Multiple flow exhaled nitric oxide

This measures exhaled nitric oxide as a biomarker of airway inflammation and distinguishes between inflammation arising from the bronchial (large airways) and alveolar (small airways) compartments in obstructive lung disease (Kharitonov et al, 2002).

2.7.1 Equipment and materials

 Niox®Flex, Aerocrine, Solna, Sweden.

 Gas cylinder certified to contain approximately 200 parts per billion nitric oxide (calibration gas), Air Liquide, Germany.

 Niox®Flex bacterial / viral filters, Air Safety Limited, Lancashire, UK.

88 2.7.2 Calibration of the equipment

Gas calibrations were performed every 14 days using gas specified above.

Before calibrations, the 3 valves on the cylinder regulator were flushed out. The breathing handle was then attached to the calibration port on the regulator and the gas pressure was fine-tuned (2.9 - 3.1 bar). Gas concentration was measured and updated. Prior to each new test the Niox®Flex performed a self-test to detect any technical problems. Ambient nitric oxide levels also had to be <200 parts per billion (ppb) before testing.

2.7.3 Performance of multiple flow exhaled nitric oxide

Subjects performed a maximal inhalation followed by controlled exhalation through Niox®Flex bacterial / viral filters which were attached to the breathing handle. The computer screen has a visual display to help guide exhalation. The tests were performed in duplicate at 4 exhalation flow rates (50, 100, 200, and 300 mls/sec) and no nose clip was needed.

The results for VNO nl/min was plotted against flow rate to provide the regression slope and intercept. VNO is the rate of NO exhaled per unit time and calculated from the product of NO concentration in nl/L and expiratory flow rate in L/min corrected to body temperature pressure saturated. The slope represents the alveolar NO concentration (Calv) and intercept represents bronchial NO flux (J’awNO).

Fractional exhaled NO at an exhalation flow rate of 50 mls (FENO50) was also reported. Units for Calv and FENO50 were ppb and J’awNO was nl/s.

89 2.8 Production of Fluticasone Propionate Monodisperse Aerosols

2.8.1 Equipment

 Aerosol Delivery system (Clinical Bio-Engineering, Royal Brompton Hospital).

 Spinning-Top Aerosol Generator, Mark II, Research Engineers Ltd., London, UK.

 Aerosol Particle Sizer (model 3310 with 3302 100:1 Diluter), TSI Inc., St. Paul, Minnesota, USA.

 Andersen Cascade Impactor, Graseby-Andersen, Smyrna, Georgia, USA.

 Agilent 1000 High-Performance Liquid Chromatography System with UV detection, Agilent Technologies, Berkshire, UK.

 Micronized Fluticasone Propionate (FP), GlaxoSmithKline, Hertfordshire, UK.

 Total trap filters, (Filtrete G-200), 3M, St. Paul, USA.

 2 L volume syringe, Jaeger, Hoechberg, Germany.

 Ethanol, Sigma-Aldrich, Dorset, UK.

 Methanol (HPLC grade), Fisons, Loughborough, UK.

 Sterile distilled water, Baxter, Northampton, UK.

 Heparinized tubes.

 Centrifuge, Haraeus Labofuge 200, DJB Labcare, UK.

 n-Hexane, BDH, VWR International Ltd, Poole, UK.

 Silicone oil DC 200 / 200, BDH, VWR International Ltd, Poole, UK.

 HPLC Agilent 1100 system, Agilent Technologies LDA UK Limited.

 Agilent 1100 Degasser, Biomax 062343.

 Agilent 1100 Pump, Biomax 062341.

 Agilent 1100 Autosampler, Biomax 065258.

 Agilent 1200 Oven, Biomax 501077.

90

 Agilent 1100 VWD, Biomax 065259

 Agilent 1100 Control Module, Biomax 066183.

2.8.2 Validation of aerosol particle sizer (APS) determined particle size

The APS used in these studies assumes that aerosol particles are spherical with uniform density. Since this is not true for all aerosols, validation tests of particle size and mass concentration were necessary prior to patient studies. The accuracy of the APS was crucial in ensuring patients inhale the correct particle size and dose.

The Andersen Cascade Impactor (ACI) is traditionally used to determine aerosol particle size and drug mass concentration. This method is the gold standard.

It was therefore important to compare the same aerosol distributions measured by the APS with those measured by the ACI. Each ACI impaction plate was coated with a solution of 1% silicone oil in hexane and allowed to dry. This prevented particle bounce within the ACI. A volume of 30 mls ethanol (99.8% ethanol, conforming to BP, EP and USP standards) was added to 100 mg of fluticasone propionate powder.

The resulting mixture was placed in a beaker in an Ultrasonic Bath for 5 – 10 minutes until the initial milky liquid became clear.

Three monodisperse aerosols (1.5, 3 and 6 µm) with a GSD <1.22 were generated from the resulting solution using the STAG with the top spinning at 2000, 1250 and 300 r.p.s. respectively. Figure 2.28 shows three typical STAG-generated FP aerosol distributions as measured by the APS. When the APS measured the target aerosol particle size and mass concentration the aerosol production was stopped. The ACI was immediately attached by connecting the USP inlet (United

91 States Pharmacopeia) to the mouthpiece on the aerosol chamber. The aerosol was drawn through the ACI at a suction flow rate of 28 L/min for 2 minutes.

The FP collected on each ACI stage and impaction plate (including the USP and base filter) was recovered by carefully washing with a solution of methanol and distilled water. High performance liquid chromatography (HPLC, Agilent 1100) was performed to calculate aerosol particle size (MMAD) for comparison to the APS. The MMAD’s of 3 aerosol distributions were measured by both the ACI and APS and were plotted in regression analysis (Figure 2.29). This showed a linear relationship between the two instruments for MMAD (r² = 0.99).

Figure 2.28: Typical monodisperse FP distributions for: A 1.5µm; B 3µm; C 6µm as displayed by the APS

92

Figure 2.29: Comparison of particle size (MMAD) measured by ACI vs. APS for FP monodisperse aerosols with SD error bars (3 replicates)

2.8.3 Validation of aerosol particle sizer (APS) measured concentration mass The aerosols were generated as described above. Then in a manner likewise to patients inhaling aerosols, quantities of aerosol were drawn through low resistance total trap filters using a 2 L volume syringe at spontaneous flows. The filters, contained within a specially designed holder, were attached directly to the aerosol chamber at the mouthpiece. Three filters were used consecutively per aerosol run.

The APS was used methodically to check aerosol particle MMAD and mass concentration of the remaining aerosol between each filter test and for up to 7

0 1 2 3 4 5 6 7 8

0 1 2 3 4 5 6 7 8

FP MMAD (m) Measured by APS

FP MMAD

(m) Measured by Andersen

93 minutes after the aerosol had formed. The FP deposition on each filter was recovered and analysed also by HPLC to quantify mass concentration for comparison to the APS measurements. The mass concentration decayed as expected due to sedimentation of the aerosol in the chamber after aerosol production ceased. The mass calculated by the APS was plotted against the mass concentration recovered from the filter for each particle size (Figure 2.30).

Figure 2.30: Regression slopes comparing FP mass concentration obtained through HPLC analysis of filters vs. APS for 1.5-, 3-, and 6 μm monodisperse aerosols

Despite the non-spherical nature of the particles a linear relationship existed between the APS and the ACI FP particle MMAD’s (Kalsi et al 2011). The APS slightly underestimated MMAD as particle size increased. The comparison of mass concentration measured by APS and filter collections also showed a linear relationship even though there was not a one to one correspondence. These measurements were reproducible for each particle size meaning that provided that the appropriate corrections factors were used, the APS can be used to give a measure of both particles size and mass concentration of a given monodisperse FP distribution. The regression slopes were used to extrapolate particle MMAD and

94 mass concentration measured by the ACI to the APS to ensure the correct particle size and drug dose was given to patients.