| Summary: | Carvedilol, 1-(9H-carbazol-4-yloxy)-3-[[2-(2-methoxyphenoxy)ethyl]amino]-2-
propanol, is a new antihypertensive drug which has recently been introduced on the
market in Canada under the trade name, COREG. It contains a chiral centre in its
structure and therefore exists as two enantiomers. The drug is marketed as the
racemate, however, the two enantiomers possess different pharmacological actions.
(-)-(S)-Carvedilol is a much more potent Pi-blocker than (+)-(R)-carvedilol, whereas
both enantiomers exhibit the same oci-adrenergic antagonism. This study consisted of
the development of sensitive stereoselective assays for carvedilol using high-performance
liquid chromatography (HPLC) and capillary electrophoresis (CE) and the
determination of the stereoselective protein binding of carvedilol.
An attempt to derivatize carvedilol enantiomers with fluorogenic reagents was
undertaken. The reaction with 2-naphthoyl chloride, 2-anthroyl chloride, and
(+)-(S)-naproxen chloride resulted in incomplete derivatization. Mass spectrometric
analysis of the reaction products revealed the formation of the mono-derivative of
carvedilol with 2-naphthoyl chloride at the secondary amine group on the side chain
and also the di-derivative at both the amine group and the hydroxyl group of carvedilol.
The reaction with (+)-(S)-1-(1-naphthyl)ethyl isocyanate was also examined and was
incomplete and produced multiple derivatives.
A direct chiral HPLC method was therefore developed, without the need for
derivatization, using (S)-indoline-2-carboxylic acid and (R)-1-(a-naphthyl)ethylamine as
the stationary phase. The assay was validated for carvedilol enantiomers in serum. A
limit of quantitation (LOQ) of 1 ng/ml for both enantiomers was obtained.
A new stereoselective CE method was also developed for the analysis of the
enantiomers of carvedilol in serum. Several types and concentrations of cyclodextrins
were tested. Near baseline resolution was obtained using 10 mM hydroxypropyl-pcyclodextrin
as the chiral selector. The electrophoretic conditions were optimized. The
chiral CE method for carvedilol was validated for the drug enantiomers in serum. An
LOQ of 50 ng/ml per enantiomer was obtained.
The HPLC and the CE assays were compared by analyzing a series of serum
samples containing racemic carvedilol in different concentrations using the two
methods. The concentrations obtained by the two assays were not found to be
significantly different.
The stereoselective binding of carvedilol enantiomers to serum proteins was
investigated by equilibrium dialysis. Carvedilol is highly bound to serum proteins
(> 98%). The free fractions obtained after dialysis of serum containing carvedilol were
0.6% for (+)-(R)-carvedilol and 0.9% for (-)-(S)-carvedilol with an R/S ratio of 0.67. The
binding of the two enantiomers was found to be significantly different, with
(-)-(S)-carvedilol being less bound. Binding to isolated serum proteins was also
determined. Using 4% human serum albumin (HSA) in isotonic phosphate buffer, the
unbound fractions obtained were 2.4% and 2.6% for (+)-(R)-and (-)-(S)-carvedilol,
respectively, with an R/S ratio of 0.92. The stereoselective binding to HSA was not
found to be significantly different. On the other hand, the binding of carvedilol to
100 mg% cti-acid glycoprotein (AAG) in isotonic phosphate buffer was found to be
highly stereoselective. The unbound fractions obtained were 1.5% for
(+)-(R)-carvedilol and 2.5% for (-)-(S)-carvedilol, with an R/S ratio of 0.60. The results
suggest that AAG is the major protein responsible for the stereoselective binding of
carvedilol enantiomers in serum.
Carvedilol enantiomers were not found to exhibit concentration-dependent
binding in human serum over the concentration range of 0.5-4 ug/ml per enantiomer.
However, binding to 4% HSA was found to be significantly different above 2 ug/ml for
each enantiomer. The free fractions increased substantially above concentrations of
3 ug/ml of each enantiomer when the binding to 100 mg% AAG was tested suggesting
that the AAG binding sites are saturable.
In order to simulate an increase in the levels of AAG as would occur in
myocardial infarction or surgery, the binding of carvedilol enantiomers to 4% HSA
combined with 100 mg% AAG or 400 mg% AAG was compared. A decrease in the free
drug concentration was observed for both enantiomers when the levels of AAG
increased from 100 to 400 mg% and the R/S ratio changed from 0.82 to 0.67.
In summary, comparative HPLC and CE assay methods were developed for the
stereoselective analysis of carvedilol enantiomers. While the fluorescent detection
used for the HPLC method allowed for lower detection limits required for determination
of free (unbound) enantiomer concentrations in serum, the CE method, with a higher
limit of quantitation, was used for the determination of total enantiomer concentrations
(free and bound). However, the actual sensitivity of the CE method, considering the
amount of sample injected, was greater by approximately 10 fold than the HPLC
method. Using a combination of both methods, the stereoselective protein binding
characteristics were established for the enantiomers of carvedilol using human serum
and purified protein fractions of human serum.
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