Enantioselective separation and online affinity chromatographic characterization of R,R- and S,S-fenoterol.
BACKGROUND
rac-Fenoterol is a beta2-adrenoceptor agonist (beta2-AR) used in the treatment of asthma. It has two chiral centers and is marketed as a racemic mixture of R,R'- and S,S'-fenoterol (R-F and S-F). Here we report the separation of the R-F and S-F enantiomers and the evaluation of their binding to and activation of the beta2-AR.


METHODS
R-F and S-F were separated from the enantiomeric mixture by chiral chromatography and absolute configuration determined by circular dichroism. Beta2-AR binding was evaluated using frontal affinity chromatography with a stationary phase containing immobilized membranes from HEK-293 cells that express human beta2-AR and standard membrane binding studies using the same membranes. The effect of R-F and S-F on cardiomyocyte contractility was also investigated using freshly isolated adult rat cardiomyocytes.


RESULTS
Chiral chromatography of rac-fenoterol yielded separated peaks with an enantioselectivity factor of 1.21. The less retained peak was assigned the absolute configuration of S-F and the more retained peak R-F. Frontal chromatography using membrane-bound beta2-AR as the stationary phase and rac-3H-fenoterol as a marker ligand showed that addition of increasing concentrations of R-F to the mobile phase produced concentration-dependent decreases in rac-3H-fenoterol retention, while similar addition of S-F produced no change in rac-3H-fenoterol retention. The calculated dissociation constant of R-F was 472 nM and the number of available binding sites 176 pmol/column, which was consistent with the results from the membrane binding study 460 +/- 55 nM (R-F) and 109,000 +/- 10,400 nM (S-F). In the cardiomyocytes, R-F increased maximum contractile response from (265 +/- 11.6)% to (306 +/- 11.8)% of resting cell length (P < 0.05) and reduced EC50 from -7.0 +/- 0.270 to -7.1 +/- 0.2 log[M] (P < 0.05), while S-F had no significant effect.


CONCLUSIONS
Previous studies have shown that rac-fenoterol acts as an apparent beta2-AR/G(s) selective agonist and fully restores diminished beta2-AR contractile response in cardiomyocytes from failing hearts of spontaneously hypertensive rats (SHR). Here we report the separation of the enantiomers of rac-fenoterol and that R-F is the active component of rac-fenoterol. Further evaluation of R-F will determine if it has enhanced selectivity and specificity for beta2-AR/G(s) activation and if it can be used in the treatment of congestive heart failure.
Beigi
Farideh
F
Laboratory of Clinical Investigation, National Institute on Aging Intramural Research Program, Baltimore, Maryland, USA.
Bertucci
Carlo
C
Zhu
Weizhong
W
Chakir
Khalid
K
Wainer
Irving W
IW
Xiao
Rui-Ping
RP
Abernethy
Darrell R
DR
D000818:Animals; D002460:Cell Line; D002846:Chromatography, Affinity; D005280:Fenoterol / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D015394:Molecular Structure; D032383:Myocytes, Cardiac / Q000187:drug effects; D009862:Online Systems; D051381:Rats; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism; D013057:Spectrum Analysis; D013237:Stereoisomerism
D018343:Receptors, Adrenergic, beta-2; D005280:Fenoterol