues for volume of distribution (Vss = 278 L), clearance(CL = 1.57 L/min), and terminal half-life (t½ = 2.5 hr) comparereasonably with literature values.(18)

For all treatments, the bioavailability of budesonide, i.e.,the typical lung deposited dose, was well determined asjudged from the low standard error (5–12% of the estimate;Table 4). The between-subject variability ranged from 11%(DPI_C) to 43% (DPI_B). The precision by which between-subject variability was estimated for these cohorts is fairlylow with standard errors around 20%. The lung depositionestimate obtained for NEB_A is very high, especially con-sidering that it does not include the exhaled fraction. To-gether, the estimated lung deposition plus the measuredexhaled fraction for this device amounts to almost 96% of theNDD. To us, this indicates that the in vitro determined NDD

may be an underestimation of the dose delivered in vivo withthis device. However, we have no observations to explainthis surprising result.

Discussion

The goal of the present study was to validate the previ-ously observed in vitro ex-cast drug mass as a predictor fortotal lung deposited dose(12) for a broad range of inhalationproducts over a broad range of degree of lung deposition.The full combination matrix of three in vitro casts, aimed toreflect an adult population, and three flow-time profiles(adjusted for each product to represent the likely range ofusage by healthy adult volunteers) was investigated. Thecenter point (medium cast–medium flow profile) was aimedto be predictive of typical lung deposition, whereas the othereight combinations were aimed to be predictive of between-subject variability in lung deposition.

Both the in vitro data and the lung deposition estimateswere originally obtained in units of mass. In both domains,the deposited mass was converted to a deposition percentageby division with the NDD (Table 2) determined in vitro foreach device. It is important to realize that this conversionfrom mass to deposition percentages does not alter the rel-ative closeness between the in vitro and in vivo estimates for aparticular device, but rather provides a convenient scale onwhich to view the results from the different devices together.

In Figure 9, the individual lung deposition estimates areshown together with the individual ex-cast determinations,grouped according to the throat model used. The residualin vitro variability (Table 3) was for all devices larger than theeffect of flow profile (Table 3), so little is lost by groupingaccording to throat model only. Generally, the difference inex-cast deposition between the medium and large cast wasminor and only seen with the DPIs (Fig. 9). The differencebetween the small and medium cast was large for the pMDIsalone, moderate for the DPIs and nebulizer, and nonexistentfor the pMDI with spacer. Compared with the distributionsof lung deposition estimates, it is clear that the aerosol fromthe pMDIs used without spacer is filtered more extensivelyby the small cast than by any of the healthy volunteers in thecohort. For the DPIs, there is a mixed picture: for DPI_A, thein vitro and in vivo distributions agree; for DPI_B, the in vivodistribution is clearly wider; whereas for DPI_C, it is theother way around (again due to the small cast filtering moreextensively than any subject in the cohort). For the pMDI

Table 3. Ex-Cast Amount of Budesonide as Percentage of NDD, plus CV and p Value from ANOVA

Parameter Geometric mean (% of NDD) CV (%) p value na

Total Flow Throat Residual Flow ThroatFlow profile Strong Strong Strong Medium Medium Medium Weak Weak Weak All All All All All All All AllThroat model Small Medium Large Small Medium Large Small Medium Large All All All All All All All All

DPI_A 43.6 55.6 60.9 46.2 54.3 62.6 43.7 51.9 59.8 52.7 14.1 2.0 16.0 4.3 0.07 0.00 3DPI_B 39.4 51.7 55.3 41.3 44.9 53.4 37.1 40.4 46.7 45.2 15.7 7.7 13.5 8.7 0.00 0.00 3DPI_C 49.8 60.8 63.2 44.0 60.3 59.8 43.6 50.5 58.9 54.1 15.5 5.9 14.6 8.0 0.01 0.00 3MDI_A 5.6 32.2 26.0 5.4 26.9 25.2 4.2 19.0 19.9 14.4 91.7 18.7 113.9 13.7 0.00 0.00 5MDI_B 20.6 55.5 55.9 18.2 47.4 53.6 12.6 31.7 41.7 33.2 58.2 23.5 65.0 11.7 0.00 0.00 5MDI_BS 56.0 60.9 62.2 56.7 59.8 61.0 52.4 51.5 50.1 56.6 12.3 8.0 0.0 10.5 0.00 0.46 5NEB_A 52.3 87.4 87.3 65.6 88.9 86.5 72.5 89.0 91.1 78.9 20.8 5.3 19.4 11.5 0.08 0.00 3

aNumber of replications.

IN VITRO APPROACH FOR PREDICTION OF LUNG DEPOSITION 9

FIG. 8. Plasma profiles after each of the seven inhaled budesonide treatments (circles and dotted line) for each of thesubjects together with typical-value fit (coarse line). At the last visit, 4 hr after inhalation, an intravenous infusion of 200 lg ofbudesonide was administered.

10 OLSSON ET AL.

with spacer and the nebulizer, the match is reasonable.Overall, the in vitro and the in vivo variability agree fairlywell, but for each product on its own, the power of thespread in the in vitro determinations to predict the in vivodistribution is limited [compare Total CV (Table 3) and BSV(Table 4)]. This is perhaps not unexpected considering thatthe cohorts were small. Further, a direct comparison betweenthe essentially random variation in vivo with the essentiallysystematic variation in vitro is not straightforward. Despite

these limitations, the in vitro data still strongly suggest that,for healthy volunteers, variation in throat anatomy is moreimportant than variation in flow profile in causing between-subject lung deposition variability. It also clearly demon-strates the potentially large effect on lung deposition of asmall throat, particularly for pMDIs. The study in which thethree casts were selected to represent the adult population(11)

suffered from having no lung deposition data and only asmall number of products for which cast filtration data weredetermined using pharmacopeial flow profiles. With thebenefit of the larger and more relevant data set in the presentstudy, it may be suspected that the large cast is too similar tothe medium to be worth the experimental effort to use both.It may also be suspected that the small cast is rather extreme,which, however, does not take away its usefulness for illu-minating differences in how inhalation products interactwith throats, but rather tempers the expectation to encountersuch an individual in a small study.

The in vitro center point—medium cast and medium flowprofile—was designed to represent the typical healthy vol-unteer. Figure 10 provides a plot of typical lung depositionversus in vitro ex-cast deposition for the center point, includ-ing the 95% confidence intervals for the mean. Included in theplot are two additional inhalation products: the CCR1 an-tagonist AZD4818 delivered by a DPI and a dosimetric neb-ulizer for which ex-cast deposition using the center point and

Table 4. Typical-Value Lung Deposition Estimates

with Between-Subject Variability (BSV)and Associated Standard Errors (SE),

and Exhaled Fractions

Device

Estimate(95% confidence

interval), %NDDSE, % ofestimate

BSV, % ofestimate

SE, %of BSV

Exhaled,%NDD

DPI_A 57 (51–63) 6 13 30 NDDPI_B 45 (35–59 13 43 18 NDDPI_C 65 (60–70) 5 11 22 0.9MDI_A 20 (17–25) 10 39 12 < 0.2MDI_B 46 (40–54) 9 28 20 < 0.2MDI_BS 59 (54–66) 7 17 23 < 0.2NEB_A 92 (77–102) 8 25 21 3.7

ND, not determined.

FIG. 9. Estimated individual lung deposition and individual in vitro ex-cast results grouped by throat model (small,medium, large).

IN VITRO APPROACH FOR PREDICTION OF LUNG DEPOSITION 11

lung deposition has been estimated (see Appendix 1 for de-tails). All results fall close to the identity line, covering a broadrange of values from about 20 to 90% lung deposition andinclude results for a broad range of pharmaceutical aerosols.This vindicates the conclusion of the pilot study (DPI_A andDPI_B)(12) that ex-cast deposition, with no postprocessing ofthe data, provides an excellent fit under conditions of a real-istic throat model, flow profile, and handling procedure. Itstrengthens the scope of the conclusion from the pilot study,using only two DPIs, to additional DPIs and three other dis-tinctly different products in terms of aerosol delivery charac-teristics: (1) two pMDIs with rather different performance, (2)a pMDI used with a spacer, and (3) a dosimetric nebulizerused with both suspension and solution formulations. Wetherefore conclude that the hypothesis that the amount ofdrug deposited in the lung may be predicted from the mass ofdrug escaping deposition in a realistic throat model in vitro isvalid, provided that the conditions for drug delivery in vitroemulate the in vivo situation. We emphasize the fact that nopostprocessing of the ex-cast deposition was used or is war-ranted in the investigated domain. From a philosophical pointof view, this is highly rewarding because, by definition, totallung deposition equals the inhaled dose minus the amountdeposited in the throat region in the absence of exhaled mass.We believe, therefore, that our results clearly demonstrate thefutility of searching for a size limit applied downstream of aninadequate throat model to predict lung deposition.

Importantly, these results are from three separate studiesof lung deposition and in vitro ex-cast deposition usingmatched samples, i.e., using unadulterated products from thesame batch for each product in both domains. This is a un-ique strength of the present investigation in that, to our

knowledge, this is the first time that a successful head-to-headcomparison of in vitro predictions and lung depositions hasbeen performed, hereby avoiding selection bias. This is incontrast to other studies using physical models (e.g., themodels from Finlay’s group(20) and Byron’s group(2)) of thethroat region to estimate the in vitro properties of a specificproduct, and where the lung deposition results used forcomparison were cited from previously published clinicalstudies and not performed as part of the published in vitrostudy. This does not imply that these other throat models areless useful or less predictive. It simply means that a solidvalidation of their predictability is lacking at this point in time.

As alluded to above, the problem of predicting the fractionexhaled remains to be addressed. In the present study, onlythe nebulized aerosols showed significant exhaled fractions, atabout 4% of the delivered dose; the handheld devices, wheremeasured, showed < 1% exhaled fraction (Table 4). In bothFigures 9 and 10, the exhaled fraction is (of course) not in-cluded in the lung deposition, and unaccounted for in the ex-cast deposition. In principle, the exhaled fraction may bepredicted from the particle size distribution, measured by theimpactor downstream of the throat model, and estimatedparameters for the population morphometry and breathing,using a computational lung deposition model. In practice,however, these models are well known to overestimate ex-haled fractions for pMDIs and DPIs, although not for nebu-lizers. As an illustration, the predicted exhaled fractions forDPI_A, MDI_A, MDI_B, MDI_BS, and NEB_A were 4.6, 0.3,0.5, 1.3, and 3.0% of NDD, respectively, using the center-pointdata of ex-cast deposition (Table 3) and mass-median aero-dynamic diameter (MMAD; 1.8, 3.6, 3.2, 2.5, and 6.4 lm, re-spectively), and the NCRP computational lung depositionmodel(21) and reasonable parameter values. For the nebulizedaerosol, the model prediction is reasonable, whereas they aretoo high for the handheld devices (see Table 4). Nevertheless,computational models for exhaled fraction, and/or in vivoexperience for similar products, may give a sufficiently accu-rate guidance as to whether exhaled fraction is important ornot, and hence illuminate the credibility of ex-cast depositionas a predictor for lung deposition for a particular formulation.For example, some solution pMDIs that deliver aerosols withMMAD less than 1 lm are known to be associated with ex-haled fractions of more than 10% of the delivered dose.(22)

Clearly, ex-cast deposition for such products would need to beadjusted for this fact in order to be predictive.

This investigation has demonstrated predictability of totallung deposition in healthy adult subjects for nine pharma-ceutical inhalation products of various kinds using the de-scribed in vitro methodology. However, from a broaderperspective, we think it is reasonable to also claim that theinvestigation has validated the use of an in vivo–mimickingin vitro approach (throat models, flow profiles, and devicehandling). The difficulty, of course, lies in knowing if themimicking is good enough for the purpose at hand. There isa huge difference between using predictions to guide prod-uct and clinical development where the risk is held by theproducer, and using predictions for clinical claims where therisk is held by the patients.

We believe that this investigation is sufficiently solid tojustify the usage of this in vitro methodology, specifically thethroat geometry, for predicting total lung deposition inhealthy adults, to DPIs, pMDIs with and without spacers,

FIG. 10. Lung deposition versus in vitro ex-cast depositionfor medium cast and medium flow profiles. Squares, pMDIs;diamond, pMDI plus spacer; circles, DPIs; triangles, dosi-metric nebulizers. Solid symbols, budesonide; open symbols,AZD4818 (see Appendix 1). Geometric mean and 95% con-fidence interval for the mean are shown. Dashed line is theidentity line.

12 OLSSON ET AL.

and dosimetric nebulizers (keeping the limitation for exhaledfraction in mind). We also believe that extrapolation to adultpatients (in addition to healthy adults) may be justified. Inthat case, flow profiles may need to be adjusted, perhapstaking inhaled volume into account, to match the usage bythe patient population of interest. For children, the need forin vivo–mimicking in vitro methodology is even greater thanfor adults because of ethical and practical issues aroundpediatric studies. The results of the present investigationsupport the efforts in this direction using pediatric throatmodels and flow profiles.(23,24)

In its most basic implementation, medium cast–mediumflow profile–ex-cast filter, the methodology is very simple,quick, and cost-effective. This provides a point prediction oftypical lung deposition. At the next level of complexity, thefull matrix of three throat models and three flow profilesprovides an indication about the expected between-subjectvariability of lung deposition. The limitation of these simpleimplementations is that only the incoming mass to the lungis predicted. When the exhaled fraction or mucociliary clear-ance may significantly distort incoming mass from beingequal to (effective) lung deposition, or when the regional de-position pattern within the lung is of interest, then the ex-castsize distribution is relevant. This significantly more complexmeasurement necessitates divorcing an unsteady flow-timeprofile in the inhaler with a steady flow in the cascade im-pactor. The mixing inlet and associated paraphernalia allowthis to be achieved with minimal losses while sampling theentire dose. How the particle size information collected in thisway can be used is outside the scope of this article, as is apossible predictability from in vitro information to clinicaloutcome, as the fine-tuned regional deposition as well as thepharmacological properties of the substance under study willinfluence the resulting clinical effect.

Conclusion

We have shown in three separate studies, for nine differ-ent inhalation products, including DPIs, pMDIs with andwithout spacer, and dosimetrically nebulized solution andsuspension formulations, that the amount of drug escapingfiltration in a realistic throat model under realistic deliveryconditions predicts the typical total lung deposition intrained healthy adult subjects in the absence of significantexhaled mass. We have further shown that, by using com-binations of throat models and flow profiles that representrealistic deviations from the typical case, variations in ex-castdeposition reflect between-subject variation in lung deposi-tion. Further, we have demonstrated that ex-cast depositioncollected either by a simple filter or by a cascade impactoroperated at a fixed flow rate using a mixing inlet, to ac-commodate an unsteady flow profile through the inhaler,predict equally well the lung deposited dose. The ex-castparticle size distribution measured by this method may berelevant for predicting exhaled fraction and regional lungdeposition by computational models.

Acknowledgments

Pia-Lena Berg, Marika Borgeke, Inger Forsbrant, RebeccaGronvall, Ann-Sofie Hansson, Asa Lefevre, and GunillaOberg, all from the AstraZeneca Lund in-house clinic, aregratefully acknowledged for running the studies, as always,

to the highest standards; Per Mandahl, Annika Kullberg, andAnnika Eklund are gratefully acknowledged for leading theclinical study teams. Sven Andersson, Taraneh Monemi,Dario Kronauer, Peter Elfman, and Fredrik Gunnarson aregratefully acknowledged for their work with the in-vitromethods and Mikael Carlsson for his development and de-sign of hardware components on the in vitro setup.

Author Disclosure Statement

All four authors were full-time employees of AstraZenecaR&D, Lund, Sweden, at the time of the performance of theinvestigations. No conflicts of interest exist.

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Received on May 16, 2012in final form, December 6, 2012

Reviewed by:Andy Clark

Warren FinlayPeter Byron

Address correspondence to:Dr. Bo Olsson

AstraZeneca R&DS-43283 Molndal

Sweden

E-mail: bo.l.olsson@astrazeneca.com

(Appendix follows /)

14 OLSSON ET AL.

Appendix 1. Estimation of Lung Deposited Dose and Ex-Cast Deposition for AZD4818 DPI and DosimetricNebulizer

AZD4818 is a small molecule C-C chemokine receptor 1(CCR1) antagonist for which clinical development has beenterminated. AZD4818 is highly soluble in water.

Investigational products

DPI_D contained a mixture of micronized lactose and21.6% micronized AZD4818. This multidose reservoir inhalerhad a flow resistance of 66 Pa0.5 sec L–1.

NEB_B was a Spira� dosimetric nebulizer set at 1-secnebulization time after start of inhalation for each breath. Anaqueous AZD4818 nebulizer solution of 12 mg/g was used.

In vitro ex-cast deposition

The same in vitro methodology as described for filter de-terminations in the main study was used. The center pointwas measured (medium flow profile–medium cast). ForDPI_D, the same flow profile as for DPI_A was used. ForNEB_B, a constant flow rate of 18 L/min was employed(same as for NEB_A).

The nominal delivered dose (NDD) was determined to be1,572 lg (CV 4.6%, n = 23) for DPI_D (4 actuations per de-termination) and 1,153 lg (CV 5.8%, n = 14) for NEB_B (14inhalations per determination).

The mean ex-cast filter deposition was 1,036 lg (65.9% ofNDD, CV 12.4%, n = 5) for DPI_D and 983 lg (85.2% of NDD,CV 2.6%, n = 3) for NEB_B.

Lung deposition study

The primary objective was to estimate oral and pulmonaryabsolute systemic bioavailability and basic systemic pharma-cokinetic parameters of AZD4818. The study was an open,single-dose, randomized (between inhalation treatments),four-way crossover study approved by the local ethics com-

mittee before start. AZD4818 was administered to healthyvolunteers via four different routes and included 12 subjectsaged 18–55 years of whom 11 completed all treatments andwere included in the evaluation. At the first treatment visit,intravenous AZD4818 was administered (480 lg during20 min). At the next two visits, a nebulized solution (1,153 lginhaled in 14 breaths) and a dry powder (1,572 lg in 4 inha-lations) was inhaled in randomized order. At the last visit,AZD4818 was given orally (1,360 lg in 30 mL of solution).Blood samples were collected for up to 48 hr post dose. Urinewas collected for up to 48 hr post dose for the intravenous andoral treatments. The minimum time between treatments was 7days. After intravenous administration, plasma samples weremeasurable up to 12–24 hr post dose, whereas all urine sam-ples were above the lower limit of quantification. After inha-lation, almost all plasma samples were measurable. The finalmodel comprised seven compartments (lung depot followedby two transit compartments into the central compartment,two peripheral compartments, and the gut). For inhaled do-ses, the model assumed that the gut fraction was 1 minus theestimated pulmonary bioavailability. The model was fitted toall data simultaneously by nonlinear mixed effect modeling(NONMEM� ver. 7.2, ICON Development Solutions, ElliotCity, MD). The 95% confidence interval for the typical pul-monary bioavailability (lung deposition) was estimated byLog-likelihood Profiling using PsN software (Perl-speaks-NONMEM ver. 3.4.2, Uppsala University, Uppsala, Sweden).

A shorter terminal half-life after intravenous administra-tion (20 hr) than after inhalation (140 hr) showed that ter-minal elimination after inhalation was absorption rate–limited (‘‘flip-flop’’). Urine data were essential to establishthe short terminal half-life after intravenous administration.Oral bioavailability was estimated as 1.6%. Most importantin the present context is that the estimated absolute pulmo-nary bioavailability (95% confidence interval) was 82% (72–93%) of NDD for NEB_B and 67% (61–72%) of NDD forDPI_D. The exhaled fraction of the dose was 4.5% of NDDfor NEB_B and 0.6% of NDD for DPI_D.

IN VITRO APPROACH FOR PREDICTION OF LUNG DEPOSITION 15