| pA2 online © Copyright 2004 The British Pharmacological Society |
091P
University of Newcastle Winter Meeting December 2004 |
The relative affinities of (R)- and (S)-enantiomers of RX821002 the selective R.J. Tyacke, D.J. Nutt and A.L. Hudson. Psychopharmacology Unit, University of Bristol, Dorothy Hodgkin Building, BS1 3NY. |
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2-adrenoceptor antagonist in rat whole brain 
Print abstract RX821002 or 2-methoxy-idazoxan is a high affinity and efficacy antagonist for the 
2-adrenoceptor
(2-AR). It has also been shown to be highly selective for these receptors but does not demonstrate any subtype selectivity (Hudson et al., 1992; Robinson et al., 2004) and is widely used in vitro and in vivo. RX821002 is a derivative of idazoxan but unlike idazoxan, it does not displays any affinity for the imidazoline(2) binding sites (Robinson et al., 2004) . RX821002 contains a chiral carbon, however, it is usually used in its racemic preparation. Little is known about the stereospecificity of the two enantiomers or their relative affinity at the a 2-AR, though there is some evidence in the literature that (+)- or (S)-enantiomer is the more selective (Pauwels et al., 2000) . For this reason presented here are data for the binding affinities of (R)- and (S)-RX821002 at the 2-AR in rat whole brain membranes.
Rat (male, Wistar, 230-250g) brains were removed and P2 membranes prepared as previously described (Robinson et al., 2004) . Membrane aliquots (300µg protein) were incubated (45min, 22 ° C) with racemic [3H]RX821002 (1nM) or the 2-AR agonist [3H]clonidine (3nM). Non-specific binding was determined using rauwolscine (10µM). (R)- and (S)-RX821002 were examined for their ability to compete with the radiolabelled ligands over the range of 0.01nM – 10µM. Bound ligands were separated by filtration and determined by liquid scintillation counting. Data were analysed using the nonlinear regression analysis supplied with GraphPad Prism version 3.03 for Windows (San Diego California USA).
These data were best fit to single site model of inhibition of binding. Table 1 shows the calculated equilibrium dissociation constants at the 2-ARs defined using racemic [3H]RX821002 and
[3H]clonidine. The (S)-RX821002 showed the highest affinity for the 2-adrenoceptor compared with the (R)-enantiomer.
Table 1. Relative affinities of (R)- and (S)-RX821002 at the 2-AR.
Ki(nM) ± S.D. (n) |
|||
( r)-RX821002 |
( s)-RX821002 |
Selectivity |
|
(R,S)-[3H]RX821002 |
139.13 ± 40.71 (4) |
0.54 ± 0.10 (4) |
258 |
[3H]clonidine |
394.58 ± 150.77 (4) |
1.55 ± 0.67 (4) |
255 |
K D’s for (R,S)-[ 3H]RX821002 and [3H]clonidine taken from Hudson et al. (1992) and U'Prichard et al. (1977) respectively.
These data clearly prove that RX821002 shows high stereoselectivity for binding to 2-ARs in vitro, with the (S)-enantiomer being the most selective. The selectivity for the (S)-enantiomer over the (R-enantiomer approximately 250 fold. In addition, this agrees with the findings of Pauwels et al., (2000) . This stereoselectivity has potential implications for the future use of RX821002 and presents a ‘new’ very high affinity ligand for the study of 2-ARs.
Hudson , A.L., et al.. (1992). Mol. Neuropharmacol., 1, 219-229.
P auwels , P.J., et al . (2000). J Pharmacol Exp Ther, 292, 654-63.
Robinson , E.S., et al. (2004). Neuropharmacology, 46, 847-55.
U'Prichard , D.C., et al . (1977). Mol Pharmacol, 13, 454-73.