Thomas H Burkey

6-isopropoxy-9-oxoxanthene-2-carboxylic acid (AH 6809), a human EP2 receptor antagonist

On studying the interaction of various ligands with the pharmacologically defined, recombinant human EP2 receptor (Regan et al., Mol Pharmacol 46: 213-220, 1994), we discovered that the putative EP1 receptor antagonist 6-isopropoxy-9-oxoxanthene-2-carboxylic acid (AH 6809) also has affinity for the human EP2 receptor. Moreover, AH 6809 behaved as an EP2 receptor antagonist and inhibited prostaglandin E2 (PGE2)-stimulated increases in cyclic AMP. These findings have significant implications for studies that employ AH 6809 to determine the pharmacological basis of PGE2-induced responses in human cells and tissues.

Relative efficacies of cannabinoid CB1 receptor agonists in the mouse brain

We measured (-)-5-(1,1-dimethylheptyl)-2-[5-hydroxy-2-(3-hydroxypropyl)cyclohe xyl]-phenol (CP 55,940)-, (-)11-OH-delta8-tetrahydrocannabinol-dimethylheptyl (HU-210)-, anandamide- and delta9-tetrahydrocannabinol-stimulated G protein activation in mouse brain using the [35S]GTPgammaS functional assay. The Ki values for these drugs were determined by agonist competition binding with the cannabinoid CB1 receptor antagonist [3H]N-(piperidin-1-yl-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4- methyl-1H-pyrazole-3-carboxamidehydrochloride ([3H]SR141716A). This information was used to calculate the efficacy for drug stimulation of G protein activity. The rank order of efficacy was CP 55,940 > HU-210 > anandamide > delta9-tetrahydrocannabinol with the latter two drugs being partial agonists. Since efficacy values relate receptor occupancy to functional responses, we believe efficacy values are a better measure of drug-mediated functional responses compared with measurements of drug potency.

δ-Opioid receptor agonists produce antinociception and [35S]GTPγS binding in μ receptor knockout mice

Abstract We examined the effects of [ d -Pen 2 , d -Pen 5 ]enkephalin (DPDPE), [ d -Ala 2 ,Glu 4 ]deltorphin (DELT), and (+)-4-[(α R )-α((2 S ,5 R )-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]- N , N -diethylbenzamide (SNC80) on [ 35 S]GTPγS binding in brain membranes prepared from μ-opioid receptor knockout (−/−) mice. The potency and maximal response ( E max ) of these agonists were unchanged compared to control mice. In contrast, while the potency of [ d -Pen 2 ,pCl-Phe 4 , d -Pen 5 ]enkephalin (pCl-DPDPE) was not significantly different, the E max was reduced as compared to controls. In the tail-flick test, intracerebroventricular (i.c.v.) or intrathecal (i.th.) DELT produced antinociceptive effects in −/− mice with potency that did not differ significantly from controls. In contrast, the antinociceptive potency of i.c.v. and i.th. DPDPE was displaced to the right by 4- and 9-fold in −/− compared to control mice, respectively. Reduced DPDPE antinociceptive potency in −/− mice, taken together with reduced DPDPE- and pCl-DPDPE- stimulated G protein activity in membranes prepared from −/− mice, demonstrate that these agonists require μ-opioid receptors for full activity. However, because DELT mediated G protein activation and antinociception were both comparable between −/− and wild type mice, we conclude that the μ-opioid receptor is not a critical component of δ-opioid receptor function.

Endomorphin-1 and endomorphin-2 are partial agonists at the human μ-opioid receptor

Recently two tetrapeptide ligands that bind preferentially to the mu-opioid receptor were identified and named endomorphin-1 and endomorphin-2. We examined the ability of these peptides to stimulate G protein activation in human mu-opioid receptor transfected B82 fibroblasts as measured by [35S]GTPgammaS binding to cell membranes. Both endomorphin-1 and -2 act as partial agonists in this assay system compared with the mu-selective agonist [D-Ala2,N-Me-Phe4, Gly-ol5]enkephalin (DAMGO). In addition, endomorphins demonstrate efficacy similar to morphine. These findings demonstrate that endomorphin peptides have similar activity at the mu-opioid receptor as morphine and suggest that these peptides have the potential to modulate neuronal activity in vivo.

AM630 IS A COMPETITIVE CANNABINOID RECEPTOR ANTAGONIST IN THE GUINEA PIG BRAIN

AM630 has been demonstrated to be a cannabinoid receptor antagonist in the mouse brain and vas deferens. Conversely, it was recently reported that AM630 acts as a cannabinoid agonist in the guinea pig ileum. This research was designed to determine whether the difference in the action of AM630 is species specific. Studies conducted in guinea pig brain reveal that AM630 antagonizes the stimulatory effect of the cannabinoid agonist WIN 55,212-2 on [35S]GTPgammaS binding suggesting that difference in AM630 activity in different tissues is not due to species variation.

Relative efficacies of δ-opioid receptor agonists at the cloned human δ-opioid receptor

The present study was conducted to determine the relative efficacies of the selective δ-opioid receptor agonists SNC80 ((+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide), pCl-DPDPE (cyclic[d-Pen2,4′-ClPhe4,d-Pen5]enkephalin) and (−)-TAN67 ((−)-2-methyl-4aα-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12aα-octahydro-quinolino-[2,3,3-g]isoquinoline). Experiments compared the abilities of the three drugs to competitively inhibit [3H]naltrindole binding and also stimulate [35S]GTPγS binding in membranes prepared from stably transfected Chinese hamster ovary (CHO) cells that express the cloned human δ-opioid receptor. Efficacy was determined according to the formula: efficacy=(Emax-A/Emax)(A′/A+1)×0.5. Results show that SNC80 and pCl-DPDPE had efficacy values that were about 6–7 times greater than that of (−)-TAN67.

Differential down-regulation of the human δ-opioid receptor by SNC80 and [d-Pen2,d-Pen5]enkephalin

Abstract We examined the contribution of the human δ-opioid receptor carboxyl terminal tail to (+)-4-[(α R )-α-((2 S ,5 R )-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]- N , N -diethylbenzamide (SNC80)- and cyclic[ d -Pen 2 , d -Pen 5 ]enkephalin (DPDPE)-mediated receptor down-regulation. Both SNC80 and DPDPE mediated down-regulation of an epitope tagged human δ-opioid receptor. Truncation of the human δ-opioid receptor after Gly 338 blocked DPDPE-mediated down-regulation. However, SNC80 mediated significant down-regulation of the truncated receptor. These findings suggest that SNC80-mediated down-regulation involves receptor domains in addition to the carboxyl terminal tail.

The role of the G protein γ2 subunit in opioid antinociception in mice

Abstract We examined the role of the γ2 subunit of G proteins (Gγ2) in the antinociception produced by c[D-Pen2,D-Pen5]enkephalin (DPDPE) in mice. DPDPE produced 84.0±9.0% antinociception in vehicle-treated mice. After intracerebroventricular (i.c.v.) treatment with an antisense phosphorothioate oligodeoxynucleotide to the Gγ2 subunit, DPDPE-mediated antinociception decreased to 24.4±7.4%. The mismatch phosphorothioate oligodeoxynucleotide-treated mice showed 65.1±10.3% antinociception, while the missense phosphorothioate oligodeoxynucleotide-treated mice showed 76.4±23.6% antinociception by DPDPE. The reduction of analgesia in antisense phosphorothioate oligodeoxynucleotide-treated mice was significant in comparison with vehicle-treated (P

(2S,3R)TMT-l-Tic-OH is a potent inverse agonist at the human δ-opioid receptor

Abstract We examined the pharmacologic effect of β-methyl-2′,6′-dimethyltyrosine- l -tetrahydroisoquinoline-3-carboxylic acid ((2S,3R)TMT- l -Tic-OH) on G protein activation in membranes prepared from Chinese Hamster Ovary cells transfected with cDNA of the human δ-opioid receptor. (2S,3R)TMT- l -Tic-OH inhibited G protein activation to 58% of basal with an EC50 of 0.72 nM as determined by [ 35 S ]GTPγS binding. These findings suggest that (2S,3R)TMT- l -Tic-OH is a highly potent inverse agonist at the human δ-opioid receptor.

The efficacy of δ-opioid receptor-selective drugs

Delta-opioid receptor-selective drugs may provide an alternative to mu-opioid-selective drugs currently used for the relief of pain. To develop improved delta-opioid receptor-selective drugs, better measures of drug activity are necessary. In this review we suggest that efficacy calculations provide a superior measure of drug activity as compared to dissociation constants and drug potencies in functional assays. Efficacy, as discussed in this review, is defined as a quantitative measurement of the ability of a drug to stimulate second messenger systems or measurable functional responses in cells or tissues under standard conditions. Efficacy values will allow medicinal chemists to understand the contributions of both the coupling efficiency and dissociation constant to drug potencies in the development of new delta-opioid receptor-selective drugs.