Terbutaline doping control using enantioselective urine assays

Code: R16R03GJ 

Beta2-agonists are among the most commonly used drugs by athletes, which is related to the high prevalence of asthma and exercise induced bronchoconstriction (EIB) in this population.
This project will determine analytical urine thresholds for two beta2-agonist asthma drugs, salmeterol and terbutaline. 
Salmeterol is used to treat asthma and is permitted in sport when taken by inhalation in accordance with the manufacturers’ recommended regimen. There is currently no urine threshold for an adverse analytical finding (AAF) and athletes can freely administer supratherapeutic doses for doping purposes with impunity. The project will provide data on urine levels of salmeterol and other compounds related to its use found after inhaled dosing at both permitted (therapeutic) and prohibited (supratherapeutic) doses over a seven day treatment period.
Urinary thresholds and decision limits are a way to avoid excessive use of beta2-agonist asthma drugs by athletes and to lessen the burden associated with therapeutic use exemption (TUE) applications. One of the most common beta2-agonists, terbutaline, is widely used in Europe but is currently prohibited unless a TUE has been granted. Terbutaline is responsible for over three quarters of AAFs associated with beta2-agonists in doping control. The project will develop a urine threshold for terbutaline to discriminate between normal therapeutic use via inhalation, high dose (supratherapeutic) inhalation and oral ingestion of terbutaline.
Both salmeterol and terbutaline are chiral compounds administered as 50:50 mixtures of two enantiomers (stereoisomers) which are molecules with non-superimposable mirror images (analogous to right and left hands). Differences in the way the body excretes enantiomers of the same drug can be used to improve discriminatory capability of urine doping detection methods – this requires the use of advanced stereoselective UPLC-MS/MS assays to distinguish between enantiomers which will be used in this project.

Main Findings: 

Background. Terbuutaline, a short acting β2-agonist, is widely used in Europe and accounts for 76% of adverse analytical findings (AAFs) of β2-agonists in doping control. Terbutaline is a chiral drug consisting of a 1:1 ratio of two enantiomers ®-terbutaline and (S)-terbutaline, and given differences in metabolism between both enantiomers, the ratio of enantiomers may afford the ability to distinguish between oral and inhaled routes of administration.

Objective. To discriminate between therapeutic inhaled dosing and both supratherapeutic inhaled administration and oral dosing of terbutaline using a chiral assya for terbutaline applied to urine.

Methods. Part Ia: The study was crossover design, with 14 well-trained men and women undertaking three intervention arms; Inhalation regimen (low dose therapeutic) consisting of 1 mg twice daily for 7 days (2 mg/day); inhalation regimen (high dose supratherapeutic) consisting of 2 mg twice daily for 7 days (4 mg/day); prohibited oral regimen consisting of 10 mg daily for 7 days; prohibited oral regimen consisting of 20 mg daily for 3 days. Urine was collected 2 h post dose on each treatment day and (R)- and (S)-terbutaline determined using a chiral UPLC-MS/MS method. Part Ib: A further investigation of 25 mg/d oral terbutaline (15 mg morning + 10 mg afternoon) was undertaken in nine trained men for 4 weeks for comparison with urine collected 1-3 hours after administration on the first day, half-way through the intervention, and the final day. Part II: The study was an acute (24 h) PK study examining urine levels and R:S terbutaline ratio following dosing with 10 mg oral or 4 mg inhaled terbutaline and 1½ h exercise in highly-trained men with asthma.  

Results. Part Ia: Racemic terbutaline urine levels were highest in the supratherapeutic inhaled group. The sulfate metabolite was the main metabolite with some evidence of enantioselective metabolism. The urine log(R:S) terbutaline enantiomer ratio demonstrated excellent diagnostic performance at 2 h post dose with the ability to distinguish between oral and inhaled dosing, with sensitivity and specificity both greater than 98% based on an arbitrary cut-off value of 0.1.  Further discrimination between therapeutic inhaled (1 mg twice daily) and supratherapeutic inhaled (2 mg twice daily) dosing, was demonstrated using a urine threshold approach set at an arbitrary 1000 ng/ml with sufficient specificity (>98%), and while sensitivity was modest at 19%, adoption of this approach could potentially disincentivize supratherapeutic inhaled doping. Part Ib: resulted in similar findings to Part 1a with respect to log(R:S) ratio, noting that maximum levels were not achieved until week 4. Part II: In the acute PK study, urine levels were higher following inhaled administration than oral but the predictive value of R:S ratio was not repeated from Part Ia. 

Conclusion. The urine enantiomer ratio method shows some promise to discriminate between permitted inhalation and oral dosing of terbutaline in sport, and if combined with a rac-terbutaline threshold for supretherapeutic inhaled dosing, could pave the way for terbutaline in sport without the need for TUEs. Further work is required to investigate why this approach did not work in an acute single dosing scenario.