Enantioselective disposition of (R,R)‐formoterol, (S,S)‐formoterol and their respective glucuronides in urine following single inhaled dosing and application to doping control

Abstract

Formoterol is a long‐acting beta2‐adrenoceptor agonist (LABA) used for the treatment of asthma and exercise‐induced bronchoconstriction. Formoterol is usually administered as a racemic (rac‐) mixture of (R,R)‐ and (S,S)‐enantiomers. While formoterol is restricted by the World Anti‐Doping Agency (WADA), inhalation of formoterol is permitted to a predetermined dose (54 μg/24 hours) and a urine threshold of 40 ng/mL. However, chiral switch enantiopure (R,R)‐formoterol is available, effectively doubling the therapeutic advantage for the same threshold. The aim of this study was to investigate whether formoterol exhibits enantioselective urinary pharmacokinetics following inhalation. Six healthy volunteers were administered a 12 μg inhaled dose of rac‐formoterol. Urine was collected over 24 hours and analyzed by enantioselective ultra‐performance liquid chromatography−tandem mass spectrometry (UPLC−MS/MS) assay. Total (free drug plus conjugated metabolite) median (min‐max) rac‐formoterol urine levels following inhalation were 1.96 (1.05–13.4) ng/mL, 1.67 (0.16–9.67) ng/mL, 0.45 (0.16–1.51) ng/mL, 0.61 (0.33–0.78) ng/mL, and 0.17 (0.08–1.06) ng/mL at 2, 4, 8, 12, and 24 hours, respectively, well below the 2019 urine threshold. The proportion of conjugation differed between enantiomers with glucuronide conjugation much greater for (R,R)‐formoterol (around 30%–60% of total) compared to (S,S)‐formoterol (0%–30%). There was clear evidence of inter‐individual enantioselectivity observed in the ratios of (R,R):(S,S)‐formoterol, where (S,S)‐ was predominant in free formoterol, and (R,R)‐ predominant in the conjugated metabolite. In conclusion, rac‐formoterol delivered by inhalation exhibits enantioselective elimination in urine following single‐dose administration. Enantioselective assays should be employed in doping control to screen for banned beta2‐agonist chiral switch products such as (R,R)‐formoterol, and total hydrolyzed rac‐formoterol is warranted to account for inter‐individual differences in enantioselective glucuronidation.

Anabolic and lipolytic actions of beta2‐agonists in humans and antidoping challenges

“…To complicate matters, single enantiomers should be considered distinct compounds because these molecules possess different pharmacodynamics and pharmacokinetics when administered as the racemic mixture or separate enantiopure formulations . Our own work has shown distinct differences in enantiomer pharmacokinetics for beta 2 ‐agonists, including urine threshold applications . The pharmacological activity of beta 2 ‐agonists mostly resides with the (R)‐enantiomers (and (R,R)‐enantiomers), while the (S)‐enantiomers are generally considered to be pharmacological inert, such as for (S)‐salbutamol .…”

Annual banned‐substance review – Analytical approaches in human sports drug testing

“…Due to the fact that routine doping controls do not differentiate between the different enantiomers of β 2 ‐agonists, switching to enantiopure products (e.g. arformoterol) would effectively double the amount of active formoterol without being tangential to the established urinary threshold, and enantioselective analytical approaches were suggested for sports drug testing purposes . One option to test for formoterol enantiomers was presented, consisting of an enzymatic hydrolysis of urine followed by LLE for subsequent LC–MS/MS analysis.…”

Chirality of β2-agonists. An overview of pharmacological activity, stereoselective analysis, and synthesis

“…Metabolism of β 2 -agonists induces sulfation and glucuronidation, which differs from the β 2 -agonist type, e.g., albuterol and salbutamol are biotransformed by sulfation into an ineffective metabolite ( Figure 15), whereas fenoterol induces glucuronidation in addition to sulfation, and the case of formoterol, it is only glucuronidation. Glucuronidation of formoterol occurs at the phenolic position as well as the formation of a benzyl glucuronide ( Figure 16) [69][70][71].…”