Comparison of Pharmacological Activities of Three Distinct Ligands (Salvinorin A, TRK-820 and 3FLB) on Opioid Receptors in Vitro and Their Antipruritic and Antinociceptive Activities in Vivo

Salvinorin A, TRK-820 (17-cyclopropylmethyl-3,14 -dihydroxy-4,5 epoxy-6 -[N-methyl-trans-3-(3-furyl) acrylamido]morphinan hydrochloride), and 3FLB (diethyl 2,4-di-[3-fluorophenyl]-3,7-dimethyl-3,7diazabicyclo[3.3.1]nonane-9-one-1,5-dicarboxylate) are structurally distinctly different from U50,488H [(trans)-3,4-dichloro-N-methyl-N[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide methanesulfonate], the prototypic selective agonist. Here, we investigated their in vitro pharmacological activities on receptors expressed in Chinese hamster ovary cells and in vivo antiscratch and antinociceptive activities in mice. All three compounds showed high selectivity for the opioid receptor (KOR) over the opioid receptor (MOR) and opioid receptor (DOR) and nociceptin or orphanin FQ receptors. In the guanosine 5 -O-(3-[S]thio)triphosphate ([S]GTP S) binding assay, all three were full agonists on the KOR. The rank order of affinity and potency for the KOR was TRK-820 U50,488H salvinorin A 3FLB. TRK-820 acted as a partial agonist on MOR and DOR, whereas salvinorin A and 3FLB showed no activities on these receptors. Salvinorin A, TRK-820, and 3FLB caused internalization of the human KOR in a dosedependent manner. Interestingly, although salvinorin A and U50,488H had similar potencies in stimulating [S]GTP S binding, salvinorin A was about 40-fold less potent than U50,488H in promoting internalization. Following 4-h incubation, all three compounds induced down-regulation of the human KOR, with salvinorin A causing a lower extent of down-regulation. Although TRK820 was potent and efficacious against compound 48/80-induced scratching, salvinorin A showed low and inconsistent effects, and 3FLB was inactive. In addition, salvinorin A and 3FLB were not active in the acetic acid abdominal constriction test. The discrepancy between in vitro and in vivo results may be due to in vivo metabolism of salvinorin A and 3FLB and possibly to their effects on other pharmacological targets. At least three types of opioid receptors, , , and , mediate pharmacological effects of opioid drugs and physiological actions of endogenous peptides (for review, see Chang, 1984; Mansour et al., 1988). Opioid receptors are coupled to Gi/Go proteins to affect several different effectors, including inhibition of adenylyl cyclase, enhancement of K conductance, decrease in Ca conductance, and activation of p42/p44 mitogen-activated protein kinases (for review, see Law et al., 2000). In addition, opioid receptors are shown to act through Gz to inhibit adenylyl cyclase and G16 to activate phospholipase C (Lai et al., 1995; Lee et al., 1998), and opioid receptors stimulate Na , H exchanger 3 activity via Na , H -exchanger regulatory factor-1/ Ezrin-radixin-moesin-binding phosphoprotein-50 independent of pertussis toxin-sensitive G proteins (Huang et al., 2004). , , and opioid receptors of several species have been cloned (for review, see Kieffer, 1995; Knapp et al., 1995). In addition, a receptor with high sequence similarity to the opioid receptors, termed the ORL1 receptor, was cloned and found to be coupled to Gi/Go proteins (for review, see Kieffer, 1995; Knapp et al., 1995). Subsequently, the endogenous ligand for the ORL1 receptor was identified and named noThis work was supported by National Institutes of Health Grants DA04745, DA11263, DA17302, P30 DA13429, and T32DA07237. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.104.073668. ABBREVIATIONS: N/OFQ, nociceptin/orphanin FQ; ( )U50,488H, (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide methanesulfonate; U69,593, ( )-(5 ,7 ,8 )-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]benzeneacetamide; TRK-820, 17-cyclopropylmethyl-3,14 dihydroxy-4,5 -epoxy-6 -[N-methyl-trans-3-(3-furyl) acrylamido]morphinan hydrochloride; HZ2, 2,4-di-2-pyridyl-3,7-dimethyl-3,7-diazabicyclo[3.3.1]nonan-9one 1,5-diester; 3FLB, diethyl 2,4-di-[3-fluorophenyl]-3,7-dimethyl-3,7-diazabicyclo[3.3.1]nonane-9-one-1,5-dicarboxylate; [S]GTP S, guanosine 5 -O-(3[S]thio)triphosphate; CHO, Chinese hamster ovary; DAMGO, Try-D-Ala-Gly-(Me)Phe-Gly-ol; DPDPE, Tyr-D-Pen-Gly-Phe-D-Pen-OH (disulfide bridge between D-Pen and D-Pen); hKOR, human opioid receptor; TBS, Tris-buffered saline; PAGE, polyacrylamide gel electrophoresis; ICI 204,448, (R,S)-N-[2-(N-methyl3,4-dichlorophenylacetamido)-2-(3-carboxyphenyl)ethyl]pyrrolidine. 0022-3565/05/3121-220–230$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 312, No. 1 Copyright © 2005 by The American Society for Pharmacology and Experimental Therapeutics 73668/1186283 JPET 312:220–230, 2005 Printed in U.S.A.