Research Article - (2015) Volume 6, Issue 2

A Pilot Study on Effect of Beta2-Adrenergic Receptor Gene Polymorphism on Response to Fluticasone/Formoterol Inhaler in Patients with Persistent Asthma

Motohiro Kurosawa1,2*, Tatsuo Yukawa3 and Eijin Sutoh1
1Department of Allergy and Respiratory Medicine, Sutoh Hospital, Annaka-shi, Gunma, Japan
2Gunma Institute for Allergy and Asthma, Gunma, Japan
3Yukawa Clinic of Internal Medicine, Tochigi, Japan
*Corresponding Author: Motohiro Kurosawa, Department of Allergy and Respiratory Medicine, Sutoh Hospital, 3532-5 Annaka, Annaka-shi, Gunma 379-0116, Japan, Tel: +81-27-382-3131, Fax: +81-27-382-6568 Email:

Abstract

Background: Safety concerns have been raised regarding the regular use of long-acting ß2-agonists (LABAs) with inhaled corticosteroids (ICSs), and some studies suggested patients with asthma who are homozygous for arginine (ArgArg) at the 16th amino acid position of the ß2-adrenergic receptor (ADRB2) gene benefit less from treatment with ICS/LABA than do those homozygous for glycine (GlyGly). Phase III trial of fluticasone/formoterol, which consisted of 8-week, multicenter randomized single-blinded parallel-group study and 52-week, multicenter open-label study, have been conducted in Japan. In the trials, we performed a pilot study on effect of ADRB2 gene polymorphisms on lung function and asthma control to regular use of the new ICS/LABA inhaler. Methods: As number of patients was limited to each institution, nine patients were allowed to participate in this pilot study. DNA in the specimens in patients with persistent asthma was extracted, and the target DNA sequence of the gene was amplified. Allelic discrimination assay for single nucleotide polymorphisms relating to the ADRB2 was carried out. Results: In the 8-week study, patients with either ArgArg or ArgGly genotype showed good changes of peak expiratory flow (PEF) values from baseline with fluticasone/formoterol treatment. In the 52-week study, the improvement of lung function tests (forced expiratory volume in one second and PEF) from baseline was observed with the treatment, and all of the daily diary-based measures of asthma control were improved from baseline in each patient over the treatment period irrespective of their genotypes of ADRB2. Conclusion: This is the first prospective pilot study, which might suggest inheritance of ADRB2 gene polymorphisms may not appear to effect clinical outcomes with fluticasone/formoterol inhaler.

Keywords: Beta2-adrenergic receptor; Gene polymorphism; ADRB2; Fluticasone; Formoterol; Combination therapy; Asthma

Introduction

Combination therapy with long-acting β2-adrenergic agonists (LABAs) and inhaled corticosteroids (ICSs) has been one of the most widely prescribed treatments for the control of asthma in the world, and this combination therapy has been endorsed in the Global Initiative for Asthma (GINA) guidelines for the treatment of moderate to severe asthma in 2006 [1]. Based on safety concerns about LABAs raised by the Food and Drug Administration (FDA) in the United States in February 2010 [2], the FDA issued a requirement on April 14, 2011 for all manufactures of LABAs that are marked for asthma in the United States to conduct controlled clinical trials to assess the safety of regimen of ICS/LABA [3].

Recent pharmacogenetic studies demonstrated that the ß2-adrenergic receptor (ADRB2) gene polymorphisms modify bronchodilating response to regular use of albuterol [4,5]. With respect to LABA therapy, it has been reported that asthmatic patients with the homozygous for arginine (ArgArg) at codon 16 of the ADRB2 gene have an impaired therapeutic response to salmeterol in either the absence or presence of ICS [6,7]. Also, ethnic-specific pharmacogenetic difference in response to salmeterol has been suggested in individuals with different ADRB2 genotypes [8]. In contrast, other studies reported that ADRB2 gene polymorphisms did not result in significant change of bronchodilating response to LABAs in the presence of ICSs [9,10]. The results of some studies seemed to be limited by their retrospective design and inability to control for specific factors.

The ICS, fluticasone propionate (fluticasone), delivers a potent and sustained anti-inflammatory effect [11], and the LABA, formoterol has a fast onset of action and prolonged bronchodilatory effect [12]. The new ICS/LABA combination of fluticasone/formoterol has become available first in Germany and the United Kingdom in September 2012, with subsequent launches in other European countries for administration via a single pressurized metered-dose inhaler (pMDI).

Also in Japan, phase III trials of the ICS/LABA pMDI have been performed. The trials in Japan consisted of two studies, study I and study II [13,14]. Namely, study I was 8-week, multicenter (88 centers in Japan) randomized single-blinded parallel-group study, and was composed of a 28-day screening, 2-week run-in (fluticasone 200 µg daily), and 8-week treatment periods. Patients aged ≥ 16 years (N=455) with persistent asthma for ≥ 6 month before screening, FEV1 50-85% predicted, and >12 % and >200 mL reversibility following albuterol inhalationwere randomized to receive fluticasone/formoterol 100/10µg twice daily (N=228) or fluticasone 100 µg twice daily (N=227) for 8 weeks (Clinical Trials Gov JapicCTI number: 101340) [13]. Study II was a long-term (52 weeks), multicenter (71 centers in Japan) open-label study assessing the safety and efficacy of fluticasone/formoterol, and was composed of a 28-day screening, 2-week run-in (fluticasone 200~1000 µg daily or equivalent), and 52-week treatment periods. Patients aged ≥ 16 years (N=244) with persistent asthma for ≥ 6 month before screening, FEV1 40-85% predicted, and >12 % and >200 ml reversibility following albuterol inhalation started to receive twice-daily treatment with fluticasone/formoterol 100/10 µg, 250/10 µg or 500/20 µg, based on the doses of ICS that had been used in a run-in period. Doses were allowed to increase or decrease according to the patients’ symptoms, and treatment was continued for 52 weeks (Clinical Trials Gov JapicCTI number: 101341) [14]. As the phase III trials showed fluticasone/formoterol pMDI had a good safety and efficacy profile in the two studies, fluticasone/formoterol pMDI has become available in Japan in November 2013.

To the best of our knowledge, there has been no report that investigated the effect of ADRB2 gene polymorphisms on response to regular use of fluticasone/formoterol pMDI. Therefore, in the phase III trials we’ve performed a prospective pilot study with patients whose ADRB2 genotypes had already been analyzed before the trials. This is the first prospective pilot study on effect of ADRB2 gene polymorphisms on response to regular use of fluticasone/formoterol pMDI.

Materials and methods

Patients and genotyping of ADRB2 polymorphism

This study was performed with the approval of the Institutional Ethics Committee of Gunma Institute for Allergy and Asthma, Gunma, Japan and written informed consent was obtained from each participant before the study commenced. Diagnosis of bronchial asthma was confirmed using the GINA guidelines. All patients showed clinical symptoms that met the criteria for asthma, and they were diagnosed by experienced pulmonologists. Forced expiratory volume in one second (FEV1) was measured with a spirometer, and airway reversibility was defined as a >12 % and >200 ml increase in volume in the first second of forced expiration from baseline after inhalation of short-acting β2-adrenergic bronchodilators. Smoking habit was ascertained by means of a questionnaire, and subjects with a smoking history that was either recent 12 months or equivalent to ≥ 10 pack-years were excluded.

DNA in the specimens obtained by rubbing buccal mucosa with a cotton swab from the patients was extracted by using QIAamp 96 DNA blood kits (Qiagen, Hilden, Germany). The target DNA sequence of the ADRB2 gene NM_000024.4 was amplified using a set of primers that were previously described [15,16] (forward, nucleotides 188-212: 5’-AGCCAGTGCGCTCACCTGCCAGACT-3’; reverse, nucleotides 406-383:5’-GCTCGAACTTGGCAATGGCTGTGA-3’) to generate an amplicon of 219bp in length. Allelic discrimination assay for single nucleotide polymorphisms (SNPs) relating to the ADRB2 expression (rs 1042713) was carried out by using previously described SNPs detective system, sequence-specific thermal-elution chromatography as reported from our laboratory [17,18].

Because the number of patients who could participate in the trials was limited for each institution, nine patients (four in study I, and five in study II, respectively), whose ADRB2 genotypes had already been analyzed at our laboratory, were allowed to be involved in the present study (Table 1). Namely, one patient with wild-type ArgArg and three with variant-type ArgGly in study I, and one with wild-type ArgArg and four with variant-type ArgGly/GlyGly in study II respectively, participated in the phase III study conducted in Japan.

Name (initial) Age (years) Gender Genotype Daily Dosea
In 8-week, multicenter randomized single-blinded parallel group study 
YM 28 female ArgArg unknownb
SW 58 female ArgGly unknownb
MT 41 female ArgGly unknownb
HK 56 male ArgGly unknown b
In a long-term (52 weeks), multicenter open-label study
SN 56 male ArgArg 1000/40 µg
HT 48 female GlyGl 1000/40 µg
TK 42 female GlyGly 1000/40 µg
NT 42 female GlyGly 200/20 µg and 500/20 µg since 28 week
EH 48   ArgGly 500/20 µg and 1000/40 µg since 28 week
adose of fluticasone/formoterol, bdaily dose was unknown until the phase III trials finished. ADRB2: ß2-Adrenergic Receptor; Arg: Arginine; Gly: Glycine

Table 1: ADRB2 genotype of nine patients in this study, which was performed in fluticasone/formoterol pressurized meter-dose inhaler phase III trials in Japan (8-week, multicenter randomized single-blinded parallel group study, and 52-week, multicenter open-label study).

Efficacy evaluations

The primary outcome variables of the phase III trials conducted in Japan were lung function values, which are recognized as useful indicators that correlate well with clinical outcomes in asthma. Morning predose FEV1 values determined by a spirometer were assessed at approximately the same time (± 1 hour) at each study visit immediately before the administration of study medication. The value of peak expiratory flow (PEF) was assessed twice daily using a Mini-Wright PEF meter (Clement Clark, Harlow, UK). Daily diary-based measures of asthma control, including asthma symptom-free days, awaking-free nights, and rescue medication use of short-acting ß2-adrenergic agonist (albuterol pMDI 90 µg/inhalation), were recorded by the patient. In study I, follow-up clinic visits were scheduled 4 and 8 weeks after randomization. In study II, follow-up clinic visit were scheduled 4, 12, 28, 36 and 52 weeks after starting treatment. The primary endpoint was the changes of lung function tests (predose PEF and FEV1 values) at each study visit. The main secondary endpoints were changes in daily diary-based measures of asthma control mentioned above. The baseline values of PEF were defined as the mean value for the last 10 days of the run-in period, excluding the day of randomization, and that at each week were the mean value for the last 28 days. A symptom-free day was defined as a 24-hour period during which a patient did not experience any asthma symptoms, and a rescue medication-free day was defined as a 24-hour period during which a patient did not use rescue medication.

Statistical analysis

The data of each lung function value of the patients were collected at each visit in the phase III trials. Statistical analysis for the changes from baseline (week 0) to each subsequent visit for lung function values, PEF and FEV1, was analyzed using a paired t-test. A p-value of <0.05 was considered statistically significant.

Results

Figure 1 shows the changes of predose PEF and FEV1 values of the patients in study I (8-week, multicenter randomized single-blinded parallel-group study). Morning and evening PEF values at baseline (week 0) were 368.5 ± 38.1 (mean ± SEM) and 369.5 ± 39.8 L/min, respectively. Morning and evening PEF values at the end of the 8-week treatment were 376.9 ± 39.8 and 382.2 ± 40.1 L/min, and the increase of morning and evening PEF values were significant (p=0.041 in morning PEF and p=0.032 in evening PEF, respectively) (Figure 1A and Figure 1B). On the other hand, no significant change of FEV1 was observed (Figure 1C). Daily diary-based measures of asthma control, including asthma symptom-free days, awaking-free nights, and rescue medication use of the patients in study I are presented in Table 2.

  Patient
Name
Symptom-Free Days (%) Awakening-Free Nights (%) Rescue Days (%) Medication Use Inhalation/day
  YM
Baseline   20 100 20 0.2
8 week   73.2 100 1.8 0
  SW
Baseline   0 100 90 2.1
8 week   0 100 46.4 1.1
  MT
Baseline   90 100 10 0.2
8 week   94.6 100 5.4 0.1
  HK
Baseline   0 100 30 0.3
8 week   0 100 47.2 0.8
The baseline values were defined as the value for the last 10 days of the run-in period, excluding the day of randomization, and that at each week were the value for the last 28 days.

Table 2: Changes in daily diary-based measures of asthma control of patients in 8-week, multicentre randomized single-blinded parallel group study

allergy-therapy-Changes-morning

Figure 1: Changes in morning PEF (A), evening PEF (B) and FEV1 (C) values of four patients in 8-week, multicenter randomized single-blinded parallel-group study of fluticasone/formoterol pressurized meter-dose inhaler phase III trials conducted in Japan. The baseline values of PEF were defined as the mean value for the last 10 days of the run-in period, excluding the day of randomization, and that at each week were the mean value for the last 28 days. Statistical analysis for the changes from baseline (week 0) to each subsequent visit was analyzed using a paired t-test. PEF: Peak Expiratory Flow; Mpef: Morning Peak Expiratory Flow; Epef: Evening Peak Expiratory Flow; FEV1: Forced Expiratory Volume in One Second; N.S.: Not Significant; *p<0.05.

After study I in the phase III study was completed, the key was opened showing that patients YM, MT and HK were controlled with twice-daily inhalation of fluticasone/formoterol 100/10 µg, and patient SW was twice-daily inhalation of fluticasone 100 µg. Morning and evening PEF values of patient SW at baseline (week 0) and 8 week were 343.0, 346.5 L/min in morning PEF, and 340.0, 345.0 L/min in evening PEF, respectively. The ADRB2 genotype of the patients treated with twice-daily use of fluticasone/formoterol 100/10 µg was either ArgArg or ArgGly genotype. Increase of symptom-free days and decrease of rescue medication use was observed in patient YM with ArgArg homozygote of the ADRB2 gene.

Figure 2 shows the changes of predose PEF and FEV1 values of the patients in study II (52-week, multicenter open-label study). The morning PEF values at baseline (week 0) and that at 36 week were 271.4 ± 26.8 and 302.8 ± 23.6 L/min, respectively. A significant increase of morning PEF values from baseline with 36-week treatment was observed (p=0.018) (Figure 2A). The evening PEF values at baseline, 36 week and 52 week were 288.8 ± 36.0, 321.8 ± 28.7 and 325.6 ± 31.0 L/min, respectively. A significant increase of evening PEF values from baseline with 36-week and 52-week treatment was observed (p=0.031 and 0.014, respectively) (Figure 2B). The FEV1 values at baseline and 36 week were 1.504 ± 0.139 and 1.668 ± 0.119 L, respectively. The change of FEV1 values from baseline with 36-week treatment was significant (p=0.018) (Figure 2C). The improvement of lung function tests (predose PEF and FEV1) from baseline with either 36- or/and 52-week treatment was present in the patients irrespective of their genotype of ADRB2 gene. Daily diary-based measures of asthma control, including asthma symptom-free days, awaking-free nights, and rescue medication use of five patients in study II are shown in Table 3. All of the daily diary-based measures of asthma control were improved from baseline in each patient over the treatment period.

Patient Name Symptom-Free Days (%) Awakening-Free Nights (%) Rescue Days (%) Medication Use Inhalation/day
SN        
Baseline 0 0 60 0.7
36 week 0 0 0 0
52 week 0 0 0 0
HT        
Baseline 60 80 40 0.4
36 week 100 100 0 0
52 week 96.4 100 3.6 0.1
TK        
Baseline 0 100 30 0.4
36 week 0 100 0 0
52 week 0 100 3.6 0
NT        
Baseline 10 100 30 0.3
36 week 85.7 100 0 0
52 week 76.2 90.5 0 0
EH        
Baseline 70 90 10 0.1
36 week 100 100 0 0
52 week 100 100 0 0
The baseline values were defined as the value for the last 10 days of the run-in period, excluding the day of randomization, and that at each week were the value for the last 28 days.

Table 3: Changes in daily diary-based measures of asthma control of patients in a long-term (52 weeks), multicenter open-label study.

allergy-therapy-Changes-evening

Figure 2: Changes in morning PEF (A), evening PEF (B) and FEV1 (C) values of five patients in a long-term (52 weeks), multicenter open-label study of fluticasone/formoterol pressurized meter-dose inhaler phase III trials conducted in Japan. The baseline values of PEF were defined as the mean value for the last 10 days of the runin period, excluding the day of randomization, and that at each week were the mean value for the last 28 days. Statistical analysis for the changes from baseline (week 0) to each subsequent visit was analyzed using a paired t-test. PEF: Peak Expiratory Flow; Mpef: Morning Peak Expiratory Flow; Epef: Evening Peak Expiratory Flow; FEV1: Forced Expiratory Volume in One Second; N.S.: Not Significant; *p<0.05.

Discussion

To the best of our knowledge, there has been no report which investigated the effect of ADRB2 gene polymorphisms on response to regular use of fluticasone/formoterol pMDI. So, we conducted a prospective pilot study on effect of ADRB2 gene polymorphisms on response to regular use of fluticasone/formoterol pMDI in its phase III trials in Japan.

The most commonly cited SNPs in the ADRB2 gene occur at nucleotide position 46 (guanine/adenine) and 79 (cytosine/guanine), and result in the substitution of glycine (Gly) for Arg at codon 16 (Arg16Gly) and glutamic acid (Glu) for glutamine (Gln) at codon 27 (Gln27Glu), respectively. Following the identification of SNPs in the ADRB2 gene that affect cell-surface expression or ligand-binding activity of the receptor, there has been particular interest in pharmacogenetic associations related to this receptor [19-22]. In two common polymorphisms, Arg16Gly and Gln27Glu of the ADRB2 gene, only the Arg16Gly polymorphism was investigated in the present study because of very low frequency of minor allele of Gln27Glu polymorphism in Japanese [23].

As the number of patients who were able to participate in the phase III trials was limited, nine patients were allowed to be involved in this study which investigated the effect of ADRB2 gene polymorphisms on lung function and asthma control to regular use of formoterol/fluticasone pMDI. In 8-week, multicenter randomized single-blinded parallel-group study of the trials, the patients with either wild-type ArgArg or variant-type ArgGly were shown to be treated with fluticasone/formoterol pMDI, and the PEF values changed from baseline were increased at the end of the treatment. In 52-week, open-label study of the trials, the improvement of lung function tests (PEF and FEV1) from baseline with the treatment was observed in all five patients, and the daily diary-based measures of asthma control were improved from baseline in each patient over the treatment period irrespective of their genotype of ADRB2.

Safety concerns have recently been raised regarding the regular use of fluticasone/salmeterol inhaler, and some studies suggested patients with asthma who are homozygous ArgArg at the 16th amino acid position of the ADRB2 gene benefit less from treatment with fluticasone/salmeterol than do those homozygous GlyGly [6,7]. In contrast with fluticasone/salmeterol, short- and long-term pharmacogenetic studies of budesonide/formoterol did not show an effect of Gly16Arg genotype on safety measures or clinical outcomes in response to budesonide/formoterol [24-26]. Possible explanation for the difference between these ICSs/LABAs involves the choice of LABA; formoterol versus salmeterol, because investigations about the effects of ADRB2 gene polymorphism on ex vivo and in vivo function of ß2-adrenoceptors in asthmatic patients suggested that ADRB2 gene polymorphism had no significant effect on response to formoterol [27]. Concerning about the differences related to using different ICSs, fluticasone versus budesonide in fluticasone/formoterol and budesonide/formoterol inhalers may mean little clinically [28,29]. In fact, one randomized, double-blind, double-dummy, multicenter 12-week phase III study demonstrated comparable efficacy of fluticasone/formoterol to budesonide/formoterol [28], and the other 12-week, open-label multicenter phase III trial reported fluticasone/formoterol inhaler showed non-inferiority to budesonide/formoterol formulation [29].

In conclusion, this prospective pilot study might suggest inheritance of ADRB2 gene polymorphisms may not appear to effect clinical outcomes with fluticasone/formoterol pMDI. Further investigations are needed to replicate this observation using a large size of study population and confirm pharmaco-ethnic differences.

References

Citation: Motohiro Kurosawa, Tatsuo Yukawa and Eijin Sutoh (2015) A Pilot Study on Effect of Beta2-Adrenergic Receptor Gene Polymorphism on Response to Fluticasone/Formoterol Inhaler in Patients with Persistent Asthma. J Allergy Ther 6:208.

Copyright: © 2015, Kurosawa M et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.