J. Aerosol Sci. Vol. 30, No. 3, pp. 289—297, 1999( 1999 Elsevier Science Ltd. All rights reserved

Printed in Great Britain0021-8502/99 $19.00#0.00PII: S0021-8502(98)00052-4

HUMAN TRACHEOBRONCHIAL DEPOSITIONAND EFFECT OF A HISTAMINE AEROSOL INHALED BY

EXTREMELY SLOW INHALATIONS

Martin Anderson,*st Magnus Svartengrenst and Per Camners

sInstitute of Environmental Medicine, Division of Inhalation Toxicology, Karolinska Institutet, Stockholm,Sweden

tDepartment of Public Health Sciences, Division of Occupational Medicine, Karolinska Hospital, Stockholm,Sweden

(First received 7 January 1998; and in final form 3 April 1998)

Abstract—Twelve subjects inhaled the same amounts of histamine aerosol in both a normalprovocation test and in a test with extremely slow inhalations (ESI), a method which givesdeposition in small airways. The purpose was to compare dose-effect relationships in large (diameter'1 mm) and small (diameter (1 mm) ciliated airways. The effect on large airways was estimatedby measurement of airway resistance (R

!8), and the effect on small airways was measured by the

single-breath nitrogen test phase III slope (N2-delta). Mouth and throat deposition of the histamine

aerosol was calculated from human experimental data, and deposition in the lower airways wascalculated using a computerised model for particle deposition in the lungs. The study showed thatonly half the dose inhaled with ESI (0.65 mg) of that inhaled with normal inhalations (1.3 mg), wasneeded to obtain the same degree of obstruction for large airways in spite of that the calculateddeposition in large airways was 40% higher for normal inhalations than for ESI. The threefoldstronger effect in large airways was probably caused by a more uniform distribution of particleswithin each generation with ESI compared to normal inhalations with particle deposition near thebifurcation. There was slightly higher N

2-delta with ESI, and the calculated dose to the smaller

airways for the same inhaled dose was 10 times higher with ESI than with normal inhala-tions. ( 1999 Elsevier Science Ltd. All rights reserved

I N T R O D U C T I O N

Optimum therapy of airway diseases as well as bronchial provocation tests requires agood knowledge of regional particle deposition. It is sometimes suggested that a use ofsmaller particles will increase deposition in small airways. If ‘‘small airways’’means ‘‘bronchioles’’ (i.e. ciliated airways with diameter less than 1 mm, correspondingto generation 12—16 in ‘‘Model A’’ (Weibel, 1963), it is evident from models of particledeposition in the lung of task group on lung dynamics (Bates et al., 1966) that deposition atnormal inhalation flow in these airways is not markedly enhanced by the use of smallerparticles.

With extremely slow inhalations (ESI) and rather large particles (6 km) it is possible toincrease deposition in the bronchioles by a factor of ten due to a decrease of depositionin large airways by impaction and to an increase by sedimentation in bronchioles (Agnew,1996; Anderson et al., 1995; Svartengren et al., 1995; 1996a,b; Falk et al., 1997; Camner et al.,1997).

The purpose of the present study was to inhale the same amounts of a histamine aerosolwith normal flow and with ESI and to compare the dose-effect relationships in large andsmall ciliated airways for the two inhalation modes. A histamine aerosol was chosenbecause it is used extensively in clinical provocation tests and although it is harmless to thesubjects, it induces marked effects.

*Author to whom correspondence should be addressed.

289

Table 1. Personal and lung function data of the 12 subjects. Predicted values were calculated according toEuropean Community for Coal and Steel (Quanjer 1993)

FEV*1

FVCs

Age Weight Height Rawt

Subject Sex (yrs) (kg) (cm) l % pred. l % pred. (kPa l~1]s~1)

1 M 24 75 189 4.9 99 7.2 122 0.112 F 24 68 173 3.8 105 4.4 106 0.173 F 49 61 157 2.1 88 3.1 111 0.254 F 21 58 164 3.4 101 3.8 99 0.095 F 43 61 163 3.2 116 3.9 121 0.156 M 27 70 173 4.1 98 5.1 104 0.217 F 26 53 163 3.6 113 4.7 129 0.198 F 22 57 170 3.0 84 3.5 86 0.209 M 26 75 182 5.8 127 6.9 126 0.10

10 M 31 55 174 3.0 86 4.7 117 0.1811 M 26 77 177 3.9 89 4.9 95 0.1912 F 26 70 182 4.9 107 5.8 106 0.09

Mean — 29 65 172 3.8 101 4.8 110 0.16SD — 8.5 8.5 9.4 1.0 13 1.3 13 0.05

*Forced expiatory volume in one second.sForced vital capacity.tAirway resistance.

MATERIAL AND METHODS

Subjects and design

Twelve healthy non-smokers inhaled a histamine aerosol generated by a Pari Inhalierboynebulizer (PARI GmbH, Starnberg, Germany; MMD"7.7 km). Personal and lung func-tion data are given in Table 1. Tests were done at three separate days; a pre-test, a normalprovocation, and a provocation with ESI. The provocations were adjusted to give the sameinhaled amount at the two types of provocation.

In the pre-test, forced expiratory volume in one second (FEV1) was used to measure the

airway response to histamine at normal inhalations. The last two concentrations of thecorresponding provocation doses given to each subject (half dose and full dose) were thenused in the normal and the ESI-provocations.

Both provocation tests started with measurements of airway resistance (R!8

) and phaseIII of single breath nitrogen washout (N

2-delta). Then the half dose was given. Three to six

minutes later R!8

and N2-delta was measured again. This procedure was then repeated with

the full dose. The orders of the normal and the ESI-provocations were randomised.Deposition in mouth and throat was calculated from our own experimental data with

radio-labelled test particles. Deposition in each generation of the lower airways andalveolar compartment was calculated using a computer-based model for particle depositionin the lung.

Provocations

In all provocations the subjects were sitting and wearing a nose clip.Pre-test, and normal provocation: Ten breaths of each concentration of histamine chloride

were inhaled from the nebuliser. In the pretest, stepwise doubled concentrations up to32 mgml~1 were used. Nebulization was controlled by a dosimeter (Spira Electro 2, SpiraRespiratory Care Center, Finland) and started after 20 ml of air had been inhaled andcontinued for another 0.8 s. Inhalation flow rate and volume were simultaneouslymonitored by the dosimeter. Each subject was instructed to inhale at least 0.6 l in eachbreath so that no aerosol should be resident in the nebulizer chamber or in the airways atthe end of inhalation. The two last concentrations and doses (half-dose, and full-dose) wereused in the normal provocation test.

290 M. Anderson et al.

Fig. 1. The accumulated masses as functions of particle diameter for inhalation flows 0.5 l s~1 (fulldrawn line) and 0.55 l s~1 (dotted line). The median volume diameter and 90% lower and uppervalues were for 0.5 l s~1 7.7, 3.0 and 17.2 km, respectively, and for 0.055 l s~1 7.8, 2.7 and 15.8km,

respectively.

ESI-provocation: Each subject inhaled both the half-dose and the full-dose with twobreaths. Nebulization was manually controlled and each breath lasted for 18.5 s, to give thesame inhaled dose as in the normal provocation. The inhalation flow rate was equal to theflow from the compressed air supply; 0.055 l s~1. The nebulizer inlet for excess air wasblocked, thus making it impossible to keep any other inhalation flow.

Aerosol measurements

Nebulizer output was measured by analysing radioactivity (99.Tc) on filters (RespigardII, Marquest Inc., U.S.A.) using the same set-up as in the provocations. For normal flowrate (0.5 l s~1) the dosimeter controlled the onset and duration of the nebulization as in thenormal provocations. The normal provocation inhalation pattern was mimicked witha syringe connected to the nebulizer via the filters. For ESI (0.055 l s~1) no syringe wasneeded as the nebulizer inlet for excess air was blocked and the nebulizer was left on withthe filters connected to the outlet.

Output measurements with a solution of 99.Tc verified that nebulisation times of 10]0.8at 0.5 l s~1 and 2]18.5 at 0.055 l s~1 gave identical outputs.

Droplet size was measured by a light scattering instrument (Malvern Mastersizer,Malvern instruments limited, Malvern, U.K.) with the nebulizer mounted close to the laserbeam (Clarke, 1995). Figure 1 shows the accumulated mass as a function of particle size. Itcan be seen from the figure that the mass distributions are practically the same for the twomodes of inhalation.

¸ung function tests

All tests were performed with the subjects sitting and wearing a nose clip. All lungfunction tests except R

!8was measured by a Pulmonary Function Laboratory Model 2100

(SensorMedics, Anaheim, CA, USA). The best of three measurements was used for FEV1

and forced vital capacity. Mean values were used for total lung capacity, N2-delta and R

!8.

R!8

was measured at a breathing frequency of approximately 2 Hz using a whole bodyplethysmograph (Model 2800, SensorMedics, Anaheim, CA, U.S.A.).

Extremely slow inhalations of histamine 291

Table 2. Deposition in mouth and throat in percent of all inhaled particles atextremely slow inhalations (Anderson et al., 1995; Camner et al., 1997)

Particle size (km) Sedimentation parameter DepositionD2/F (kml~1]s~1) (%)

6 923.1 21.88 1391 33.7

10.2 2168 51.4

Calculations of deposition

Mouth and throat deposition at 0.5 l s~1 was calculated from data on the relationshipbetween deposition and the impaction parameter D2F on 85 subjects described earlier(Anderson et al., 1993). For 0.055 l s~1 the relationship between mouth and throat depos-ition and the sedimentation parameter D2/F was used. Deposition of 6, 8, and 10.2 kmradiolabelled test particles inhaled at 39, 46, and 48 ml s~1, respectively is given in Table 2(6 km: Anderson et al., 1995, 8 and 10 km: Camner et al., 1997). The equation of theregression line from these data was

d"0.243#1.42(D2/F),

where d is deposition in mouth and throat, D is the aerodynamic diameter in km, and F isinhalation flow rate in 1 min~1. The equation was used to calculate deposition in mouthand throat for each of the 63 size classes of the inhaled aerosol given by the light scatteringinstrument.

The new size distribution representing the aerosol after mouth and throat for each flowrate was divided into five size classes and deposition in each airway generation for each classand flow rate was calculated using a computer based model on particle deposition in thelung. This model is based on equations for impaction and sedimentation (Bates et al., 1966),and on the human model ‘‘A’’ (comprising generations 0—23 in a lung volume of 4800 ml;(Weibel, 1963). In the model the dimensions of the airways can be altered so that bothR

!8and deposition can be calculated at various degrees of constriction (Svartengren et al.,

1995). We chose not to alter the diameter of generation 0—3 in the model since these havecartilage rings or plates and are not expected to alter in diameter much. Neither weregenerations 17—23 altered since these represent the alveolar compartment. In the depositioncalculations the measured R

!8values before the provocations, after half-dose and after

full-dose were used to determine the diameters of generations 4—16.

Statistical methods

Comparisons between lung function values were done using Wilcoxons signed rank test.A level of p(0.05 (two tailed) was considered to be significant.

RESULTS

Provocation and lung function

Median values and ranges of airway resistance (R!8

) and phase III of single-breathnitrogen washout (N

2-delta) for the two provocation tests are given in Table 3. Both lung

parameters were significantly higher than baseline values after half- and full-dose (p(0.05).Figure 2 shows the median increase in percent of the baseline values for R

!8and N

2-delta

in the two provocations. The percentage R!8

was significantly higher at the ESI-provoca-tion than at the normal provocation at half-dose (p(0.05), but not at full-dose. N

2-delta

was significantly higher for the ESI-provocation than for the normal provocation atfull-dose (p(0.05) but not at half-dose (p(0.1).

292 M. Anderson et al.

Table 3. Values (median and range) of airway resistance and slope three of single breath nitrogen washout beforeprovocation, after half dose, and after full dose at the two flow rates

R!8

(kPal~1]s~1 N2-delta, (% l~1)

Before After After Before After AfterFlow rate (l s~1) provocation half-dose full-dose exposure half-dose full-dose

0.5 0.17 0.24 0.36 0.87 1.15 1.72(0.09—0.27) (0.12—0.51) (0.18—0.71) (0.27—2.37) (0.53—2.40) (0.40—3.17)

0.055 0.18 0.32 0.43 1.02 1.52 2.17(0.08—0.22) (0.20—0.60) (0.25—0.92) (0.30—2.0) (0.50—3.0) (0.80—4.0)

Fig. 2. Values of airway resistance (R!8

) and slope of phase three of the single breath nitrogen test(N

2-delta) in percent of base-line values after half-dose, and after full-dose at inhalation flow rates of

0.5 and 0.055 l s~1.

Calculations of deposition and dose

Figure 3 shows the droplet mass distribution for the aerosol entering the mouth andthe calculated distribution after passing through mouth and throat, at both flowrates. Mouth and throat deposition was calculated to be 53 and 38% for 0.5 and 0.055 l s~1,respectively.

The calculated deposition patterns in the lower airways and alveolar region at 0.5 l s~1

differed markedly from those at 0.055 l s~1. Figures 4a and 4b shows the deposition at threelevels of R

!8, before, after half-dose, and after full-dose, at the normal provocation and the

ESI-provocation. At median R!8

measured before provocation only 3% of the aerosol massdeposited in the small airways (generations 12—16) at 0.5 l s~1, while at 0.055 l s~1 23% ofthe aerosol mass deposited in these airways (Table 4). From data on nebulizer output andconcentration, the median inhaled amount can be calculated. The median inhaled amountwas 1.3 mg histamine chloride at the full dose. From the regional deposition values togetherwith inhaled amounts, regional doses can be calculated. The calculations at the R

!8before

and after the provocation determine the upper and lower limits of the regional deposition,as R

!8increases during the provocation (Table 4).

Extremely slow inhalations of histamine 293

Fig. 3. Aerosol size distribution of aerosol entering the mouth and calculated size distributions forthe aerosol entering trachea at inhalation flow rates of 0.5 and 0.055 l s~1.

Table 4. Theoretical deposition in percent of inhaled particles at different flow rates, before provocation, after halfdose, and after full-dose

Deposition of inhaled amount (%)

Before provocation After half-dose After full-dose

MouthFlow and Gen. Gen. Gen. Gen. Gen. Gen. Gen. Gen. Gen.(l s~1) throat 0—11 12—16 17—23 Exhaled 0—11 12—16 17—23 Exhaled 0—11 12—16 17—23 Exhaled

0.5 52.8 28.5 2.7 11.6 4.4 31.9 2.1 8.9 4.3 34.8 1.5 6.7 4.10.055 38.0 20.5 22.9 15.6 2.9 22.8 19.2 17.2 2.8 24.5 17.1 17.6 2.7Ratio 1.39 1.39 0.12 0.74 1.5 1.40 0.11 0.52 1.5 1.42 0.09 0.38 1.5

DISCUSSION

At 0.5 l s~1 deposition in mouth and throat for particles larger than one or a few km canbe well predicted from human experimental data using the relationship between theimpaction parameter D2F and deposition (Anderson et al., 1993). At ESI, impaction is lowand the D2F parameter irrelevant. Instead deposition depends on sedimentation and shouldbe dependent on the sedimentation parameter D2/F. We therefore used a regression linefrom human experiments with 6, 8, and 10 km Teflon particles inhaled by ESI. Thecalculation of deposition in mouth and throat is thus for both inhalations based on relevanthuman experimental data. In order to take the polydispersity into account, mouth andthroat depositions were calculated for all the 63 size classes. The particle size distributionwas different for the two flow rates when the aerosols entered trachea.

To take the polydispersity into account, each aerosol (after mouth and throat) wasdivided into five size classes and regional lung deposition was calculated for each class. Theregional deposition within the lung was calculated using a model in which the airwaydiameters can be altered and deposition calculated at any chosen particle size, airwayresistance, and flow. This model gives similar results for the fractions of particles depositedin the tracheobronchial and alveolar regions whether the modified anatomic model of

294 M. Anderson et al.

Fig. 4. (a) Calculated deposition of the histamine aerosol in generations 0—23 of model ‘‘A’’according to Weibel at the two inhalation flow rates of 0.5 (a) and 0.055 l s~1 (b). Values for themeasured three degrees of airway resistance (before, after half-dose and after full-dose) are given foreach flow. The deposition is presented as the percentage of particles entering the lower airways.

Findeisen, which is used by Task Group on Lung Dynamics (1966) (Bates et al., 1966), or themodel of Weibel (1963) is used. The model is based on a uniform, symmetrical ventilation,but in vivo the filling of the lung is more complex. However, at normal flow our model givesrelationships between R

!8and tracheobronchial deposition which are highly similar to

relationships between measured R!8

and 24 h percentage retention found in experimentalstudies (Ericsson et al., 1995, Svartengren et al., 1995, 1996a,b).

Deposition at ESI is due almost solely to sedimentation, which makes a theoreticalprediction less complex. However, one problem here is that limited experimental deposition

Extremely slow inhalations of histamine 295

and clearance data are available, which makes the validation of the theoretical modelmore difficult than for the normal inhalation. At the normal inhalation when particlesare deposited mainly in large airways or in the alveolar region, the 24 h retention canbe used as a measure of tracheobronchial deposition (Camner and Philipson, 1978;Morrow et al., 1967; Smaldone et al., 1990, Stahlhofen et al., 1986; Yeates et al., 1981).However, deposition in small airways is not possible to estimate from 24 h retentionvalues since clearance continues at a relative fast rate for several weeks (Anderson et al.,1995; Falk et al., 1997), and long-term clearance studies using the ESI has not yet beenpublished.

Concerning the effect on the large airways we found that the median R!8

after thenormal provocation with full dose was about the same as the median R

!8of the ESI-

provocation with half-dose. Furthermore, at the same R!8

values, the calculatedregional dose to the large airways at the normal provocation was 39—42% higher (depend-ing on airway resistance) than for the ESI-provocation. R

!8is a measure well related

to the calibre of larger airways (Pedley et al., 1970). The large non-cartilaginous airways,i.e. airways that can be narrowed by smooth muscle contraction, (generation 4—11 inWeibel’s Model A) was calculated to contribute to about 80% of the airway resistance. Thismeans that R

!8should be a relevant parameter for the effects in the large airways in the

present study.In spite of the uncertainties in the dose calculations it seems reasonable that they can not

explain the three-foldly higher effect per dose to the large airways at the ESI provocationthan at the normal provocation. One explanation to the larger effect may be a differentdeposition pattern within each airway generation. At normal flow, impaction is the mostimportant deposition mechanism and deposition occurs mainly near the airway bifurcation.At the ESI, deposition by sedimentation gives a more uniform pattern within the airwaygenerations and also between the various generations. This might cause a more generalconstriction and a higher resistance. An alternative explanation might be that smoothmuscle contraction in large airways to a considerable extent was caused by the histaminedeposited in the small airways, but this seems not probable from the results of the effects onthe small airways (see below).

For small airways, N2-delta is a much less distinct measure than R

!8is for large ones. It

measures non-uniformity in alveolar ventilation and has been used clinically to detectchanges in small airways (Buist, 1975), but it is probably affected also by constriction inlarge airways. In the present study there was only slightly larger effects on N

2-delta with the

ESI than with the normal provocation.It doesn’t seem plausible that uncertainties in the calculated dose to the small airways can

explain the large differences in dose (a factor of 10) to the small airways between the twoprovocations (Table 4). This large difference in dose might have caused the significantdifference in the effect on N

2-delta.

In the present study, a new method was used to deliver a large fraction of inhaledhistamine to small ciliated airways and a standard method to deliver a large fraction tolarge ciliated airways. The results indicate that a uniform distribution of particles in largeairway generations induces a stronger effect than if the particles are located just near thebifurcations. Concerning the small airways, calculations from models of particle depositionshow large differences in deposition pattern between normal and extremely slow inhala-tions. The main problem is still to find a reliable measure of small airway narrowing that isnot affected by narrowing of the large airways. One advantage to use the ESI-technique ina provocation test is that regional doses are less dependent of airway obstruction than innormal provocation tests. We believe that our approach to use ESI for small airwaydeposition can be of value for evaluation of effects of other agents in large and smallairways.

Acknowledgements—The authors thank Lars Goran Persson and Ulla Bergsten for skilful technical assistance.This study was supported by grants from the Swedish Asthma and Allergy Association, the National SwedishEnvironment Protection Agency, the Swedish Heart Lung Foundation and the funds of the Karolinska Institute.

296 M. Anderson et al.

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