Pharmacokinetics of indacaterol, glycopyrronium and mometasone furoate administered as an inhaled fixed-dose combination in Japanese and Caucasian healthy subjects

Background A once-daily (o.d.) fixed-dose combination of indacaterol acetate (IND), glycopyrronium bromide (GLY), and mometasone furoate (MF) delivered via the Breezhaler® device (IND/GLY/MF) is being developed for treatment of asthma. This study compared steady-state pharmacokinetics of IND, GLY and MF between Japanese and Caucasian male subjects after multiple inhalations of IND/GLY/MF o.d. Methods This was a single-center, open-label, 2-treatment crossover study with a 21-day washout period. Japanese and Caucasian subjects received IND/GLY/MF 150/50/80 μg (inhaled corticosteroid [ICS] medium-dose) or 150/50/160 μg o.d. (ICS high-dose) for 14 days in each period. Pharmacokinetics were characterized up to 24 h post-dose on Days 1 and 14. Results In total, 16 Japanese (median age 31 years [range 20–40 years], mean weight 68.3 kg) and 17 Caucasian subjects (median age 27 years [range 21–43 years], mean weight 75.0 kg) were randomized. Geometric mean ratios (Japanese/Caucasian) [90% confidence interval (CI)] for Cmax for IND, GLY and MF at the high ICS dose on Day 14 were 1.31 [1.13, 1.51] 1.38 [1.13, 1.69] and 1.07 [0.969, 1.18], respectively. Geometric mean ratios (Japanese/Caucasian) [90% CI] for AUC0–24h on Day 14 for IND, GLY and MF at the high ICS dose were 1.17 [1.01, 1.35], 1.05 [0.920, 1.20] and 1.15 [1.05, 1.27] respectively. Similar trends were noted for all components for the medium ICS dose treatment. IND/GLY/MF was safe and well tolerated; no AEs suspected to be study drug-related were observed. Conclusion Pharmacokinetics of IND, GLY and MF (high and medium dose) when delivered as a fixed-dose combination were comparable between Japanese and Caucasian subjects. The IND/GLY/MF combination at the administrated doses was safe and well tolerated in both ethnic groups. Trial registration Japan Registry of Clinical Trial: jRCT2031200227, retrospectively registered on 04, December, 2020.

recurrent episodes of wheezing, breathlessness, chest tightness, and coughing, particularly at night or in the early morning [1]. As of 2017, 358 million individuals suffer from asthma worldwide, including an estimated 30 million people in Europe and 17.7 million people in the US [2]. In Japan, the Ministry of Health, Labor, and Welfare reported approximately 1,177,000 (515,000 men and 662,000 women) from asthma sufferers in 2014 [3]. The proportion of adult Japanese asthma patients with severe asthma and receiving step 4 treatment as per Japanese guidelines or step 5 as per GINA guideline was 10.6% [3]. Asthma patients who are uncontrolled on lowdose inhaled corticosteroid (ICS)/long-acting β agonist (LABA), despite good adherence and correct technique, may benefit from increasing the maintenance dose to medium-dose ICS-LABA or use of add-on long-acting muscarinic antagonist (LAMA) or add-on leukotriene receptor antagonists (LTRA) or high-dose ICS/LABA (Step 4) [4].
Non-adherence to medication is a major cause of poor control of asthma and may be related to several factors including difficulty using inhalers properly, complicated regimens (e.g. multiple times per day, multiple different inhalers) and misunderstanding of the role of controller medications [4]. Improvements in medication adherence could lead to significant improvements in asthma outcomes [5]. Therefore, the once daily combination of LABA/LAMA/ICS in a single inhaler can be an effective treatment option for patients with persistent asthma, while improving patient adherence to asthma therapy [6,7]. Indacaterol acetate (IND), glycopyrronium bromide (GLY), and mometasone furoate (MF) delivered via the Breezhaler ® device (IND/GLY/MF) is a fixed-dose combination (FDC) of IND, a LABA, GLY, a LAMA, and MF, an ICS, in development for o.d.-maintenance treatment of asthma GINA (Step ≥ 4). Each of these three monocomponents, IND [8][9][10], GLY [11,12] and MF [13,14] have previously been approved as individual drugs for either chronic obstructive pulmonary disease (COPD) or asthma. Ethnic factors are known to contribute to differences in pharmacokinetics (PK) and pharmacodynamics of a number of drugs, thereby, resulting in variations in the treatment response [15][16][17]. Studies have reported no differences in the systemic exposure of IND and GLY between Japanese and Caucasian subjects upon administration via the Breezhaler ® device [18,19]. However, for MF delivered with the Twisthaler ® inhalation device, the maximum plasma concentration (C max ) and area under the curve (AUC) values were about two to threefold higher for Japanese patients with asthma compared with Caucasian patients with asthma [20].
The present Phase I study aims to investigate a potential impact of ethnic factors on the PK of orally inhaled IND/GLY/MF 150/50/80 μg (medium-dose ICS) or 150/50/160 μg (high-dose ICS) o.d., delivered via the Breezhaler ® device in healthy Japanese and Caucasian subjects.

Methods
This was a single-center, open-label, two-treatment crossover study with a 21-day washout period. Eligible Japanese and Caucasian subjects were randomized to one of two treatment sequences and received  (Fig. 1). All subjects were trained in the use of the Breezhaler ® device at the screening visit and the baseline visits of both treatment periods. All treatments were followed by post-inhalation mouth rinsing using two rinses of 30 mL water. Water used for mouth rinsing was to be spat out. Subjects were instructed to not swallow the water. The particle size for MF in both treatments was similar, while the in vitro fine particle mass (FPM) increased in a dose proportional manner between the two MF doses (80 μg and 160 μg). The FPM is a measure of the quantity of smaller (≤ 5 µm) particles delivered from the inhaler that generally deposit in the airway and can be considered as an indicator of lung deposition [21]. The study consisted of a screening visit, two treatment sequences, each with a baseline and treatment period, a washout period and an end of study (EOS) visit. Eligible subjects were randomized to one of two treatment sequences and received IND/ GLY/MF (high-or medium-dose, Fig. 1) for 14 days in treatment period 1 after an initial 2-to 28-day screening period and baseline evaluation (Day-1).
Following this, there was a 21-day washout period between the last treatment of treatment period 1 and the first treatment in treatment period 2, prior to treatment administration in period 2. The subjects remained at the study center during each treatment period. The study completion visit was performed on Day 21 of period 2, at which time drug safety was evaluated. At the baseline visit of both treatment periods, all subjects were trained in the appropriate use of the Breezhaler ® device. PK was evaluated up to 24 h post-dose on Days 1 and 14 of each period. On Day 1, 14 of each treatment period, all subjects fasted (i.e. no food and liquid except water) for at least 10 h prior to administration of study treatment and continued to fast for at least 4 h thereafter. For the other dosing days (Day 2 to 13 of each treatment period), study medication was administered before breakfast and no overnight fast was required. On Day 1 and 14 of each treatment period, no fluids were permitted, apart from the fluid given for mouth rinsing following the inhalations, for 1 h prior to and 1 h after study drug administration. Otherwise, subjects had a fluid intake of approximately 200 mL every 4 h during waking hours in addition to fluid taken with meals. There were no fluid restrictions on any other dosing days (Day 2-13 of each treatment period).
The study was conducted in accordance with the principles of the Declaration of Helsinki, the International Conference on Harmonization Guideline for Good Clinical Practice, and the Japanese Standards for the Implementation of Clinical Trials on Pharmaceutical Product.
The study protocol was reviewed and approved by the Institutional Review Board of P-one clinic.

Study objectives
The primary objective was to compare the steady-state PK of IND/GLY/MF between Japanese and Caucasian healthy male subjects after multiple o.d. oral inhalation of IND/GLY/MF medium and high dose. The key secondary objective was to compare the PK of IND/GLY/ MF between Japanese and Caucasian healthy male subjects after single-dose oral inhalation of IND/GLY/MF medium or high dose, via Breezhaler ® on Day 1. The exploratory objective was to compare the PK of MF between high-dose ICS (MF dose: 160 μg) and mediumdose ICS (MF dose: 80 μg) on Day 1 and Day 14, respectively. MF Twisthaler and MF Breezhaler ® are different in regards to both the inhalation device and the formulations they deliver. The medium and high MF doses (80 μg and 160 μg) as part of the IND/GLY/MF combination delivered via the Breezhaler ® device are comparable to the 400 μg and 800 µg doses of MF delivered by the Twisthaler device, respectively. The high MF dose (320 μg) as part of the IND/MF combination delivered via the Breezhaler ® device is comparable to the 800 μg dose of MF delivered by the Twisthaler device [22]. The high MF dose (160 μg) as part of the IND/GLY/MF combination is comparable to the high MF dose (320 μg) as part of the IND/MF combination in terms of systemic exposure [23].

Study population
A total of 32 subjects (16 Japanese and 16 Caucasian) were planned to be randomized in this study. Each Caucasian subject was to be matched pair wise to his Japanese counterpart by age (± 10 years) and weight (± 20% [kg]). All subjects provided written informed consent prior to any assessments being performed. As per the key inclusion criteria, participants were healthy male subjects, aged 20-45 (inclusive) years of age, a BMI of 18-30 kg/m 2 and in good health as determined by past medical history, physical examination, vital signs, electrocardiogram, and laboratory tests at screening and baseline of treatment period 1. Japanese subjects were to be of first-generation Japanese origin (Japanese subjects of first generation are defined as subjects living in Japan and having both parents and grandparents of Japanese descent); Caucasian subjects of first-generation Caucasian origin (Caucasian subjects of first generation are defined as subjects having both parents and grandparents of Caucasian descent). Key exclusion criteria were: (1) use of other investigational drugs at screening, or within five half-lives of enrollment, or within 4 months, whichever was longer; (2) a history of clinically significant ECG abnormalities; (3) significant illness which had not resolved within 2 weeks prior to initial dosing; (4) history of acute or chronic bronchospastic disease (including asthma and COPD, treated or not treated); (5) any surgical or medical condition which might significantly alter the absorption, distribution, metabolism, or excretion of drugs, or which could jeopardize the subject in case of participation in the study; (6) inability to use the Breezhaler ® device at screening. All blood samples were taken by either direct venipuncture or an indwelling catheter inserted in a forearm vein. At specified time points, 4 mL blood was collected in lithium heparin tubes for determination of IND and GLY, and 6 mL blood was collected in K2-EDTA tubes for determination of MF. The plasma samples of each compound were assayed using three separate validated liquid chromatography tandem mass spectrometry methods. Briefly, for IND determination, 0.2 mL plasma was processed using solid phase extraction and then separated on an ACQUITY UPLC BEH C 18 column using acidified water and acetonitrile mobile phase at a flow rate of 0.750 mL/min within 3.30 min. The lower limit of quantification (LLOQ) of IND was 5.00 pg/mL. For GLY determination, sample processing was performed by means of solid phase extraction using a volume of 0.2 mL plasma. Separation was obtained within 2.5 min on an ACQUITY UPLC BEH C 18 analytical column at a flow rate of 0.750 mL/min with binary mobile phases made of water and acetonitrile both acidified 0.1% formic acid. The LLOQ of the method was 1.00 pg/mL.

Pharmacokinetic analysis
For the MF determination, the method consisted of liquid-liquid extraction using 0.8 mL of plasma, and the separation of the extracted sample was achieved on an ACQUITY UPLC BEH C 18 column at a flow rate of 1.00 mL/min within 3.51 min, using a binary mobile phase made of 0.05% ammonia in water and acetonitrile. The LLOQ of the method was 0.250 pg/mL. Additional analytical parameters and performances of the three methods used for the determination of the three compounds are summarized in Additional file 1: Table 1. Concentrations below the LLOQ were treated as zero in summary statistics of concentration data as well as PK parameter calculations. PK parameters (AUC 0-24 h , C max and T max ) were determined by non-compartmental analysis using WinNonlin Phoenix (version 6.4; Certara, Princeton, NJ, USA).

Safety
Safety was monitored in terms of adverse events (AEs) and serious adverse events (SAEs). There was regular monitoring of hematology, blood chemistry, urinalysis, and ECGs, and regular assessments of vital signs, physical condition, and bodyweight.

Statistical analysis
All subjects who received at least one dose of study medication were included in the safety analysis set. The PK analysis set included all subjects with at least one available valid PK concentration measurement, who received any study drug and had no protocol deviations that were expected to impact the PK analysis. Log-transformed primary PK parameters (AUC 0-24 h and C max ) for IND, GLY and MF on Day 1 and Day 14 were analyzed by day using a linear mixed-effects model. The model included ethnic group, sequence, period, treatment and the interaction between ethnic group and treatment as fixed effects. Subject and matched pairs were considered as random effects. All matched pairs with evaluable PK parameters for at least one subject were included in the analysis. The above analysis was also repeated by including baseline age and bodyweight as covariates into the model.

Study participants
A total of 33 subjects (16 Japanese and 17 Caucasian subjects) were randomized to one of the two IND/GLY/MF treatment sequences. Twenty nine out of 33 subjects (14 Japanese and 15 Caucasian subjects) completed the study while four subjects (2 Japanese and 2 Caucasian subjects) discontinued the study. Three subjects (one Japanese and two Caucasian subjects) receiving IND/GLY/ MF 150/50/80 μg discontinued the study due to the withdrawal of consent by subject in treatment period 1. One Japanese subject receiving IND/GLY/MF 150/50/80 μg was discontinued from the study by the Investigator due to AEs of gastroenteritis and headache, in treatment period 2.
All the subjects were men; the baseline characteristics are presented in Table 1. The study population of healthy Japanese and Caucasian subjects was consistent with the key inclusion and exclusion criteria as outlined under study population in the Methods section. The median age of Japanese subjects was 31 years and median age of Caucasian subjects was 27 years. Mean bodyweight was higher in Caucasian subjects than in Japanese subjects.

Pharmacokinetics Indacaterol
Overlapping mean plasma concentration-time profiles of IND were observed between treatment groups (IND/ GLY/MF medium and high-dose) in both Japanese and Caucasian subjects. The mean plasma concentrations were slightly higher in Japanese subjects than in Caucasian subjects (Fig. 2a)

Glycopyrronium
Comparable mean GLY concentration time profiles were observed between treatment groups (IND/GLY/MF medium and high dose) in both Japanese and Caucasian subjects. The mean concentrations over 24 h were slightly higher in Japanese subjects than in Caucasian subjects (Fig. 2b) (Table 3). Based on the results of the exploratory statistical analysis including age and bodyweight as covariates, the geometric mean ratios (90% CI) (Japanese/Caucasian) for C max on Day 14 in IND/GLY/ MF medium-and high-dose treatment groups for GLY were 1.33 (90% CI 1.09-1.61) and 1.20 (90% CI 0.98-1.46), respectively. The mean ratios (90% CI) (Japanese/ Caucasian) for AUC 0-24 h on Day 14 in IND/GLY/MF medium-and high-dose groups were 0.95 (90% CI 0.84-1.09) and 0.95 (90% CI 0.83-1.08), respectively.

Mometasone furoate
After single and multiple doses of IND/GLY/MF (either medium or high-dose) in Japanese and Caucasian subjects, the median time to reach T max of MF ranged from 1 to 2 h both on Day 1, and on Day 14. There was no difference in T max between Japanese and Caucasian subjects (overall range: 0.25 to 3 h both on Day 1 and Day 14) (Fig. 2c, Table 2). A twofold increase in dose of MF from 80 μg to 160 μg led to an approximately twofold increase in exposure (C max and AUC 0-24 h ) in both Japanese and Caucasian subjects (Fig. 2c). The accumulation ratios (R acc for AUC 0-24 h and C max ) after o.d. dosing of IND/GLY/MF medium dose for 14 days were 1.37 (AUC 0-24 h ) and 1.35 (C max ) in Japanese subjects, and were 1.33 (AUC 0-24 h ) and 1.28 (C max ) in Caucasian subjects. The accumulation ratios (R acc for AUC 0-24 h and C max ) after  (Table 4).

Safety
IND/GLY/MF was well tolerated and had a similar safety profile at both doses in Japanese and Caucasian subjects.
No AEs reported during the study was considered to be related to the study drug by the Investigator and all AEs were resolved by the end of the study. There were no deaths or SAEs reported in the study. One Japanese subject receiving IND/GLY/MF medium-dose reported two AEs (gastroenteritis and headache) that led to discontinuation of study treatment.

Discussion
We

Analysis of individual data across treatments and analytes
Individual concentration-time profiles and individual PK parameters across analytes (IND, GLY or MF) and treatments (150/50/80 μg or 150/50/160 μg) were explored as part of data analysis. There was no correlation between the exposure of the different analytes as evidenced by the

Table 3 Summary of the analysis of plasma PK parameters (Japanese vs Caucasian) on Day 1 and Day 14
Model: log (pk) = ethnic group + sequence + period + treatment + ethnic group * treatment, with random effects for subject and matched pair.  lack of any systematic trends for higher exposure for all analytes in an individual subject. These data confirmed generally consistent use of inhalation technique across subjects. Similar accumulation was noted in an individual subject for a particular analyte in both treatments; however, the extent of accumulation was different for different analytes. This observation is consistent with the slightly different mean accumulation ratios for AUC and C max for the 3 analytes (  administered via the Breezhaler ® device was evaluated in Japanese and Caucasian subjects [23]. IND and MF are primarily metabolized via the CYP3A4 [24] whereas GLY is predominantly metabolized by CYP2D6. No ethnic variation has been reported for the expression and polymorphisms of CYP3A4, the observed difference in the IND and MF exposure was not considered to be due to any ethnic differences in a metabolic process.
Single and multiple doses of IND/GLY/MF medium and high dose were safe and well tolerated in Japanese and Caucasian healthy subjects. Drug-related AEs or SAEs were not reported in the study.
Overall, no clinically relevant ethnic effects on the systemic exposure to IND, GLY and MF was observed after multiple-dose administration of IND/GLY/MF in healthy subjects. The observed differences were small in magnitude and were not considered clinically relevant in view of the observed exposure variability, challenges with characterizing C max for inhaled drugs and the available safety data at doses higher than proposed doses of IND [25], GLY [26] and MF [27] as part of IND/GLY/MF. In the Phase 3 clinical study for IND/GLY/MF in asthma patients [28], while the study was not powered to demonstrate differences by subgroups, there were no observed differences in clinical efficacy or safety profile according to recorded ethnicity. In addition, during the development of IND/GLY/MF in Japan, 52 weeks treatment of IND/GLY/MF was well tolerated in Japanese patients with inadequately controlled asthma [29]. Overall adverse event profile was generally consistent with that known to occur with one or more of the components. No new or unexpected safety signals were observed in the study. Furthermore, a literature search to assess the potential risk of safety concerns in Japanese subjects resulting from an increased exposure for IND, GLY or MF did not reveal any safety concerns specific to the Japanese population following single or multiple dose administration of IND [18,30], GLY [31] or the IND/GLY combination [32,33] in COPD patients. The observed adverse events were expected in the patient population studied and were similar to those seen in studies conducted in the Caucasian population. Similarly, there were no safety concerns specific to the Japanese population in a single dose study with IND [34] or a multiple dose study with MF in Japanese asthma patients [35]. Overall, based on the literature evidence, the safety profile for IND, GLY and MF in monotherapy or combination products was similar between Japanese and Caucasian populations. No potential risk of safety concerns was identified in Japanese subjects due to increased exposure of IND, GLY or MF.
This study was conducted as a parallel group evaluation in Japanese and Caucasian subjects in a single center with demographically matched subjects from both ethnic groups. Assessment of PK in both ethnic groups at the same site within the same study avoids inter-study or inter-center variability, which may contribute to an apparent difference in PK of inhaled compounds due to potential site-specific differences in inhalation technique used by study volunteers. In prior studies for MF delivered with the Twisthaler ® inhalation device, the C max and AUC values were about two to threefold higher for Japanese patients with asthma compared with Caucasian patients with asthma [20]. This difference in approach to study conduct (single center vs. cross-study comparisons based on separate studies in Caucasian and Japanese patients) may explain the differences noted between prior data and our analysis we noted no clinically relevant difference between MF AUC and C max between ethnicities.
The mean total and peak exposures of IND and GLY observed in this study were higher than noted in previous studies [38]. Mean C max and AUC 0-24 h of GLY on Day 14 following administration of GLY alone in asthma patients (Caucasian) were 166 pg/mL and 464 h × pg/mL, respectively. Mean C max and AUC 0-24 h of IND on Day 14 following administration of IND alone or as part of the IND/MF fixed-dose combination were 307-317 pg/mL and 1910-1950 h × pg/mL, respectively, in healthy subjects. However, acknowledging limitations of cross-study comparisons, overall the observed exposure levels are consistent with those observed in previous PK studies.

Conclusions
IND, GLY and MF were systemically available shortly after the inhalation of single or multiple doses of IND/ GLY/MF at the medium and high ICS dose strengths in both Japanese and Caucasian subjects. Median plasma T max for IND, GLY and MF across both dose groups was 15 min, 5 min and 1-2 h, respectively. Slightly higher exposure of each individual drug in Japanese subjects as compared with Caucasian subjects was observed following single or multiple doses of IND/GLY/MF medium or high dose but is not considered clinically relevant. For MF, C max and AUC 0-24 h increased in a dose-proportional manner between IND/GLY/MF medium and high dose on both Day 1 and Day 14 for both ethnicities. Single and multiple doses of IND/GLY/MF medium and high dose were safe and well tolerated in Japanese and Caucasian healthy subjects.
Additional file 1. Analytical parameters and performances of the three methods used for IND, GLY and MF.