Pharmacokinetics and Pharmacodynamicsof GSK961081, a Novel Inhaled MuscarinicAntagonist b2‐Agonist, and FluticasonePropionate Administered Alone,Concurrently and as a Combination BlendFormulation in Healthy Volunteers

Virginia Norris, Claire Ambery, and Trevor Riley

Abstract

Objective: To investigate the pharmacokinetics and pharmacodynamics of inhaled GSK961081 and fluticasonepropionate (FP) given alone, concurrently and as a combination blend formulation.Methods: The study was double‐blind, double‐dummy, four‐way crossover. Twenty‐four healthy volunteers took singledoses of the following in randomized order: (1) GSK961081 800mg; (2) FP 500mg; (3) GSK961081 800mg and FP 500mgas a blend formulation; and (4) GSK961081 800mg and FP 500mg concurrently via separate inhalers. The eLung breathingsimulator was also used for the in vitro characterization of the formulations.Results: There was no pharmacokinetic interaction when GSK961081 and FP were administered concurrently. MeanCmax and AUC(0�t) of GSK961081 were lower (�20%) and mean Cmax and AUC(0�t) of FP were higher (two fold)following GSK961081/FP blend formulation compared to concurrent or the individual components alone. There was anincrease in the FP in vitro ex‐throat dose for theGSK961081/FP blend from the eLung breathing simulator. Serum cortisolsuppression was greater with GSK961081/FP blend, with lower (�10%) cortisol levels than after GSK961081þ FPconcurrent or FP alone.Conclusion: GSK961081/FP blend formulation was associated with an increase in FP systemic exposure and greaterserum cortisol suppression.

Keywords

muscarinic receptor antagonist and b2‐adrenoreceptor agonist, bronchodilator, fluticasone propionate, bi‐functionalmolecule, GSK961081

It is recommended that treatment of chronic obstructivepulmonary disease (COPD) should be based on an

individualized, combined assessment that considers both

current disease, determined by assessment of symptomsand activity limitation, and future risk, determined from

airflow limitation or exacerbation history.1 Inhaled

bronchodilators are the mainstay of the symptomatictreatment of COPD, and both long-acting b2-agonists

(LABAs) and long-acting muscarinic antagonists

(LAMAs) are frequently prescribed as maintenancetherapy. The complementary activity of LAMA and

LABA has been shown to provide an additive effect in

COPD, giving rise to a greater degree of bronchodilationthan either of the components given alone.2,3 A

combination of these agents can provide greater efficacy

for patients who remain symptomatic on a monotherapywith a LABA or LAMA.1,4 The addition of an inhaled

corticosteroid (ICS) to dual bronchodilator therapy is

indicated for patients with high levels of symptoms andwho are at risk of frequent exacerbations.

Clinical Pharmacologyin Drug Development3(4) 305–313

© 2014, The AmericanCollege of ClinicalPharmacologyDOI: 10.1002/cpdd.105

GlaxoSmithKline, UK

Submitted for publication 15 April 2013; accepted 5 January 2014

Corresponding Author:Virginia Norris, GlaxoSmithKline, GSK House, 980 Great West Road,Brentford, Middlesex TW8 9GS, UK(e‐mail: virginia.h.norris@gsk.com)ClinicalTrials.gov identifier: NCT01449799.

Original Article

GSK961081 is a novel bifunctional molecule (or dualpharmacophore) that combines muscarinic antagonism

and b2-agonism in a single molecule5 (Figure 1) and is in

development for the treatment of COPD. In trials inpatients with COPD, GSK961081 showed clinically

meaningful bronchodilation with rapid onset of action

and a good safety and tolerability profile.6,7

The combination of the LABA and LAMA compo-

nents in a single, bifunctional molecule simplifies

formulation development and ensures the pharmacologi-cal and pharmaceutical compatibility of the active

ingredients.8 Combination of an ICS with a bifunctional

bronchodilator molecule has the potential to provide asimpler route to a triple therapy.

In this study, the systemic pharmacokinetics (PK),

systemic pharmacodynamics (PD), and safety of singledose GSK961081 given as a combination dry powder

blend with fluticasone propionate (FP) via a single

inhaler, concurrently with FP via separate inhalers, andeach component alone, were assessed. This is the first

study in which GSK961081 and FP have been adminis-

tered as a combination blend formulation to humans.The report also describes how comparative in vitro

data at healthy subject-relevant flow rates were generated

using the Electronic Lung (eLung)9,10 breathing simula-tor to compare the aerodynamic particle size distribution

(APSD) of the delivered dose for the investigational dry

powder inhaler (DPI) products used in this study.

Subjects and MethodsStudy PopulationSixteenmale and 8 female healthy volunteers participated

in the study at Hammersmith Medicines Research,London, UK between July 13, 2011 and September 21,

2011. All subjects provided written informed consent

before participation and the protocol was approved by theNational Research Ethics Committee London, Brent,

UK. The study was conducted in accordance with the

Declaration of Helsinki and Good Clinical Practiceguidelines.

Study DesignThis was a double-blind, single-center, single-dose,

double-dummy, four-way crossover study (GlaxoS-

mithKline study number MFP113423; www.clinical-trials.gov registration number NCT01449799). Subjects

received each of the following four treatments in

randomized order: (1) GSK961081/FP 800/500mg givenas a combination blend formulation (GSK961081/FP

blend); (2) GSK961081 800mg and FP 500mg given

concurrently via separate inhalers (GSK961081þFPconcurrent); (3) GSK961081 800mg; and (4) FP 500mg.The doses were selected to ensure adequate systemic

exposure for PK analysis. Subjects were admitted to theclinical unit the evening before dosing and were

discharged 24 hours after dosing. Each of the four

treatment periods was separated by a washout period of7–10 days. A follow-up visit was completed 10–14 days

after the final dose of study medication.

All treatments were administered by inhalation fromthe DISKUS1 device, which contains a foil blister strip in

which each blister contains 12.5mg of a blend of the

micronized drug substance(s) with lactose monohydrate.The following investigational DPI products were used:

GSK961081/FP combination DPI 400/250mg,GSK961081 DPI 400mg, FP DPI 250mg, and a placebolactose inhaler (PL). The treatments were administered as

follows, each by four inhalers: GSK961081/FP 800/

500mg blend (GSK961081/FP 400/250mg, GSK961081/FP 400/250mg, PL, PL); GSK961081 800mg and FP

500mg concurrently via separate inhalers (GSK961081

400mg, GSK961081 400mg, FP 250mg, FP 250mg);GSK961081 800mg (GSK961081 400mg; GSK961081400mg, PL, PL) and FP 500mg (FP 250mg, FP 250mg,PL, PL). Dosing of the four inhalers was completedwithin 2minutes. Further details of the dosing protocol

are provided in the Online Supplement.

The primary endpoints were plasma concentrationsand derived PK parameters (maximum plasma concen-

tration [Cmax] and area under the concentration curve

[AUC]) for GSK961081 and FP. The secondary endpointswere weighted mean serum cortisol (0–24 hours), urinary

cortisol excretion (0–24 hours), minimum and weighted

mean change from baseline potassium (0–4 hours),maximum and weighted mean change from baseline

glucose (0–4 hours), maximum and weighted mean

change from baseline heart rate (0–4 hours) and maxi-mum and weighted mean change from baseline QT

interval corrected for heart rate using Fridericia’s formula

(QTcF [0–4 hours]), adverse events (AEs) and othersafety measures including spirometric forced expiratory

volume in 1 second (FEV1).

PK AssessmentsBlood samples for the determination of plasma

GSK961081 and FP were collected via an indwellingFigure 1. Molecular structure of GSK961081.

306 Clinical Pharmacology in Drug Development 3(4)

cannula or by direct venepuncture pre-dose and at 0.25,0.5, 0.75, 1, 2, 3, 4, 6, 8, 10, 12, 16, and 24 hours post-

dose. GSK961081 and FP bioanalysis was performed by

Aptuit, Inc. (Verona, Italy) and York BioanalyticalSolutions, Ltd (York, UK), respectively. The lower limit

of quantification (LLQ) was 25 and 3 pg/mL for

GSK961081 and FP, respectively (see Online Supple-ment for additional analytical methods).

PD AssessmentsBlood samples for the determination of serum potassium

and blood glucose, were collected pre-dose and at 0.5, 1,

2, and 4 hours post-dose. Serum potassium and glucosemonitoring was performed using iSTAT portable chemi-

cal analysers at Hammersmith Medicines Research

(London, UK).Blood samples for the determination of serum cortisol

were collected pre-dose and at 1, 2, 3, 4, 6, 8, 10, 12, 16,

and 24 hours post-dose. Urine samples for the determina-tion of cortisol were collected pre-dose and continuously

up to 24 hours post-dose. Serum and urinary cortisol

analyses were performed by Simbec Research (MerthyrTydfil, UK) using an HPLC–MS/MS method. Further

details of assay methodology are provided in the Online

Supplement.

Safety AssessmentsAEs and serious AEs were documented by the investiga-tor from first dosing until the final follow-up visit via the

InForm data management system (Phase Forward,

Waltham, MA). Other safety assessments, performed atthe clinical site, included hematological, clinical chemis-

try and urinalytic laboratory analyses, vital signs, and

spirometry.

In Vitro Assessment of APSDThe eLung is a breathing simulator designed tocharacterize the APSD of an emitted aerosolized dose

at specified inhalation flow rates.10 An inhalation profile

with a peak inspiratory flow rate (PIFR) of 92 L/min wasselected to simulate that generated by healthy subjects

using the DISKUS inhaler.11,12 The eLung was used in

combination with a coated, anatomically correct humanthroat cast of average dimensions13 to provide a better

estimate of oropharyngeal deposition of the emitted dose.

The aerosolized dose passing beyond the throat cast iscaptured in a holding chamber and then drawn through a

next generation impactor (NGI) at a constant flow rate of

60 L/min to characterize the APSD of the aerosolizeddose.14 The throat cast, holding chamber and individual

stages of the NGI apparatus were washed with 50:50

acetonitrile/water to recover the deposited drug. Thesolutions were then analyzed by HPLC. The individual

stage drug deposition profiles and mean ex-throat dose

(sum of the chamber, pre-separator, and stages 1 through

to the micro orifice collector) were determined intriplicate.

Statistical MethodsSample Size. A formal sample size calculation was not

performed; an estimation approach was used. The sample

size required for this study was estimated on the basis ofpreviously observed estimates of within-subject variabil-

ity of log (AUC) and log (Cmax) for GSK961081

(unpublished data, GlaxoSmithKline, London, UK) andFP15 such that the bounds of the 90% confidence interval

(CI) for the primary treatment comparison would be

within 20% of the observed ratio. Based on theseestimates, 20 subjects would provide the study with

sufficient power for equivalence testing based on a 30%

equivalence range. Twenty-four subjects were enrolled toensure that at least 20 subjects completed all treatment

periods and all critical assessments.

PK AnalysesFrom the GSK961081 and FP plasma concentration–time

data, the following parameters were determined: Cmax,time at which Cmax occurs (tmax), area under the plasma

concentration–time curve to last quantifiable concentra-

tion (AUC(0�t)), area extrapolated to infinity(AUC(0�1)), or area to the last quantifiable concentration

within a subject across all treatments (AUC(0�t’)) and

time of last quantifiable concentration (tlast). To evaluatethe primary PK objectives, loge-transformed AUC and

Cmax for GSK961081 and FP were separately analyzed

using a mixed effects model. Period and treatment werefitted as fixed effects, and subject was fitted as a random

effect. For each comparison of interest, a point estimate

and associated 90%CI was constructed for the difference.The point estimate and associated 90% CI were then

back-transformed to provide a point estimate and 90% CI

for the ratio.

PD AnalysesStatistical analysis was performed on the loge-trans-formed weighted mean 0–24-hour serum cortisol and the

loge-transformed total 24-hour urinary cortisol excretion,

separately. A mixed effects model was fitted for each ofthe endpoints with treatment and period fitted as fixed

effects, and subject as a random effect. Subject and period

baseline were fitted as continuous covariates. Subjectbaseline was defined as the mean of the subject’s logebaseline measurement across all the periods. Period

baseline was defined as the difference between the logebaseline value for that period and the subject baseline.

Similar statistical analyses were conducted on:

minimum and weighted mean change from baselinepotassium (0–4 hours); maximum and weighted mean

change from baseline glucose (0–4 hours); maximum and

weighted mean change from baseline heart rate (0–

Norris et al 307

4 hours); and maximum and weighted mean change frombaseline QTcF (0–4 hours).

ResultsSubject Disposition and Baseline CharacteristicsTwenty-four healthy volunteers participated in the study.

All subjects completed the study as planned. All subjectswere included in the PK, PD, and safety populations.

Summary demographic data and details of eligibility

criteria are provided in the online supplement.

PK ResultsPeak plasmaGSK961081 concentrations occurred around1 hour post-dose (Figure 2) for all three GSK961081

containing treatments (tmax range: 0.25–1.02 hours),

following which concentrations rapidly declined andwere below the LLQ by 3–4 hours post-dose (tlast range:

0.28–10.0 hours; Table 1). Based on statistical analysis

results GSK961081 systemic exposure was lower afteradministration of the GSK961081/FP combination blend

formulation than after GSK961081þ FP concurrent

(AUC(0�t’) was 27% lower; AUC(0�t) was 32% lower)or GSK961081 alone (AUC(0�t’) was 13% lower;

AUC(0�t) was 29% lower). Similarly, maximal

GSK961081 systemic exposure was lower after adminis-tration of the GSK961081/FP blend than after

GSK961081þFP concurrent or GSK961081 alone

(Cmax was 14% lower after both treatments).GSK961081 systemic exposure was similar after

GSK961081þFP concurrent, and GSK961081 alone in

terms of Cmax and AUC(0�t); however, AUC(0�t’) was20% higher after GSK961081 and FP concurrent.

Peak plasma FP concentrations occurred around

0.5 hours post-dose for all three FP containing treatments(tmax range: 0.25–2.02 hours; Figure 2). Plasma FP

concentrations were quantifiable to the last sampling

time point (24 hours) in the majority of subjects (tlastrange: 2.00–24.0; Table 1). Based on statistical analysis

results FP systemic exposure was higher after adminis-

tration of the GSK961081/FP blend than afterGSK961081þFP concurrent (AUC(0�t) was 93% higher;

AUC(0�1) was 48% higher) or FP alone (AUC(0�t) was

82% higher; AUC(0�1) was 40% higher). Similarly,maximal FP systemic exposure was higher after

administration of the GSK961081/FP blend than after

GSK961081þFP concurrent (Cmax was 98% higher) orFP alone (Cmax was 102% higher). FP systemic exposure

was similar after GSK961081þ FP concurrent and FP

alone.

PD ResultsAll treatments containing FP suppressed serum cortisolcompared with GSK961081 alone (considered placebo

for this endpoint; Table 2).Weighted mean serum cortisol

(0–24 hours) was similar after GSK961081þFP concur-rent and FP alone. However, FP suppression of serum

cortisol, although not statistically significant, was slightly

greater after administration of the GSK961081/FP blendthan after FP alone or GSK961081þFP concurrent;

weighted mean serum cortisol after GSK961081/FP

blend was 6% lower than FP alone (adjusted geometricmean ratio (95% CI)) 0.938 (0.838, 1.050), and 10%

lower than GSK961081þ FP concurrent (adjusted geo-

metric mean ratio (95% CI)) 0.905 (0.810, 1.011). Totalurinary cortisol (0–24 hours) data were consistent with

serum cortisol results (Table 2), that is, cortisol was lower

with the GSK961081/FP blend compared withGSK961081þFP or FP alone.

A summary of heart rate, serum potassium, blood

glucose, and QTcF is provided in Table 2. Maximum andweighted mean heart rate change from baseline (0–

4 hours) were similar after GSK961081/FP blend,

GSK961081þFP concurrent, and GSK961081 alone.Maximum and weighted mean heart rate changes from

baseline (0–4 hours) were greater after GSK961081/FP

blend and GSK961081þFP concurrent, compared withFP alone Maximum and weighted mean QTcF (0–

4 hours) results were consistent with heart rate results.

Minimum and weighted mean serum potassium change

Figure 2. (a) Arithmetic mean (SE) plasma concentration–time plots for GSK961081. (b) Arithmetic mean (SE) plasmaconcentration–time plots for FP.

308 Clinical Pharmacology in Drug Development 3(4)

from baseline (0–4 hours) was similar after GSK961081/

FP blend, GSK961081þFP concurrent, and GSK961081

alone. Minimum and weighted mean serum potassiumchanges from baseline (0–4 hours) were greater after

GSK961081/FP blend and GSK961081þFP concurrent

compared with FP alone (there were no differencesbetween any of the treatments for maximum or weighted

mean blood glucose change from baseline (0–4 hours).

Safety ResultsTwenty-one AEs were reported by 13 (54%) subjects. All

AEs were of mild or moderate intensity. There were noserious AEs and no AEs that resulted in premature

discontinuation of treatment. Five AEs were considered

by the investigator to be potentially related to studymedication: four instances of headache (GSK961081/FP

blend: two; GSK961081þFP concurrent: one; FP: one),

and one of dizziness (GSK961081þFP concurrent). Noclinically meaningful treatment effects on clinical safety

laboratory tests, vital signs, or 12-lead electrocardiogram

readings were seen. Spirometry showed a slight increase

from baseline FEV1 after all GSK961081 containing

treatments. No change from baseline in mean FEV1 was

seen after treatment with FP alone.

In Vitro APSD ResultsThe mean ex-throat doses for the investigational DPIproducts containing GSK961081 and FP tested by the

eLung are presented in Table 3. The drug deposition

profiles including the throat cast, holding chamber andindividual NGI stages are shown in Figure 3.

The GSK961081 individual stage deposition profile

for the GSK961081/FP combination blend DPI wassimilar to the GSK961081 single drug substance

comparator. A decrease of approximately 10% in the

GSK961081 ex-throat dose was observed for theGSK961081/FP blend relative to GSK961081 alone.

In contrast, a decrease in the throat cast deposition and

corresponding increase in deposition in NGI stages 3–6was observed for the FP component of the GSK961081/

FP blend DPI relative to the FP single drug substance

comparator. The increase in the FP ex-throat dose for the

Table 1. Summary of GSK961081 and FP PK Parameters

Treatment

GSK961081 (N¼ 24) FP (N¼ 24)

Arithmeticmean (SD)

Geometricmean (95% CI)a

Arithmeticmean (SD)

Geometricmean (95% CI)a

Cmax (pg/mL)081/FP blend 97.7 (63.0) 75.0 (51.6, 109) 201 (112) 137 (80.0, 234)081þ FP 111 (67.8) 86.8 (59.9, 126) 112 (64.3) 69.2 (37.6, 128)Alone 108 (64.6) 87.5 (64.4, 119) 108 (73.8) 67.8 (39.6, 116)

AUC(0�t) (pg�h/mL)081/FP blend 202 (173) 102 (52.5, 197) 1,077 (558) 726 (403, 1,308)081þ FP 267 (194) 150 (77.9, 287) 692 (402) 376 (176, 802)Alone 261 (241) 143 (78.9, 259) 711 (457) 399 (206, 775)

AUC(0�t’) or AUC(0�1)b (pg�h/mL)

081/FP blend 185 (158) 151 (86.4, 264) 1,163 (706) 1,092 (732, 1,631)081þ FP 213 (183) 205 (138, 305) 755 (489) 615 (389, 973)Alone 218 (201) 172 (101, 293) 674 (553) 615 (368, 1,028)

tmax (hour)081/FP blend 0.60 (0.31) 0.75 (0.25, 1.02) 0.39 (0.18) 0.50 (0.25, 0.75)081þ FP 0.76 (0.33) 1.00 (0.50, 1.00) 0.52 (0.27) 0.50 (0.25, 1.02)Alone 0.80 (0.28) 1.00 (0.28, 1.02) 0.74 (0.39) 0.75 (0.25, 2.02)

tlast (hour)081/FP blend 2.81 (1.95) 3.00 (0.50, 8.00) 21.9 (5.95) 24.0 (10.0, 24.1)081þ FP 3.63 (2.26) 4.00 (1.00, 10.00) 20.8 (7.94) 24.0 (2.00, 24.2)Alone 3.61 (2.50) 4.00 (0.28, 10.00) 20.2 (7.80) 24.0 (4.02, 24.0)

AUC(0�t), area under the plasma concentration–time curve to last quantifiable concentration; AUC(0�t’), area under the plasma concentration–timecurve to last quantifiable concentration within a subject across all treatments; AUC(0�1), area under the plasma concentration–time curve extrapolatedto infinity; CI, confidence interval; Cmax, maximumplasma concentration; 081¼GSK961081; FP, fluticasone propionate; n, number of subjects with non‐missing observations, including imputed non‐quantifiable values; tlast, time of last quantifiable concentration; tmax, time at which Cmax occurs. Forarithmetic mean (SD) missing values were imputed with zero. For geometric mean (95% CI) missing values were imputed as follows: AUC(0�t) andAUC(0�1) with half the lowest respective observed value; Cmax with half the LLQ; AUC(0�t’), tmax, and tlast no imputation.aGeometric mean (95% CI), except for tmax and tlast where median (minimum–maximum) is presented.bAUC(0�t’) for GSK961081 and AUC(0�1) for FP.

Norris et al 309

GSK961081/FP blend relative to FP alone was approxi-

mately 45%.

DiscussionThis was the first study in which GSK961081 and FP have

been administered as a combination blend formulation.

This study in healthy subjects was performed to

investigate the systemic PK, PD, and safety ofGSK961081 and FP when administered alone, concur-

rently, and as a blend, to assess the suitability of the blend

formulation for further development as a potential singleinhaled triple therapy for the treatment of COPD.

Systemic exposure to GSK961081 following adminis-

tration as the GSK961081/FP blend was �20% lowercompared with GSK961081þ FP concurrent and

GSK961081 alone. This minimal reduction in systemic

exposure did not alter the expected GSK961081 systemicPD response, with comparable potassium, heart rate,

glucose, and QTcF results obtained for GSK961081/FP

blend and GSK961081 alone. Systemic exposure to FPfollowing administration as the GSK961081/FP blend

was 50–100% higher than compared to GSK961081þFP

concurrent and FP alone. Variability associated with FPPK parameters was comparable when FP was adminis-

tered as the combination blend or alone. Associated with

the increased systemic FP exposure for the GSK961081/FP blend was �10% greater serum cortisol suppression

compared to FP alone or FP in combination with

GSK961081.Cortisol suppression is an ICS class effect16,17 and in

clinical investigation serum and urine cortisol suppres-

sion has been shown to be directly associated with FPAUC.15,18 In this study the increased systemic FP

exposure and cortisol suppression in healthy subjects

for the GSK961081/FP blend is likely to represent themost extreme scenario on the basis that plasma FP levels

are demonstrated to be lower in patients with COPD as

compared to healthy subjects.19 FP 500mg twice daily isthe approved regimen for the treatment of COPD in

Europe in combination with salmeterol. Treatment with

this regimen for periods of up to 3 years is not associatedwith any significant risk of adverse effects commonly

associated with prolonged systemic exposure to cortico-

steroids such as cataracts, osteoporosis or bone frac-tures.20 Clinical data also indicate that, in combination

with salmeterol, a 500mg/day FP dose may have utility in

COPD.21,22 Based on the systemic PK (and PD) results ofthis study, a GSK961081/FP blend containing 250mg of

FP could be an appropriate twice-daily formulation in

terms of safety and efficacy for exploration in furtherclinical studies.

The systemic PK (and PD) findings for the blend were

not observed for GSK961081 and FP given concurrently,

Table 2. Summary of Arithmetic Mean (SD) GSK961081 andFP PD Parameters

TreatmentMaximuma

(N¼ 24)Weighted

mean (N¼ 24)

Serum cortisol (0–24 hours) (ng/mL)081/FP blend N/A 51.8 (21.5)081þ FP N/A 58.9 (32.2)081 alone N/A 64.4 (31.2)FP alone N/A 56.3 (25.0)

Urinary cortisol (0–24 hours) (mg)081/FP blend N/A 20.2 (10.3)081þ FP N/A 28.5 (29.7)081 alone N/A 28.8 (10.5)FP alone N/A 21.0 (9.82)

Heart rate (0–4 hours) change from baseline (bpm)081/FP blend 4.15 (5.52) 0.25 (3.53)081þ FP 4.21 (3.72) 0.74 (3.28)081 alone 4.43 (4.06) 0.97 (3.19)FP alone 2.13 (5.46) �1.55 (4.78)

Serum potassium (0–4 hours) change from baseline (mmol/L)081/FP blend �0.31 (0.22) �0.17 (0.17)081þ FP �0.27 (0.22) �0.15 (0.19)081 alone �0.28 (0.17) �0.16 (0.18)FP alone �0.17 (0.19) �0.05 (0.16)

Blood glucose (0–4 hours) change from baseline (mmol/L)081/FP blend 0.08 (0.17) �0.09 (0.17)081þ FP 0.08 (0.20) �0.06 (0.21)081 alone 0.06 (0.20) �0.11 (0.18)FP alone 0.05 (0.17) �0.13 (0.19)

QTcF (0–4 hours) change from baseline (ms)081/FP blend 6.90 (8.14) 1.73 (5.85)081þ FP 8.43 (7.17) 1.26 (4.99)081 alone 8.77 (7.35) 2.02 (5.52)FP alone 4.10 (7.34) �3.02 (6.17)

bpm, beats per minute; CI, confidence interval; 081¼GSK961081; FP,fluticasone propionate; N/A, not applicable; QTcF,QT interval correctedfor heart rate using Fridericia’s formula. No values were imputed.aMinimum presented for potassium (0–4 hours).

Table 3. Summary of Arithmetic Mean (SD) eLung Ex‐Throat DoseData forGSK961081 400mg and FP 250mg Delivered ViaDISKUSInhaler at a Healthy Subject‐Representative Flow Rate of 92 L/min

Device GSK961081 ex‐throat dose, mg/blister FP ex‐throat dose, mg/blister

GSK961081 DISKUS 400mg/blister 102.2 (3.7) N/AFP DISKUS 250mg/blister N/A 38.4 (3.8)GSK961081/FP DISKUS 400/250mg/blister 92.5 (4.0) 55.8 (2.2)

FP, fluticasone propionate; N/A, not applicable.

310 Clinical Pharmacology in Drug Development 3(4)

with PK being comparable following GSK961081 and FP

given concurrently and the individual components givenalone. FP is a substrate for CYP3A4 and GSK961081 is a

substrate of CYP3A4 and P-glycoprotein with in vitro

studies with human liver microsomes indicating lowpotential for CYP450 inhibition. Therefore the absence of

any PK interaction when GSK961081 and FP were

administered concurrently was as expected.The findings in this study, whereby GSK961081 and

FP systemic exposure correlated with the in vitro APSD

differences observed in the eLung analysis, may beattributed in part to the use of the same inhalation device

(DISKUS) for all treatments. The aerosolization of

carrier-based blends on inhalation is influenced by thecomposition and surface properties of the blend con-

stituents as well as the mixing process.23 Based on

hypotheses presented in the literature as to how fineparticles influence the performance of carrier-based

DPIs, two potential mechanisms may be proposed to

explain the observed increase in FP ex-throat dose for thecombination blend inhaler.24 The micronized

GSK961081 particles either preferentially occupy areas

of high adhesion on the surface of the coarse lactosecarrier particles and/or formmixed agglomerates with the

FP particles which are more easily dispersed on

inhalation. Given that there is only a modest correspond-ing decrease in the GSK961081 ex-throat dose for the

combination blend, co-agglomeration of the drug sub-

stances is speculated to be the dominant mechanism bywhich enhancement of the respirable FP particles occurs.

In principle, in vitro APSD data could be used to guide

dose selection for any future GSK961081/FP combina-tion blend in consideration for clinical investigation,

given the correlation of systemic exposure differences

and ex-throat dose in this study. However this would notreplace thorough clinical investigation.

All treatments were well tolerated, with no serious

AEs or withdrawals and no clinically meaningful

treatment effects on safety laboratory parameter tests,

vital signs or 12-lead electrocardiogram readings. This isconsistent with those from earlier studies with

GSK961081 in patients with COPD.6,7

The doses of GSK961081 (800mg) and FP (500mg)used in this study were selected to enable the single-dose

PK profiles to be adequately characterized and were

expected to be well tolerated. The dose of FP used(500mg) was selected because it had previously been

shown to have a measurable effect on serum cortisol

following single-dose administration.15 The PK and PDresults for FP alone and GSK961081 alone in this study

were consistent with those previously reported. Steady-

state systemic exposures, although not investigated in thisstudy, can be predicted from single-dose data and then

assessed in PDmodel such as the cortisol model proposed

by Mackie and Bye.18

ConclusionsAdministration of GSK961081 and FP as a blend was

associated with an increase in FP systemic exposurerelative to GSK961081þFP concurrent or FP alone,

and greater serum cortisol suppression. The PK findings

correlate with an increase in the FP in vitro ex-throatdose for the blend as determined using the eLung

breathing simulator. There was no PK or PD interaction

when GSK961081 and FP were administered concur-rently and so these finding suggest that the increase in

FP systemic exposure for the combination formulation

could be due to physical interactions in the blendinfluencing oropharyngeal deposition following deliv-

ery via the DPI.

Acknowledgments

We wish to acknowledge Hiran Cooray for operational support,

and the investigators, staff and subjects at the study site,

Figure 3. (a) Comparison of arithmetic mean (SE) eLung‐determined deposition of GSK961081 in throat cast, holding chamber andNGI stages 1–MOC for GSK961081/FP 400/250mg and GSK961081 400mg delivered via DISKUS inhaler at a healthy subject‐representative flow rate of 92 L/min. (b) Comparison of arithmetic mean (SE) eLung‐determined deposition of FP in throat cast, holdingchamber and NGI stages 1–MOC for GSK961081/FP 400/250mg and FP 250mg delivered via DISKUS inhaler at a healthy subject‐representative flow rate of 92 L/min.

Norris et al 311

Hammersmith Medicines Research, London, UK. We thank

Richard Martin, Daniel Alexander, Paul Hopkins, and Melanie

Hamilton for the generation of in vitro Electronic Lung data, and

David Prime for his helpful input to the data interpretation.

Editorial support (in the form of development of a draft outline

in consultation with the authors, assembling tables and figures,

collating author comments, copyediting, fact checking,

referencing and graphic services) was provided by Ian Grieve

at Gardiner-Caldwell Communications and was funded by

GlaxoSmithKline.

Declaration of Conflicting Interests

V.N., C.A., and T.R. are employees of and hold stock in

GlaxoSmithKline.

Funding

All authors meet the criteria for authorship set forth by the

International Committee for Medical Journal Editors. All

authors had full access to the data and confirm the accuracy

and completeness of the data and the data analysis. All authors

developed and revised the manuscript for important intellectual

content. V.N. and C.A. designed the clinical study, contributed

to the analysis including approval of the statistical plan, and

interpreted the data. T.R. interpreted the clinical and in vitro

data. This study was funded by GlaxoSmithKline (study number

MFP113423; ClinicalTrials.gov identifier NCT01449799).

References

1. The Global Initiative for Chronic Obstructive Lung disease

(GOLD) revised 2011. http://www.goldcopd.org/guide-

lines-global-strategy-for-diagnosis-management.html. Ac-

cessed February 6, 2013.

2. Sansores R, Ramirez-Vanegas A, Reddy C,Mejia-Alfaro R.

Effect of the combination of two bronchodilators on

breathlessness in patients with chronic obstructive pulmo-

nary disease. A crossover clinical trial. Arch Med Res.

2003;34(4):292–297.

3. Celli BR, MacNee W, Agusti A, et al. Standards for the

diagnosis and treatment of patients with COPD: a summary

of the ATS/ERS position paper. Eur Respir J. 2004;

23(6):932–946.

4. Cazzola M, Molimard M. The scientific rationale for

combining long-acting b2-agonists and muscarinic antag-

onists in COPD. Pulm Pharmacol Ther. 2010;23(4):257–

267.

5. Steinfeld T, Hughes AD, Klein U, Smith JA, Mammen M.

THRX-198321 is a bifunctional muscarinic receptor

antagonist and beta2-adrenoreceptor agonist (MABA)

that binds in a bimodal and multivalent manner. Mol

Pharmacol. 2011;79(3):389–399.

6. Wielders PL, Ludwig-Sengpiel A, Locantore N, Baggen S,

Chan R, Riley JH. A new class of bronchodilator improves

lung function in COPD: a trial with GSK961081.Eur Respir

J. 2013;42(4):972–981.

7. Norris V, Zhu C, Ambery C. The pharmacodynamics of

GSK961GSK961081 in patients with COPD. Eur Respir J.

2011;38(Suppl 55):138s.

8. Hughes AD, Jones LH. Dual-pharmacology muscarinic

antagonist and b2-agonist molecules for the treatment of

chronic obstructive pulmonary disease. Future Med Chem.

2011;3(13):1585–1605.

9. Burnell PKP, Malton A, Reavill K, Ball MHE. Design,

validation and initial testing of the Electronic LungTM

device. J Aerosol Sci. 1998;29(8):1011–1025.

10. Brindley A, Sumby BS, Smith IJ. The characterisation of

inhalation devices by an inhalation simulator: The

electronic lung. J Aerosol Med. 1994;7(2):197–200.

11. Broeders MEAC, Molema J, Vermue NA, Folgering HTM.

Peak inspiratory flow rate and slope of the inhalation

profiles in dry powder inhalers. Eur Respir J.

2001;18(5):780–783.

12. de Koning JP, van der Mark TW, Coenegracht PMJ, Tromp

TFJ, Frijlink HW. Effect of an external resistance to airflow

on the inspiratory flow curve. Int J Pharm. 2002;234(1–

2):257–266.

13. Burnell PKP, Asking L, Borgstrom L, et al. Studies of the

human oropharyngeal airspaces using magnetic resonance

imaging IV—the oropharyngeal retention effect for four

inhalation delivery systems. J Aerosol Med. 2007;20(3):

269–281.

14. Marple VA, Roberts DL, Romay FJ, et al. Next generation

pharmaceutical impactor (a new impactor for pharmaceuti-

cal inhaler testing). Part I: Design. J Aerosol Med.

2003;16(3):283–299.

15. Mollmann H, Wagner M, Krishnaswami S, et al. Single-

dose and steady state pharmacokinetic and pharmacody-

namic evaluation of equipotent doses of inhaled fluticasone

propionate and budesonide in healthy subjects. J Clin

Pharmacol. 2001;41(12):1329–1338.

16. Bernstein DI, Allen DB. Evaluation of tests of hypothalam-

ic-pituitary-adrenal axis function used to measure effects of

inhaled corticosteroids. Ann Allergy Asthma Immunol.

2007;98(2):118–127.

17. Dahl R. Systemic side effects of inhaled corticosteroids in

patients with asthma. Respir Med. 2006;100(8):1307–1317.

18. Mackie AE, Bye A. The relationship between systemic

exposure to fluticasone propionate and cortisol reduction in

healthy male volunteers. Clin Pharmacokinet. 2000;39

(Suppl 1):47–54.

19. Dalby C, Polanowski T, Larsson T, Borgstrom L, Edsbacker

S, Harrison TW. The bioavailability and airway clearance

of the steroid component of budesonide/formoterol and

salmeterol/fluticasone after inhaled administration in

patients with COPD and healthy subjects: a randomized

controlled trial. Respir Res. 2009;10(1):104.

20. Calverly PMA, Anderson JA, Celli B, et al. Salmeterol and

fluticasone propionate and survival in chronic obstructive

pulmonary disease. N Engl J Med. 2007;356(8):775–789.

312 Clinical Pharmacology in Drug Development 3(4)

21. Anzueto A, Ferguson GT, Feldman G, et al. Effect of

fluticasone propionate/salmeterol (250/50) on COPD ex-

acerbations and impact on patient outcomes. COPD.

2009;6(5):320–329.

22. Ferguson GT, Anzueto A, Fei R, Emmett A, Knobil K,

Kalberg C. Effect of fluticasone propionate (250/50mg) or

salmeterol (50mg) on COPD exacerbations. Respir Med.

2008;102(8):1099–1108.

23. de Boer AH, Chan HK, Price R. A critical view on lactose-

based drug formulation and device studies for dry powder

inhalation: which are relevant and what interactions to

expect? Adv Drug Deliv Rev. 2012;64(3):257–274.

24. Jones MD, Price R. The influence of fine excipient particles

on the performance of carrier-based dry powder inhalation

formulations. Pharm Res. 2006;23(8):1665–1674.

Supporting InformationAdditional supporting information may be found in the

online version of this article at the publisher’s web-site.

Norris et al 313