Safety, Tolerability and Pharmacokinetics and Pharmacodynamics of Inhaled Once-Daily Umeclidinium in Healthy Adults Deficient in CYP2D6 Activity: A Double-Blind, Randomized Clinical Trial

Clinical Drug Investigation

Anthony Cahn • Rashmi Mehta • Andrew Preece • James Blowers • Alison Donald

A. Cahn (corresponding author): Medicines Research Centre, GlaxoSmithKline, Stevenage, UK

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Online Resource 1

Supplementary Information

Plasma concentrations of GSK57319 (umeclidinium) were determined using a validated analytical method based on protein precipitation of plasma sample (100 µL) using acetonitrile (300 µL) containing isotopically labelled [13C12]-GSK573719 at a concentration of 2.5 ng/mL as an internal standard. The extraction tubes were thoroughly vortex mixed and then centrifuged at 3000g for 10 min at ambient temperature, prior to the supernatant being transferred to clean tube evaporated to dryness under a stream of nitrogen and reconstituted in water (75µL). The reconstituted residue was injected (10µL) on to a high performance liquid chromatography system utilising a Synergi Polar RP 80Å (5 micron packing, 50 x 2.0mm) column (supplied by Phenomenex, Macclesfield, UK), eluted using a gradient of aqueous ammonium acetate (10mM) containing 0.2% formic acid (A) and methanol (B) as shown in Table.

Time (min) / %A / %B
0 / 55 / 45
0.9 / 10 / 90
0.95 / 55 / 45
1.5 / 55 / 45

GSK573719 has a retention time of approximately 1.2 min, with detection using tandem mass spectrometry on a Sciex 3000 (Applied Biosystems, Warrington UK) using TurboIonSpray in positive polarity mode. Mass transition monitored was 96 from precursor ions of 428 and 440 for GSK573719 and internal standard. The assay had a linear dynamic range of 0.2to 10ng/mL and quantification was performed using peak area ratios with 1/x2 weighted linear regression.

The assay was validated and shown to be specificity, reliable and reproducible with a precision (%CV) of 11.2% and an accuracy (%bias) varying from -11.3% to 1.8% over the range of 0.02 to 10 ng/mL.

Within each analytical run Quality Control samples (QC), prepared at 3 different analyte concentrations and stored with study samples, were analyzed with each batch of samples against separately prepared calibration standards. For the analysis to be acceptable, no more than one-third of the total QC results and no more than one-half of the results from each concentration level were to deviate from the nominal concentration by more than 15%. The applicable analytical runs met all predefined run acceptance criteria.

Supplementary Tables

Table S1 Dose proportionality assessed using the power model in healthy CYP2D6 normal metabolizers (HVT-NM) and healthy CYP2D6 poor metabolizers (HVT-PM)

HVT-NM / HVT-PM
Parameter / Slope (90 % CI) / Day / Slope (90 % CI)
AUC0.25 (ng•h/mL) / 1.317 (1.229, 1.405) / 1 / 1.215 (1.063, 1.368)
AUC1 (ng•h/mL) / - / 7 / 1.201 (1.102, 1.300)
Cmax (ng/mL) / 1.265 (1.182, 1.347) / 1 / 1.172 (1.021, 1.323)
7 / 1.108 (0.962, 1.255)

AUC, area under the concentration-time curve; AUC1, area under the concentration-time curve over 1h; CI, confidence interval; Cmax, maximum concentration; CYP2D6, cytochrome P450 2D6; HVT-NM, healthy CYP2D6 normal metabolizers; HVT-PM, healthy CYP2D6 poor metabolizers

Table S2 Summary urine pharmacokinetic statistics; healthy CYP2D6 normal metabolizers (HVT-NM) versus healthy CYP2D6 poor metabolizers (HVT-PM); single dose (Day 1) versus repeat dose (Day 7)

UMEC / HVT-NM / HVT-PM
Day 1 / Day 7 / Day 1 / Day 7
Parameter / Dose (µg) / N/n / Geometric mean
95 % CI/CV (%) / N/n / Geometric mean
95 % CI/CV (%) / N/n / Geometric mean
95 % CI/CV (%) / N/n / Geometric mean
95 % CI/CV (%)
Ae24 (ng) / 100 / 16/16 / 600.104
(496.609, 725.167)/36.7 / - / 6/6 / 573.980
(430.362, 765.525)/28.0 / 6/6 / 3194.947
(2526.311, 4040.549)/22.7
500 / 16/16 / 5055.417
(4293.014, 5953.217)/31.4 / 8/8 / 16130.340
(13232.766, 19662.395)/24.0 / 12/12 / 5846.184 (5112.129,6685.642)/21.4 / 11/11 / 16614.831
(14107.423, 19567.898)/24.7
1000 / 16/16 / 12578.789
(11111.074, 14240.383)/23.6 / 8/8 / 30316.365
(20936.129, 43899.327)/46.5 / 6/6 / 13307.637
(9615.202, 18418.042)/31.7 / 6/6 / 40845.998
(31502.707, 52960.387)/25.1
Fe24 (%)a / 100 / 16/16 / 0.633 (0.25, 1.04)/NA / - / - / 6/6 / 0.592 (0.38, 0.82)/NA / 6/6 / 3.258 (2.14, 4.04)/NA
500 / 16/16 / 1.061 (0.64, 2.11)/NA / 8/8 / 3.301 (1.99, 4.27)/NA / 12/12 / 1.193 (0.79, 1.70)/NA / 11/11 / 3.413 (2.14, 4.91)/NA
1000 / 16/16 / 1.291 (0.85, 1.88)/NA / 8/8 / 3.305 (1.84, 5.55)/NA / 6/6 / 1.383 (0.91, 1.88)/NA / 6/6 / 4.188 (3.05, 5.53)/NA
Clr (l/h) / 500 / 16/16 / 6.357 (5.430, 7.441)/27.8 / 8/8 / 6.646 (4.549, 9.710)/47.8 / 12/12 / 7.671 (6.636, 8.867)/23.1 / 11/11 / 6.740 (5.762, 7.884)/23.7
1000 / 16/16 / 6.672 (5.564, 8.001)/35.1 / 8/8 / 7.823 (6.446, 9.495)/23.5 / 6/6 / 6.717 (5.087, 8.869)/27.0 / 6/6 / 8.885 (6.882, 11.471)/24.7
t1/2 (h) / 100 / 16/15 / 18.802 (14.916, 23.701)/43.7 / - / - / 6/6 / 17.413 (8.944, 33.901)/57.8 / 6/6 / 34.955 (18.527, 65.948)/66.5
500 / 16/16 / 19.323 (15.326, 24.364)/45.6 / 8/8 / 31.044 (22.281, 43.252)/41.3 / 12/12 / 17.415 (12.970, 23.384)/49.0 / 11/11 / 25.882 (20.626, 32.477)/34.8
1000 / 16/16 / 18.499 (14.997, 22.819)/41.0 / 8/8 / 25.635 (16.553, 39.7)/56.1 / 6/6 / 23.228 (11.649, 46.314)/73.6 / 6/6 / 40.090 (19.143, 83.958)/80.1
aArithmetic mean value (range)
Ae24, urinary recovery of unchanged drug over 24 h; CI, confidence interval; Clr, renal clearance; CV, coefficient of variation; CYP2D6, cytochrome P450 2D6; Fe24, fraction of dose excreted unchanged in urine over 24 h; HVT-NM, healthy CYP2D6 normal metabolizers; HVT-PM, healthy CYP2D6 poor metabolizers; NA, not applicable; t1/2, elimination half-life; UMEC, umeclidinium

Table S3 Assessment of accumulation in plasma and urine (Day 7 versus Day 1)

HVT-NM / HVT-PM
Parameter / Treatment (µg) / Ratio of adjusted geometric means
(90 % CI) / Ratio of adjusted geometric means
(90 % CI)
AUC0.25 (ng•h/mL) / UMEC 100 / - / 1.586 (1.011, 2.488)
AUC4 ( ng•h/mL) / UMEC 500 / 2.217 (1.834, 2.680) / 2.085 (1.952, 2.227)
AUC24 (ng•h/mL) / UMEC 1000 / 2.098 (1.713, 2.570) / 2.410 (2.190, 2.653)
Cmax (ng/mL) / UMEC 100
UMEC 500
UMEC 1000 / -
2.174 (1.076, 2.771)
1.149 (0.902, 1.465) / 1.508 (1.142, 1.991)
1.448 (1.183, 1.772)
1.174 (0.890, 1.551)
Ae24 (ng) / UMEC 100
UMEC 500
UMEC 1000 / -
3.221 (2.700, 3.844)
2.387 (2.001, 2.848) / 5.566 (4.791, 6.467)
2.855 (2.559, 3.184)
3.069 (2.642, 3.566)

Ae24, urinary recovery of unchanged drug over 24 h; AUC, area under the concentration-time curve; CI, confidence interval; Cmax, maximum concentration; CYP2D6, cytochrome P450 2D6; HVT-NM, healthy CYP2D6 normal metabolizers; HVT-PM, healthy CYP2D6 poor metabolizers; UMEC, umeclidinium

Supplementary Figures

Fig. S1a Semi-log plots of (a) individual change from baseline maximum (0–4 h) heart rate (HR) versus umeclidinium (UMEC) maximum concentration (Cmax) (by treatment group and day) for healthy CYP2D6 normal metabolizers (HVT-NM) (repeat dose, Day 1); (b) individual change from baseline maximum (0–4 h) HR versus UMEC Cmax (by treatment group and day) for HVT-NM (repeat dose, Day 7); and (c) individual change from baseline maximum (0–4h) HR versus UMEC Cmax (by treatment group and day) for HVT-PM (repeat dose)

CYP2D6, cytochrome P450 2D6; HVT, healthy volunteer

Fig. S1b

Fig. S1c

bpm, beats per minute; Cmax, maximum concentration; UMEC, umeclidinium