(2S,3R) -Methyl-2 ,6 -dimethyltyrosine-L-tetrahydroisoquinoline-3- carboxylic acid [(2S,3R)TMT-L-Tic-OH] Is a Potent, Selective - Opioid Receptor Antagonist in Mouse Brain

The constrained opioid peptide (2S,3R) -methyl-2 ,6 -dimethyltyrosine-L-tetrahydroisoquinoline-3-carboxylic acid [(2S,3R)TMT-LTic-OH] exhibits high affinity and selectivity for the -opioid receptors (Liao et al., 1997). In the present study, we examined the pharmacological properties of (2S,3R)TMT-L-Tic-OH in mouse brain. A 5 -O-(3-[S]thiotriphosphate) ([S]GTP S) binding assay was used to determine the effect of (2S,3R)TMT-L-Tic-OH on G protein activity in vitro, in mouse brain membranes. (SNC80; ( )-4-[( R)-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3methoxy-benzyl]-N,N-diethyl-benzamide) or (DAMGO; [D-Ala, Me-Phe,Gly(ol)]enkephalin) selective opioid full agonists stimulated [S]GTP S binding in mouse brain membranes 150 4.5% and 152 5.7% over the basal level, respectively. (2S,3R)TMTL-Tic-OH did not influence basal [S]GTP S binding in mouse brain membranes but dose dependently shifted the doseresponse curve of SNC80 to the right, with a Ke value of 3.6 0.7 nM. In contrast, (2S,3R)TMT-L-Tic-OH had no effect on the doseresponse curve of the -selective opioid agonist, DAMGO. Warm water (55°C) tail-flick and radiant heat paw-withdrawal tests were used to determine the in vivo nociceptive properties of (2S,3R)TMT-L-Tic-OH in the mouse. Intracerebroventricular injection of (2S,3R)TMT-L-Tic-OH had no significant effect on withdrawal latencies in either nociceptive tests. (2S,3R)TMT-L-Tic-OH (30 nmol/mouse) attenuated deltorphin IIbut not DAMGOmediated antinociception (40 13 and 100% of maximal possible effect, respectively) when administered intracerebroventricularly 10 min before the agonist. Taken together these results suggest that (2S,3R)TMT-L-Tic-OH is a potent highly selective neutral -opioid antagonist in mouse brain. Three major opioid receptor types, -, -, and -, have been identified by pharmacological assays and by molecular cloning (Knapp et al., 1995). Each of the three opioid receptor types mediates analgesia (Quock et al., 2001). The unique physiological role of the individual opioid receptor types, however, is not fully recognized, mainly due to the paucity of highly selective antagonists. Selective -opioid receptor antagonists could be important as pharmacological tools to identify the physiological role of the -opioid receptors, but may also serve as therapeutic agents to regulate -opioid receptor function in various clinical disorders (Cowell et al., 2002). We have previously synthesized a sterically constrained pseudopeptide opioid ligand, (2S,3R)TMT-L-Tic (Liao et al., 1997), and investigated its pharmacological properties in a recombinant Chinese hamster ovary cell line, stably expressing the human -opioid receptor (hDOR/CHO) (Malatynska et al., 1995). We found that (2S,3R)TMT-L-Tic behaved as a potent inverse agonist in the recombinant hDOR/CHO cells (Hosohata et al., 1999). To identify the physiologically relevant mechanism of (2S,3R)TMT-L-Tic-OH action however, it is important to determine its pharmacological properties in tissues expressing physiological receptor concentrations, particularly in the central nervous system. In the present study, we examined the effect of (2S,3R)TMT-L-Tic-OH on [S]GTP S binding in mouse brain membrane preparations. We also determined This work was supported in part by grants from the U.S. Public Health Service and from the National Institute of Drug Abuse (DA06284 and DA13449). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. DOI: 10.1124/jpet.102.042929. ABBREVIATIONS: (2S,3R)TMT-L-Tic-OH, (2S,3R) -methyl-2 ,6 -dimethyltyrosine-L-tetrahydroisoquinoline-3-carboxylic acid; hDOR, human -opioid receptor; CHO, Chinese hamster ovary; [S]GTP S, guanosine-5 -O-(3-[-[S]thio)triphosphate; SNC80, ( )-4-[( R)-((2S,5R)-4-allyl2,5-dimethyl-1-piperazinyl)-3-methoxy-benzyl]-N,N-diethylbenzamide; DAMGO, [D-Ala,Me-Phe,Gly(ol)]enkephalin; Ke, dissociation constant from Schild analysis; MPE, maximal possible effect; DPDPE, [D-Pen,D-Pen]-enkephalin; ICI 174,864, N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH; SR 141,716, N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride. 0022-3565/03/3042-683–688$7.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 304, No. 2 Copyright © 2003 by The American Society for Pharmacology and Experimental Therapeutics 42929/1034553 JPET 304:683–688, 2003 Printed in U.S.A. 683 at A PE T Jornals on M ay 7, 2017 jpet.asjournals.org D ow nladed from the effect of (2S,3R)TMT-L-Tic-OH on the potencies and intrinsic activities of and -selective opioid receptor agonists (SNC80 and DAMGO, respectively) in the [S]GTP S binding assay in mouse brain membranes. A warm water (55°C) tail-withdrawal (tail-flick) test was used to investigate the in vivo effect of (2S,3R)TMT-L-Tic-OH on nociception in mice. In addition, the more sensitive radiant heat paw-withdrawal test was also employed to test the hyperalgesic properties of the compound. Finally, the ability of (2S,3R)TMT-L-Tic-OH to antagonize the antinociception mediated by (DAMGO) and -selective (deltorphin II) opioid receptor agonists was also tested. Materials and Methods Chemicals. (2S,3R)TMT-L-Tic-OH was synthesized at the University of Arizona, in the laboratory of Dr. V. J. Hruby (Liao et al., 1997). SNC80 and deltorphin II were obtained from Tocris Pharmaceuticals (Baldwin, MO); DAMGO was purchased from Sigma/RBI (Natick, MA). SNC80 was dissolved in 40% ethanol to obtain a 1 mM stock solution. Deltorphin II was initially prepared as 4 mM stock solution in 20% dimethyl sulfoxide. All other compounds were dissolved in distilled water (tail-flick test) or assay buffer ([S]GTP S binding assay). Further dilutions were made by distilled water (tailflick test) or assay buffer ([S]GTP S binding assay). [S]GTP S (1250 Ci/mmol) was purchased from PerkinElmer Life Sciences (Bos-