Validation of a New Breathing Simulator Generating

JOURNAL OF AEROSOL MEDICINE Volume 13, Number 2, 2000 Mary Ann Liebert, Inc. Pp. 139–146

Validation of a New Breathing Simulator Generating and Measuring Inhaled Aerosol with Adult Breathing Patterns K. NIKANDER, B.A.,1 J. DENYER, B.Sc.,2 M. EVERARD, M.D., Ph.D., 3 and G.C. SMALDONE, M.D., Ph.D.4

ABSTRACT The use of breathing simulators for the in vitro determination of the inhaled mass of drug from nebulizers has become widely accepted. Their use is, however, based on the assumption that there is a correlation between the in vitro and in vivo inhaled mass of drug. The aim of the study was therefore to investigate whether a new breathing simulator—the MIMIC Breathing Emulator (Medic-Aid Limited, Bognor Regis, UK)—could accurately emulate the in vivo inhaled mass of budesonide suspension for nebulization. Eight adult healthy subjects were included. Each subject inhaled for 2 min from a Spira Module 1 jet nebulizer (Respiratory Care Center, Hämeenlinna, Finland), charged with 1.0 mg of budesonide suspension for nebulization (0.5 mg mL 2 1 , 2 mL suspension, AstraZeneca, Sweden) and supplied with an inhaled mass filter between the nebulizer and the subject. The breathing patterns were recorded during the nebulization and simulated in vitro at two different experimental sites (simulations A and B) with the breathing simulator. With the patients breathing through the filters (in vivo test), inhaled mass of budesonide averaged 103.6 m g. The two in vitro experiments using the simulator revealed similar results with in vitro simulation A equal to 101.0 m g and simulation B 99.1 m g. There were no statistically significant differences between the in vivo results and those of in vitro simulation A. Results were significantly different for simulation B (p 5 0.032) although the difference was less than 4.5%. These data indicate that the breathing simulator can be used to accurately simulate sine waveforms, human breathing patterns, and the in vitro and in vivo inhaled mass of budesonide suspension for nebulization. Key words: breathing simulator, inhaled mass, nebulizer in vitro–in vivo validation, budesonide suspension for nebulization INTRODUCTION

I

that aerosol delivery to a patient can be estimated by havT H AS BEEN K N OW N FO R SO M E TIM E

ing the patient breathe the aerosol into a filter located between the mouthpiece or face mask and the device itself.(1–3) The quantity of drug on the filter represents the particles that would have

1

AstraZeneca R & D Lund, Lund, Sweden and Department of Clinical Physiology, Malmö General Hospital, Malmö, Sweden. 2 Medic-Aid Limited, Bogmor Regis, United Kingdom. 3 Sheffield Children’s Hospital, Sheffield, United Kingdom. 4 Pulmonary/Critical Care Division, Health Sciences Center, Stony Brook, New York.

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been inhaled by the patient if the filter was not there. This quantity of aerosol has been called the “delivered dose” or, more recently, the “inhaled mass”.(1) The latter term evolved as a result of attempts to avoid using the term “dose” because the aerosol inhaled is often not the actual dose to the lung. Inhaled mass can be measured in vitro by using a breathing simulator and sine waveforms to replace the patient.(3–5) This is obviously more convenient than in vivo measurements and allows multiple bench measurements to be performed reproducibly during the development of aerosol delivery systems. Some simulators have been built to mimic a single human inspiration and are used for in vitro tests of dry powder inhalers.(6) A number of simulators are run continuously using sine waveforms that in terms of mean tidal volume (V T ), breathing frequency (f ) and duration of inspiration as a fraction of the total breath (t i/t tot ) mimic a human tidal breathing pattern.(7–9) The latter, combined with a filter at the mouthpiece or face mask can be used to assess the function of nebulizers and pressurized metered dose inhalers with valved holding chambers. The use of breathing simulators is, however, based on the assumption that there is a correlation between the in vivo and in vitro inhaled mass of drug. This means that the breathing simulator should provide a reliable means for recording important characteristics of human breathing patterns, for replication of human breathing patterns and for testing the impact of breathing on the inhaled mass of drug. The aim of the study was therefore to investigate whether a breathing simulator—the MIMIC Breathing Emulator (10)— could accurately simulate the in vivo inhaled mass of budesonide suspension for nebulization.

MATERIALS AND METHODS Overall study design The study was divided into three parts. Part I used a standard instrument (Harvard pump, Harvard Apparatus, S Natik, MA) to test the accuracy of the breathing simulator to record and reproduce sine waveform air flow simulating human breaths. Part II extended the test to measuring the inhaled mass of nebulized budesonide using the Harvard pump and the breathing simulator. Finally, in Part III the breathing simulator

was applied to measurement of actual human breaths and to measurement of the amount of nebulized budesonide that would be inhaled with those breaths.

Breathing simulator The breathing simulator set-up consists of a pump (MIMIC Breathing Emulator), a pneumotachograph with transducer (MIMIC Breathing Monitor, Medic-Aid) and software (MIMIC Applications, Medic-Aid). The pump consists of a cylinder, a motor drive, a printed circuit board (PCB) and a power supply. It is controlled by a computer and can be used to reproduce patients’ breathing patterns with tidal volumes ranging from 150 to 2800 mL.(10) The pump is calibrated with a 1-L syringe. The transducer attached to the pneumotachograph converts the pressure differential into an electrical signal. Collected data can be evaluated regarding mean V T , f and t i/ttot.

Part I—Recording in vitro created waveforms Using a Harvard pump, five sine wave tracings were generated to reflect the breathing patterns of children and adults in terms of tidal volume (V T ), breathing frequency (f ) and duty cycle (t i/t tot ), that is, V T 500 mL, f 12, ti/ttot 0.5; 500 mL, 12, 0.4; 250 mL, 20, 0.5; 100 mL, 30, 0.5 and 100 mL, 30, 0.4. A t i/ttot of 0.5 was classified as “normal” and 0.4 as “obstructed.” A V T of 100 mL was chosen to test the breathing simulator below the specification of 150 mL. The sine wave tracings were simultaneously recorded by the breathing simulator and a strip chart Grass recorder, that is, an analog system (Grass Instrument Co., Braintree, MA). The instantaneous volume tracing was transferred to the Grass recorder via a potentiometer attached to the pump piston. The electrical signal was reproduced by the Grass recorder as a voltage adjusted proportionately to the volume. The voltage signal was differentiated electronically to give a relative flow tracing which provided an independent measure of the t i/ttot . The analog recorder had a timer tracing that allowed an accurate measure of the breathing frequency. By directly reading from the Grass recorder printout, a measure of V T , f, and t i/ttot for each sine waveform was obtained. We measured eight complete cycles for each waveform which corresponded to the software sub-routine of the breathing simulator which isolated eight consecutive breaths. By directly read-

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ing from the Grass recorder printout, a measure of V T , f, and t i/ttot for each of the five waveforms produced by the Harvard pump was obtained (“Recorded by Grass,” Table 1). These five waveforms were simultaneously recorded with the breathing simulator (“Recorded by simulator,” Table 1). Thereafter, the recorded waveforms were simulated with the breathing simulator (“Simulated,” Table 1).

Part II—Simulation of in vitro inhaled mass The aim of the second part of the investigation was to test whether there would be any differences in the inhaled mass of budesonide when using either a standard Harvard pump- or a breathing simulator-nebulizer set-up. The Harvard pump was set to produce a sine waveform with a V T of 500 mL, a f of 12 and a ti/ttot of 0.5. A constant output Spira Module 1 jet nebulizer (Respiratory Care Center, Hämeenlinna, Finland) was charged with 1.0 mg of budesonide suspension for nebulization (0.5 mg mL2 1 , 2 mL suspension, AstraZeneca, Sweden). Pari filter housings (Pari-Werk GmbH, Starnberg, Germany) with electrostatic filter pads (Filtrete TM Media, 3M Corporation, St. Paul, MN) with a diameter of 67 mm were connected to the nebulizer’s inspiratory and expiratory ports and the nebulizerfilter set-up was connected to the Harvard pump. The nebulizer was connected to a Schuco compressor (Schuco Incorporated, NY), operated at a

pressure of 2.0 bar with a flow rate of 7.5 L min2 1 through the nebulizer and run for 4 min. The test of the in vitro inhaled mass of budesonide was repeated with eight Spira nebulizers. The sine waveform was recorded using the breathing simulator with the pneumotachograph connected at the expiratory filter during the actual tests with the Harvard pump. During the second part of the test the Harvard pump was replaced by the breathing simulator. The test set-up was identical in all other respects using the same eight Spira nebulizers, the same amount of budesonide and running the nebulizers for 4 min. The simulated sine waveform was again recorded with the pneumotachograph connected at the expiratory filter for a comparison between the original and the simulated sine waveform in terms of V T , f, and t i/t tot . Budesonide was eluted from the filters with ethanol and analyzed by reversed-phase high-performance liquid chromatography (HPLC; Analytical Chemistry Department, AstraZeneca R&D, Lund, Sweden) for comparison of the amounts of budesonide deposited on the filters.

Part III—In vivo–in vitro correlation The aim of the third part of the investigation was to test whether there would be any differences in the inhaled mass of budesonide when using either healthy adult subjects or the breathing simulator. The breathing simulator was used

T A BLE 1. T H E M EA N T ID A L V O LU M ES (V T ), BR EA T H IN G F R EQ U EN C IES (f ) A N D D UT Y C Y C LES (ti /ttot ) O F F IV E SIN E W A V E T R A C IN G S C R EA TED W ITH A H AR V A R D P U M P , SIM U LT A N EO U SLY R EC O R D ED BY A G R A SS R EC O R DE R A N D TH E BR EA T H IN G S IM U LA TO R , A N D T H EN S IM UL A TED W IT H TH E B R EA TH IN G S IM U LA TO R Mean V T (mL) Recorded by Recorded by Simulated Recorded by Recorded by Simulated Recorded by Recorded by Simulated Recorded by Recorded by Simulated Recorded by Recorded by Simulated

Grass simulator Grass simulator Grass simulator Grass simulator Grass simulator

508 543 532 503 532 521 262 270 265 103 127 110 102 111 102

95% confidence limits 506, 541, 531, 501, 530, 519, 261, 269, 263, 103, 127, 109, 101, 110, 101,

510 545 535 504 534 522 264 272 266 104 128 111 103 112 103

Mean f

Mean ti /ttot

14.9 15.0 14.8 14.0 14.0 14.1 20.9 20.9 20.9 30.8 31.0 30.6 29.6 29.6 29.1

0.5 0.5 0.5 0.4 0.4 0.4 0.5 0.5 0.5 0.5 0.5 0.4 0.4 0.4 0.4

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FIG. 1. In the in vivo test set-up the subject’s breathing pattern was recorded with a pneumotachograph and transducer—the MIMIC Breathing Monitor. An inhalation filter attached to the inspiratory port of the jet nebulizer was used for catching the inhaled mass of drug.

to replicate in vitro the breathing patterns recorded with the pneumotachograph in the in vivo part of the study. Each nebulizer was used for one pair of breathing, that is, an in vivo and in vitro replicate. Eight healthy adult subjects with a mean age of 38 years (range 27 to 50), a mean height of 171.4 cm (160–186) and mean weight of 70.8 kg (54–88) were included. The study was performed in accordance with the principles stated in the Declaration of Helsinki and was approved by the South Sheffield Research Ethics Committee. All subjects gave written informed consent. The test set-up used for the in vivo measurements is shown in Fig. 1. A Low Dead Space (LDS; Medic-Aid) filter housing with an electrostatic filter pad (Filtrete TM Media) with a diameter of 67 mm was connected to the Spira nebulizer’s inspiratory and expiratory ports. The breathing pattern was recorded in real time with the breathing simulator. A wash-out system was used to ventilate the system and reduce the equipment dead space introduced by the filters. The vacuum “wash-out system” was calibrated so that the deviation from zero flow through the pneumotachograph was , 6 0.1 L min2 1 when the nebulizer was operated at 2.0 ( 6 0.1) Bar from a

medical air supply and the mouthpiece was sealed. The nebulizers were labelled with numbers from 1–8. The medical air supply was disconnected and the nebulizer was charged with 1.0 mg of budesonide suspension for nebulization. The subjects breathed through the nebulizer setup for 30 sec to allow their breathing pattern to stabilize. At 30 sec the medical air supply was again connected to the nebulizer. The subjects continued to breathe on the nebulizer set-up for 2 min, at which point the nebulizer was switched off, that is, 2 min 30 sec total time. Budesonide was eluted from the filters and analyzed as described above. In the in vitro set-up the subjects were replaced by the breathing simulator as shown in Fig. 2. The software was used to select the appropriate file with the subjects recorded breathing pattern from the in vivo test, that is, 30 sec to 2 min 30 sec. The nebulizer matching the file was selected for the in vitro test. The procedures regarding the nebulizers, the nebulizer charge with budesonide and filters were identical to the ones in the in vivo test. The in vitro tests were repeated at two different locations, that is, in vitro simulations A and B in replicates of three.

VALIDATION OF A NEW BREATHING SIMULATOR

143

FIG. 2. In the in vitro test set-up the subjects were replaced by a breathing simulator—the MIMIC Breathing Emulator. The simulator is made of a pump that consists of a cylinder, motor drive, printed circuit board (PCB), and power supply. The pump is controlled by a computer.

Statistical analyses Data were evaluated by analysis of variance (ANOVA) technique. All tests were two-sided on a 5% significance level. In Part I an ANOVA model with a single fixed factor source, that is, V T s from the Grass recorder (“Recorded by Grass,” Table 1), the breathing simulator (Recorded by simulator, Table 1) and the simulated waveforms (“Simulated,” Table 1), was used for comparisons on each of the five waveforms separately. In Part II an ANOVA model with fixed factors source (i.e., Harvard pump or breathing simulator) and nebulizer was used for comparisons. In Part III an ANOVA model with fixed factors source (simulation A, simulation B) and subject was used.

RESULTS Part I—Waveform analysis The V T s from the Grass recorder printout (“Recorded by Grass,” Table 1) and the simultaneous breathing simulator recording (“Recorded by simulator,” Table 1) differed by 3.1% (250 mL V T ), 5.8% (500 mL V T , t i/t tot 0.4) and 6.9% (500

mL V T , ti/ttot 0.5), whereas the V T s from the breathing simulator recording and the simulation (“Recorded by simulator” and “Simulated,” Table 1) only differed by 1.9–2.2% for these three waveforms (250–500 mL V T s). For the two 100 mL V T s outside the breathing simulator specification the differences ranged from 8.1 to 23.3%. The variability was low within each set of waveforms (95% confidence limits, Table 1) and all differences were therefore statistically significant (p , 0.001).

Part II—In vitro versus in vitro The selected Harvard pump sine waveform was recorded by the breathing simulator as V T 521 mL, f 12.28, and ti /t tot 0.5. This was simulated and once again recorded by the breathing simulator as V T 521 mL, f 12.48, and ti/ttot 0.5. The inhaled mass of budesonide, the amount of drug on the expiratory filters, the gravimetric suspension output, the initial nebulizer weight, and the charged nebulizer weight for both systems is shown in Table 2. There were no statistically significant differences between the Harvard pump and the breathing simulator.

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NIKANDER ET AL. T A B LE 2.

SU SP EN SIO N O U TP U T M EA SU R ED G R AV IM ETR IC A LL Y AN D A M O UN T O F B U D ESO N ID E O N I N SP IR A TO R Y E X P IR A TO R Y F ILT ER S F O R TH E H A R V A R D PU M P (H) A N D T H E B R EA TH IN G S IM U LA TO R (M)

AND

Spira jet nebulizer 1 2 3 4 5 6 7 8 Mean S.D.

Initial weight (g) H M 39.3 39.3 39.3 39.2 39.3 39.3 39.4 39.3 39.3

39.4 39.4 39.5 39.4 39.4 39.5 39.4 39.4 39.4

Charged weight (g) H M 41.2 41.3 41.4 41.1 41.2 41.3 41.4 41.2 41.3

41.4 41.4 41.3 41.3 41.4 41.4 41.4 41.4 41.4

Part III—In vivo versus in vitro The breathing patterns were successfully recorded from all eight adults during the nebulization (Table 3). The results of the deposition of budesonide on the inspiratory filters from the in vivo experiment is shown in Table 4 with the results from the two sets of in vitro simulations. The mean inhaled mass for the in vivo test was 103.6 m g, for in vitro simulation A 101.0 m g and for in vitro stimulation B 99.1 m g. There were no statistically significant differences between the in vivo and the in vitro simulation A results. The small difference between the in vivo and the in vitro simulation B results was, however, statistically significant (p 5 0.032) due to the low variability within each breathing pattern. The actual mean difference was less than 4.5%. Differences between the two in vitro tests were not statistically significant.

DISCUSSION The first set of in vitro experiments demonstrated that the breathing simulator facilitated accurate recordings and analyses of clinically relevant sine waveforms within the system’s specified range of V T s. The differences between the Grass recordings and the breathing simulator recordings were probably due to a combination of calibration errors and electronic integration errors. The Grass standard tracings were defined by a direct potentiometer readout on the recording paper whereas the breathing simulator integrated a flow tracing. In our experience differences of approximately 5% are typical for these

Vehicle output (g) H M 1.3 1.3 1.4 1.3 1.2 1.3 1.3 1.3 1.3

1.4 1.3 1.3 1.3 1.3 1.3 1.3 1.3 1.3

Inhaled mass filter ( m g) H M 154 186 203 209 182 177 156 181 181 19.5

166 168 161 163 178 192 167 181 172 10.7

Exhaled mass filter (m g) H M 196 196 222 166 206 198 173 233 199 22.4

203 197 179 187 218 223 193 224 203 17.0

recording methods. The breathing simulator faithfully replicated the tracings with negligible differences. The second set of in vitro experiments demonstrated that the breathing simulator could accurately reproduce the in vitro inhaled mass of budesonide for sine waveforms. This waveform analysis is a simplified representation of breathing that defines the basic parameters important for continuously operating nebulizers, that is, the tidal volume, breathing frequency, and duty cycle. For breath-enhanced jet nebulizers, that are directly affected by the patient’s own breathing, a closer representation of the flow pattern may be necessary to accurately estimate inhaled mass. The third group of experiments demonstrated that the breathing simulator can measure and reproduce in vivo waveforms such that in vivo/in vitro estimates of inhaled mass of budesonide are reproducible. The small difference between the two sets of in vitro simulations performed at different laboratories indicated that the breathing simulator was robust enough to facilitate reT A BL E 3. T H E B R EA TH IN G P AT TER N C H A R A C TER IST IC S O F TH E H EA LTH Y A DU LT S UB JEC TS G IV EN A S M E A N T ID A L V O LU M ES (V T ), M EA N IN SP IR A TO R Y M IN U TE V O L UM ES (V I), A N D M EA N D U TY C Y CL ES (t i /ttot ) A N D I N SP IR A TO R Y T IM ES Subject No. 1 2 3 4 5 6 7 8

Mean V T (mL)

Mean V I (L)

Mean t i/ttot

Inspiratory time (sec)

680 626 571 1103 964 458 407 1369

9.24 8.28 7.84 15.03 6.44 5.86 6.48 11.19

0.47 0.45 0.41 0.40 0.38 0.40 0.36 0.44

56.3 54.5 49.7 48.1 48.1 49.6 43.0 56.9

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VALIDATION OF A NEW BREATHING SIMULATOR T A BLE 4.

T H E R ESUL TS O F TH E D EP O SITIO N O F B U D ESO N IDE In Vivo E XP E RIM E N T A N D F R O M TH E T W O S ETS

FROM THE

I N SP IR A TO R Y F IL TER S In Vitro SIM U LA T IO N S

ON THE OF

Subject No.

In vivo (m g)

( m g)

In vitro simulation A ( m g)

(m g)

(m g)

In vitro simulation B (m g)

(m g)

1 2 3 4 5 6 7 8 Mean S.D. Median

138 109 106 108 84 87 84 113 103.6 18.4 107

134 104 97 101 81 80 87 122 100.8 19.2 99

126 99 109 108 84 85 86 116 101.6 15.7 104

130 96 107 101 85 89 83 115 100.8 16.1 99

127 97 99 100 89 83 88 102 98.1 13.5 98

132 99 116 100 85 82 89 105 101.0 16.8 100

138 98 97 104 91 81 74 102 98.1 19.1 98

peated in vitro emulations using the original human breathing patterns. The breathing simulator provides a reliable means for recording the important characteristics of human breathing patterns and subsequently testing the impact of breathing on the inhaled mass of drug on the bench. This approach facilitates the testing of different delivery systems in an efficient cost effective manner before embarking on expensive clinical trials. The breathing simulator is presently limited to breathing patterns with V T s . 150 mL with large errors introduced for V T s below 100 mL. To solve this problem, which is important for pediatrics, a Micro MIMIC Breathing Emulator has recently been developed for V T s , 150 mL (Personal communication with John Denyer, Medic-Aid). The breathing simulator has been designed for the evaluation of devices that are operated during tidal breathing such as nebulizers as well as pressurized metered dose inhalers with valved holding chambers, that is, low resistance devices. If used with higher resistance devices more powerful pump motors are required to maintain waveform accuracy. It has become increasingly recognized that in vitro nebulizer tests should incorporate simulated human breathing patterns.(1,3–5,7,9,11) A single sine waveform has been suggested as a standard for “CE” regulatory purposes and marking.(12) It is, however, recognized that the use of a single sine waveform is limited and an oversimplification for all clinical applications. For example, a recent study in 165 children demonstrated a large intersubject variability and age dependency of the inhaled mass of budesonide.(13) The variability was mainly attributed to differences in the childrens’

breathing patterns. If the objective of in vitro testing of nebulizers is to compare different nebulizers, under clinically relevant conditions, then a number of breathing patterns will be necessary to highlight clinically important differences in aerosol delivery. To characterize more complex nebulizers in vitro, a broad range of waveforms or breathing patterns may be necessary as shown by Knoch et al.(7) and Coates et al.(14) The former showed that using a breath enhanced Pari LC Plus jet nebulizer (Pari GmbH, Starnberg, G) and a piston pump the inhaled mass of sodium fluoride increased with increases in V T until a plateau was reached at about 300 mL. The latter study demonstrated that the inhaled mass of tobramycin was independent of the inspiratory flow with the Hudson nebulizer but highly inspiratory flow dependent with the Pari LC Plus nebulizer. The results of these studies as well as our study were obviously dependent on the specifics of the experimental set-up (i.e., devices) and the breathing patterns chosen. To fully utilize the potential of breathing simulators, we feel the test set-up should be standardized with minimal equipment dead space(10) and the breathing patterns or sine waveforms selected to represent human breathing patterns important for the clinical use of the device. The increase in number of new inhalation devices being introduced on the market combined with the need to better understand the properties of these devices have made extrapolations between in vitro and in vivo results desirable. The present results are the first to show that in vitro tests of drug delivery from nebulizers with a breathing simulator can replace in vivo filter tests. A unique aspect of this study was the demon-

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stration that breathing patterns recorded during in vivo filter tests for a characteristic population can be repeatedly used for in vitro breathing simulator tests during development and characterization of inhalation devices. The advantages of the in vitro testing are its reproducibility (i.e., independence from biologic variation), convenience and low cost, which makes it an ideal tool for device development. In vitro testing cannot, however, assess patient–device issues such as convenience and compliance of day-to-day usage.

CONCLUSION The results of the present study indicate that the breathing simulator—the MIMIC Breathing Emulator—can be used to accurately simulate sine waveforms, human breathing patterns, and the in vitro and in vivo inhaled mass of budesonide suspension for nebulization.

ACKNOWLEDGMENTS The study was supported, in part, by AstraZeneca R&D Lund, Lund, Sweden and MedicAid Limited, Bognor Regis, UK.

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6. Burnell, P.K.P., A. Malton, K. Reavill, and M.H.E. Ball. 1998. Design, validation and initial testing of the Electronic LungT M device. J. Aerosol Sci. 29:1011– 1025. 7. Knoch, M., and E. Wunderlich. 1995. Effect of age related breathing parameters on the performance of a new nebulizer system: An in-vitro study. J. Aerosol Med. 8:285– 288. 8. Mitchell, J.P., and M.W. Nagel. 1997. In vitro performance testing of three small volume-holding cham bers under conditions that correspond with use by infants and small children. J. Aerosol Med. 10:341–349. 9. Smaldone, G.C., M. Cruz-Rivera, and K. Nikander. 1998. In vitro determination of inhaled mass and particle distribution for budesonide nebulizing suspension. J. Aerosol Med. 11:113–125. 10. Nikander, K., J. Denyer, and G.C. Smaldone. 1999. Effects of equipment deadspace and pediatric breathing patterns on inhaled mass of nebulized budesonide. J. Aerosol Med. 12:67– 73. 11. O’Callaghan, C., and P.W. Barry. 1997. The science of nebulised drug delivery. Thorax. 52(suppl 2):S31–S44. 12. PrEN13554-1 Respiratory therapy equipment. Part 1— Nebulizing System and their Components. BSI, Chiswick, London, W4 4AL. 13. Nikander, K., and H. Bisgaard. 1999. The impact of constant and breath-synchronized nebulization on the inhaled mass of nebulized budesonide in infants and children. Pediatr Pulmonol. 28:187–193. 14. Coates, A.L., C.F. MacNeish, L.C. Lands, D. Meisner, S. Kelemen, and E.B. Vadas. 1998. A comparison of the availability of tobramycin for inhalation from vented vs unvented nebulizers. Chest 113:951– 956.

Article received on September 7, 1999 in final form, December 2, 1999 Reviewed by: John H. Dennis, Ph.D. William S. Beckett, M.D. Address reprint requests to: Kurt Nikander, B.A. AstraZeneca R & D Lund S-221 87 Lund Sweden E-mail: [email protected]

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