To the Editor:

P2X3 receptors are found on sensory nerves and are activated by ATP. The recent discovery that P2X3 antagonism is effective in treating patients with refractory chronic cough has prompted recent studies into the effects of inhaled ATP [1–4], but few data exist pertaining to its safety and tolerability. We recently studied inhaled ATP in healthy volunteers, identifying adverse effects not previously reported.

The aim of this study was to compare the Provo.X dosimeter (Ganshorn, Germany) for evoking cough with the recommended, but now obsolete, KoKo dosimeter (n-spire, UK) [5, 6]. The study was approved by an accredited research ethics committee (21/NS/0119). 13 healthy volunteers performed ATP cough challenge with both devices in a random order 2–10 days apart. Participants were all never- or ex-smokers (<10 pack-years) with normal spirometry, no history of respiratory or other relevant disease and normal physical examination. Any participants taking medications that could affect the cough reflex, or recent (<4 weeks) upper or lower respiratory tract infections were excluded. Participants inhaled 10 µL of doubling ATP concentrations (0.125–512 mg·mL−1; Thermo Fisher, UK); four single inhalations per concentration, 30 s apart, interspersed with placebo. Each device was calibrated to deliver 10 µL per single breath inhalation. Coughs within the first 15 s were recorded. Participants performed spirometry before and after ATP inhalation at each visit, as is our routine practice as cough challenge agents (e.g. capsaicin and citric acid) can cause bronchoconstriction. The primary outcome measures were maximal cough response (Emax) and dose required to evoke 50% of the maximal response (ED50) using both devices.

13 patients were recruited to the study. 11 patients completed the study with one withdrawal after the first visit for unrelated reasons, and one screening failure due to no coughing being evoked on the first visit. Therefore, a total of 24 ATP challenges were performed. Participants were sex-matched (7:6, female:male), with median (interquartile range) body mass index of 24.5 (21.5–26.5). Median (interquartile range) age was 36 (33–50) years, forced expiratory volume in 1 s (FEV1) was 104 (93–110)% predicted, forced vital capacity (FVC) was 108 (102–116)% predicted, and FEV1/FVC 0.78 (0.74–0.85). Emax and ED50 were comparable (Emax Provo.X versus KoKo, median (range) 13 (1–27) versus 9 (0–37) coughs; p=0.2; and ED50 128 (8–512) versus 192 (8–512) mg·mL−1; p=0.9, respectively). The intraclass correlation coefficient for Emax suggested moderate agreement between devices (0.517, p<0.0001). Spearman's correlation coefficient demonstrated strong correlation in cough response between the KoKo and Provo.X (r=0.64, p<0.0001). However, of the first six subjects, one developed chest tightness, therefore the challenge was stopped. Spirometry was performed, demonstrating bronchoconstriction (≥20% FEV1 reduction). This occurred after the last inhalation of the 512 mg·mL−1 dose. A further participant had asymptomatic bronchoconstriction on post-challenge spirometry at both visits; a third participant experienced chest tightness after the final inhalation of the 512 mg·mL−1 dose. Additional safety measures were then incorporated in the latter half of the study, including continuous pulse oximetry and chest auscultation between ATP concentrations. In the next seven subjects, one participant had a sustained fall in oxygen saturations to 93% after the first inhalation of the 512 mg·mL−1 dose. The challenge was stopped, and spirometry was performed demonstrating an FEV1 fall ≥20%. Salbutamol was administered (400 μg via a metered dose inhaler and spacer) and oxygen saturations returned to normal with 5 min. A further subject had an audible wheeze after the 256 mg·mL−1 dose using the Provo.X and symptomatic chest tightening at the 512 mg·mL−1 dose using the KoKo dosimeter. Two further subjects became tight-chested with audible wheezing (requiring bronchodilation) at the end of the 512 mg·mL−1 dose; this happened with both devices in one of the participants and only with the KoKo device with the other. Another participant had a sustained drop in oxygen saturations to 91% at the end of the 256 mg·mL−1 dose using both devices. This returned to normal within 5 min of administration of 400 μg of salbutamol. Adverse events were unrelated to the dosimeter make (table 1). All participants responded to a 200 μg salbutamol metered dose inhaler delivered via spacer. In total, eight out of 13 participants (62%) had an adverse event within the study. This resulted in termination of the cough challenge at one of the two highest concentrations (256 mg·mL−1 or 512 mg·mL−1) in all but one case. One subject had an asymptomatic FEV1 fall that was only identified on post-challenge spirometry at each visit, making it unclear when exactly this occurred within the challenge.

Adverse events and % change in FEV1 post challenge for each participant and dosimeter

The first studies of inhaled ATP suggested it induced bronchoconstriction in patients with asthma, COPD and healthy smokers, but reports on healthy volunteers were not consistent, with one study reporting bronchoconstriction while two others did not [7–9]. More recent publications have not reported bronchoconstriction in either healthy controls or patients with chronic cough; however, these studies did not measure lung function after ATP exposure [10, 11] and therefore asymptomatic bronchoconstriction could have been missed.

The maximum concentration of ATP used has varied between studies (100–1101 mM, 50–512 mg·mL−1), with more recent studies using lower concentrations than in the current study, at 100–300 mM (50–152 mg·mL−1). The exact dose of ATP at which bronchoconstriction occurred is unknown in the current study as spirometry was performed routinely at the end of the challenge, or if patients developed symptoms which may have occurred after bronchoconstriction. Furthermore, the precise dose of ATP delivered to the airways is difficult to compare between studies as this will also be influenced by: 1) the aerosol particle size and density produced by different devices; 2) flow limitation (if used); 3) delivery through a mouth piece or mask; 4) whether single breaths or tidal breathing from a nebuliser was used; and finally, 5) if tidal breathing, the rate and depth of breathing.

The likelihood of bronchoconstriction appears to vary between different diagnostic groups. The two studies reported by Basoglu et al. [7, 9], using the same concentrations of ATP as our study, found that all 10 asthma patients had a fall in FEV1 of ≥20%, whereas this was only seen in just under half of the smokers and COPD patients. Breathlessness, captured by a Borg score, increased significantly in smokers, COPD patients and asthma patients [7, 9]. These observations have important implications for studies of patients with chronic cough inhaling ATP, in whom asthma is one of the most common comorbidities, and therefore the risk of bronchoconstriction is significant and thus lung function should be measured. Moreover, it is plausible that dyspnoea/chest tightness induced by ATP may act as confounding factors that should be taken account of in brain imaging studies of the urge to cough; in one study exercise-induced breathlessness was found to inhibit the urge to cough [12].

An alternative explanation for the difference in adverse effects between our findings and those of other recent studies could be the instability of ATP in solution. ATP solutions are stable for approximately 1 week at 0°C, but stability at room temperature is unclear. Our ATP solutions were made fresh before each challenge but if other investigators kept diluted ATP solutions for longer periods of time or at different temperatures prior to use, the effective dose and resultant adverse effects may be reduced.

This was a small pilot study intended to validate a new piece of equipment to be used for cough challenge and the findings were unexpected. Therefore, the study was not designed to determine the concentrations of ATP at which bronchoconstriction occurred and airway responsiveness to other agents (e.g. to methacholine) was not tested. It would be valuable to validate our findings in a larger study of healthy volunteers in whom airway hyper-responsiveness has been excluded and with spirometry testing after each ATP concentration to evaluate the safest range.

In conclusion, despite providing useful insights into cough mechanisms, these data suggest that inhaled ATP may be capable of inducing significant bronchoconstriction in some healthy volunteers. Individual perception of bronchoconstriction is variable and therefore pre- and post-challenge spirometry and pulse oximetry monitoring should be considered in studies utilising ATP inhalation.