Polymorphism at codon 16 of the beta2-adrenoceptor (beta2-AR) affects the responsiveness to salmeterol in asthmatics. Data concerning formoterol are more controversial in the literature. The aim of this study was to verify whether homozygous for arginine-16 (ArgArg16) and homozygous for glycine-16 (GlyGly16) genotypes differently influence the long-term responsiveness to formoterol. Twenty-nine patients with mild-to-moderate asthma, in stable clinical conditions, underwent genotyping at codon 16 of the beta2-AR by RFLP-PCR assay. The effects of a 4-week monotherapy with formoterol (12
Genetic factors, mainly the polymorphism at codons 16 and 27 of the beta2-adrenoceptor (beta2-AR) on chromosome 5q31, are known to modulate the bronchodilatory effects of beta2-agonists [
Further complicating the issue are findings pertaining to formoterol: asthmatics carrying the GlyGly16 but not the ArgArg16 genotype had a greater risk of desensitisation [
These contrasting results might reflect differences in study design, duration of the exposition to the beta2-agonist, and main outcome. Furthermore, in order to comply with current therapeutic guidelines for asthma, in some studies inhaled corticosteroids (ICS) and LABA have been coadministered [
We report the preliminary results of a study designed to test the hypothesis that GlyGly16 asthmatics, who are otherwise more responsive to chronically used salmeterol, were more prone than ArgArg16 and/or ArgGly16 asthmatics to develop tolerance to formoterol in an experimental design free from the confounding effect of concurrent steroids administration.
We genotyped 29 Caucasian patients, 10 males and 19 females, with mild-to-moderate asthma. None had experience of athletics but 8 of them reported to perform a moderate weekly physical activity. None were smokers and all were in stable clinical condition at the time of recruitment. According to the Global Initiative for Asthma (GINA) guidelines, they had no chronic symptoms, no limitations on activities, and minimal need for “as needed” use of beta2-agonists [
All patients gave a written informed consent for the study which was in agreement with the guidelines approved by the local ethical committee.
Each patient underwent the following procedures (Figure
Study design.
Spirometry was performed according to the American Thoracic Society criteria [
Bronchial reactivity to inhaled methacholine was measured in patients with a FEV1/FVC ratio ≥70%, using a dosimeter providing a calibrated output of 9.0
This test was used to verify the acute response to a bronchodilator before and after the chronic treatment with formoterol, independently from the presence or not of bronchial obstruction at baseline. Cumulative doses of 200, 400, and 800
PEF was monitored during the 4-week treatment period by using a mini-Wright peak-flow meter (Clement Clarke Int. Ltd, UK). Home measurements of PEF were performed twice daily: the patients were instructed to record their morning and evening peak flow prior to taking each dose of formoterol, recording the best of three readings. The results were expressed as daily variability as follows:
Genomic DNA was extracted from peripheral blood leukocytes using the standard salting-out procedure. For beta2-AR ArgGly16 genotyping, DNA was amplified by PCR in a final reaction volume of 25
Differences among genotypes in terms of clinical and respiratory function parameters at baseline were analyzed by analysis of variance and unpaired
According to the genotypic analysis, 14 patients were GlyGly16, 5 were ArgArg16, and 10 were ArgGly16. All the patients remained clinically stable during the run-in period and only 5 of them occasionally used salbutamol as needed. Moreover they did not report significant respiratory symptoms during the treatment period and at the last visit performed 1 week after the end of the study protocol.
According to the working hypothesis, a comparison was made between the GlyGly16 and pooled ArgArg16 and ArgGly16 patients. No significant difference in baseline clinical and functional data was found between the two groups (Table
Comparison of baseline clinical and functional data between the two groups of patients.
GlyGly16 | ArgArg16 + ArgGly16 |
|
|
---|---|---|---|
Number | 14 | 15 | |
Age (years) | 34.28 ± 14.97 | 32.73 ± 8.34 | 0.73 |
Duration of disease (years) | 16.50 ± 8.10 | 15.87 ± 9.98 | 0.92 |
FVC (% pred) | 111.36 ± 15.30 | 105.93 ± 14.78 | 0.34 |
FEV1 (% pred) | 100.27 ± 13.07 | 91.93 ± 14.19 | 0.11 |
FEV1/FVC (%) | 76.87 ± 9.46 | 75.07 ± 8.34 | 0.59 |
|
72.14 ± 23.14 | 61.80 ± 23.67 | 0.24 |
FEV1 changes after salbutamol (%) | 9.43 ± 5.40 | 9.73 ± 7.63 | 0.90 |
PC20FEV1 (mg/mL)* | 1.93 ± 1.10 | 1.54 ± 1.26 | 0.53 |
Data are expressed as mean ± SD.
*The methacholine challenge test was performed in 11 patients of the GlyGly16 group and in 10 of the ArgArg16 + ArgGly16 group.
FVC: forced vital capacity; FEV1: forced expiratory volume in 1 sec;
The results derived from the dose-response curve to salbutamol performed before and after the 4-week treatment with formoterol are reported in Figure
FEV1 slope of the dose-response curve to salbutamol before and after the treatment period in the GlyGly16 (continuous line) and in pooled ArgArg16 and ArgGly16 patients (dashed line). Values are expressed as means with error bars. Between-groups differences:
In GlyGly16 patients, PEF variability remained substantially unchanged during the first 3 weeks, but it dramatically increased in the last week of treatment (Figure
PEF variability during the 4-week treatment period in the GlyGly16 (continuous line) and in pooled ArgArg16 and ArgGly16 patients (dashed line). Values are expressed as means with error bars. Between-groups differences:
We found that the GlyGly16 genotype is associated with a well evident liability to develop tolerance during chronic treatment with the LABA formoterol. This finding confirms those of Tan et al. and Aziz et al, but does not suffer from the confounding effect due to concurrent steroid administration [
As noted previously, conflicting results are reported in the literature about the relationship between different genotypes and chronic use of different LABAs alone or added to ICS. In the LARGE study, both ArgArg16 and GlyGly16 patients experienced an improved airway function with salmeterol added to moderate dose of ICS [
On purely pharmacological ground, salmeterol and formoterol have different structures, mechanism of action, and pharmacodynamic properties [
Our and most other previous studies on the genetic determinants of tolerance to beta2-agonists have focused on the Arg-Gly polymorphic site at codon 16 which seems to have a more important role in beta2-AR desensitisation, compared to the other polymorphic sites at codon 27 (Gln-Glu) [
Prevalence of the ArgArg16 and GlyGly16 patterns in our patients was 17% and 48%, respectively. These figures are comparable to those reported by most of previous studies of Caucasian populations for ArgArg16 pattern, while the prevalence of GlyGly pattern was slightly higher in our sample than in other studies [
This study has important limitations. First, the sample size was small. However, the fact that a significant association between GlyGly16 genotype and tolerance to formoterol emerged in a small sample is consistent with this association being biologically plausible. Second, a 4-week period might not be long enough to disclose genotype-dependent differences in tolerance to formoterol. However, it would be technically difficult and ethically unsound to maintain on LABA therapy selected subjects likely to benefit from concurrent inhaled steroids. Third, the boundary between onset of tolerance and mild exacerbation is not so straightforward, and this might result in overestimating the incidence of tolerance. This limitation affects any study aimed at providing an unbiased assessment of the risk of beta2-AR desensitisation during any pharmacological therapy in asthmatics. Eventually, asthma is a heterogeneous disease involving several pathogenetic mechanisms. Accordingly, any putative pharmacogenomic association can be variously confounded by simultaneous genetic factors and nongenetic host-related and environmental factors [
Our results support the hypothesis that the GlyGly16 genotype, which protects from tolerance to salmeterol and SABAs, qualifies as a risk factor for tolerance to formoterol. This preliminary finding needs to be confirmed in properly sized placebo-controlled studies including also the analysis of codon 27. If confirmed, it would affect asthma management and would be the rationale for synthesizing other LABAs with different molecular structures in order to enlarge the spectrum of LABA-beta2-AR interactions and, thus, tailoring the choice of LABAs to individual genotypes.
No financial or any other potential conflict of interests exists for each author.
This study is dedicated to Professor Christina Brahe.