2-Methoxymethyl-Salvinorin B Is a Potent Opioid Receptor Agonist with Longer Lasting Action in Vivo Than Salvinorin A

Salvinorin (Sal) A is a naturally occurring, selective opioid receptor (KOPR) agonist with a short duration of action in vivo. Pharmacological properties of a C(2) derivative, 2-methoxymethyl (MOM)-Sal B, were characterized. MOM-Sal B bound to KOPR with high selectivity and displayed 3-fold higher affinity than U50,488H [(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide methanesulfonate] and Sal A. It acted as a full agonist at KOPR in guanosine 5 -O-(3[S]thio)triphosphate binding and was 5and 7-fold more potent than U50,488H and Sal A, respectively. In Chinese hamster ovary cells stably expressing KOPR, all three agonists internalized or down-regulated KOPR to similar extents, with MOM-Sal B being the most potent. In mice, MOM-Sal B (0.05–1 mg/kg s.c.) caused immediate and dose-dependent immobility lasting 3 h, which was blocked by norbinaltorphimine. In contrast, ambulation in a Y-maze was increased when rats received MOM-Sal B (1–5 mg/kg s.c.). In addition, MOM-Sal B (0.5–5 mg/kg i.p.) produced antinociception (hot-plate test) and hypothermia in a dose-dependent manner in rats. MOM-Sal B was more potent than U50,488H in both tests and more efficacious than U50,488H in the hot-plate test. These latter two in vivo effects were blocked by norbinaltorphimine, indicating KOPR-mediated actions. Sal A at 10 mg/kg elicited neither antinociception nor hypothermia 30 min after administration to rats. In summary, MOM-Sal B is a potent and efficacious KOPR agonist with longer lasting in vivo effects than Sal A. Activation of the opioid receptor (KOPR), one of three major types of opioid receptors, produces many effects including analgesia, dysphoria, antipruritis, water diuresis, and hypothermia (Liu-Chen, 2004). Many selective nonpeptide KOPR agonists have been synthesized; most are arylacetamide compounds, including U50,488H, U69,593, ICI 204,448, and asimadoline. Nalfurafine is an exception, being an epoxymorphinan. Chewing or smoking the leaves of Salvia divinorum or drinking juice of crushed leaves causes hallucinations in humans (Siebert, 1994). Salvinorin (Sal) A (Fig. 1), a neoclerodane diterpene, was isolated and identified as the main active ingredient (Valdés, 1994). In a large scale screening of G protein-coupled receptors, transporters, and ion channels by radioligand binding assays, Roth et al. (2002) found that Sal A was a highly potent and selective agonist for the KOPR. Since then, several pharmacological studies on Sal A have been performed. Sal A binds to KOPR with a high affinity similar to U50,488H, a prototypic selective KOPR agonist. It does not show significant affinities to and opioid receptors or the nociceptin/orphanin FQ receptor (Wang et al., 2005). Sal A is reported to be more selective for KOPR than U50,488H or U69,593 (Beguin et al., 2008). Sal A acts as a very potent full agonist at KOPR in [S]GTP S binding, intracellular calcium mobilization, and potassium conductance assays (Chavkin et al., 2004; Wang et al., 2005). Sal A This study was supported by National Institutes of Health Grants R01DA019688 and R01-DA019688 (to D.Y.W.L.), R01-DA022694 (to S.M.R.), and R01-DA17302 to (L.-Y.L.-C.). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.107.132142. ABBREVIATIONS: KOPR, opioid receptor; ( )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; ICI 204,448, (RS)-[3[1-[[(3,4-dichlorophenyl) acetyl]methylamino]-2-(1-pyrrolidinyl)ethyl]phenoxy]acetic acid; asimadoline, N-[(1S)-2-[(3S)-3-hydroxypyrrolidin-1-yl]1-phenylethyl]-N-methyl-2,2-di(phenyl)acetamide; nalfurafine, 17-cyclopropylmethyl-3,14 -dihydroxy-4,5 -epoxy-6 -[N-methyl-trans-3-(3-furyl) acrylamido]morphinan hydrochloride; Sal, salvinorin; norBNI, norbinaltorphimine; MOM, 2-methoxymethyl; [S]GTP S, guanosine 5 -O-(3[S]thio)triphosphate; CHO, Chinese hamster ovary; CHO-FLAG-hKOPR, CHO cells with stable expression of FLAG-tagged human opioid receptor; ANOVA, analysis of variance; %MPA, percentage of maximum possible analgesia; enadoline, 2-(1-benzofuran-4-yl)-N-methyl-N[(5R,7S,8S)-7-pyrrolidin-1-yl-1-oxaspiro[4.5]decan-8-yl]acetamide; Tb, change in body temperature. 0022-3565/08/3243-1073–1083$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 324, No. 3 Copyright © 2008 by The American Society for Pharmacology and Experimental Therapeutics 132142/3311241 JPET 324:1073–1083, 2008 Printed in U.S.A. 1073 at U C L A B om eical ib S eials on July 9, 2008 jpet.asjournals.org D ow nladed fom also promotes internalization and down-regulation of the human KOPR in cultured cells (Wang et al., 2005). Sal A produces many in vivo effects characteristically mediated by KOPR, such as sedation (Fantegrossi et al., 2005), antinociception (Ansonoff et al., 2006; John et al., 2006; McCurdy et al., 2006), hypothermia (Ansonoff et al., 2006), and depression-like effects (Carlezon et al., 2006). It decreases dopamine levels in caudate putamen that can be blocked by norbinaltorphimine (norBNI), a selective KOPR antagonist (Zhang et al., 2005). In mice, Sal A causes conditioned place aversion (Zhang et al., 2005), decreased locomotor activity (Zhang et al., 2005), and impaired climbing behavior on an inverted screen task (Fantegrossi et al., 2005). It is noteworthy that the duration of in vivo effects of Sal A is short regardless of species, behavioral endpoint, or administration route. The hallucinogenic effect of Sal A in humans is brief when inhaled, reaching full effect in 30 s, lasting 5 to 10 min, and subsiding in 20 to 30 min (Siebert, 1994). In mice, the antinociceptive effect of Sal A diminished within 20 min after i.p. injection in the acetic acid abdominal constriction test (Wang et al., 2005; McCurdy et al., 2006). However, in the mouse tail-flick test, its antinociceptive effect was undetectable 20 min after i.t. injection or 30 min after i.c.v. injection (John et al., 2006). Intravenous administration of Sal A increased prolactin levels in rhesus monkeys, but the duration was only 30 min, shorter than that of U69,593 (90 min) at the same dose (Butelman et al., 2007). A pharmacokinetic study in nonhuman primates showed that the elimination t1/2 of Sal A in the serum was less than 1 h (Schmidt et al., 2005a). The chemical structure of Sal A suggests that it may be hydrolyzed at the C(2) position by esterase to Sal B (Fig. 1), which has a much lower affinity for the KOPR than Sal A (Chavkin et al., 2004; Béguin et al., 2005; Lee et al., 2005b). Sal B has been determined as the principal metabolite of Sal A ex vivo (Schmidt et al., 2005b). However, Sal B was not detected in the blood after i.v. injection of Sal A in rhesus monkeys (Schmidt et al., 2005a). Sal B may be accumulated in the extravascular compartment or be excreted via other routes. Sal A represents a new class of KOPR ligand with a unique non-nitrogen structure. Efforts have been devoted to characterize its structure-activity relationship and to develop more metabolically stable agonists and antagonists for use as pharmacological tools and perhaps as therapeutic agents. We have synthesized a series of C(2) derivatives of Sal A (Béguin et al., 2005; Lee et al., 2005a), one of which, 2-methoxymethyl (MOM)-Sal B (Fig. 1), is a selective KOPR full agonist more potent than Sal A or U50,488H (Lee et al., 2005a). In this study, we further characterized in vitro and in vivo pharmacological properties of this compound. Materials and Methods Materials. Sal A was isolated from leaves of S. divinorum, and MOM-Sal B was synthesized in Dr. David Y.W. Lee’s laboratory as described previously (Lee et al., 2005a). U50,488H was obtained from the National Institute on Drug Abuse (Bethesda, MD). [15,16H]Diprenorphine (56 Ci/mmol) and [S]GTP S (1250 Ci/mmol) were purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). GDP and anti-FLAG M1 monoclonal or polyclonal (F7425) antibodies were purchased from Sigma-Aldrich (St. Louis, MO). The following reagents were obtained from the indicated companies: GTP S (Roche Diagnostics, Indianapolis, IN); geneticin (G418; Cellgro Mediatech, Inc., Herndon, VA); goat anti-mouse IgG (H L) conjugated with Alexa Fluor 488 (Molecular Probes, Eugene, OR); SuperSignal West Pico Chemiluminescent Substrate kit, Restore Western Blot Stripping Buffer (Pierce Chemical, Rockford, IL); and Opti-MEMI reduced serum (Invitrogen, Carlsbad, CA). Animals. Male Sprague-Dawley rats (Zivic-Miller, Pittsburgh, PA) weighing 200 to 250 g (age, 60–80 days) were housed two per cage. Male Swiss-Webster mice (Ace Laboratories, Boyertown, PA) weighing 25 to 30 g were housed five per cage. The animals were maintained on a 12-h light/dark cycle with free access to food and water. Experimental procedures were approved by the Temple University Institutional Animal Care and Use Committee. Cell Lines. CHO cells with stable expression of the human KOPR (CHO-FLAG-hKOPR) were established previously (Zhu et al., 1997). Cells were cultured in 100-mm culture dishes in Dulbecco’s modified Eagle’s medium/Ham’s F-12 medium supplemented with 10% fetal calf serum, 0.4 mg/ml geneticin, 100 units/ml penicillin, and 100 g/ml streptomycin in a humidified atmosphere consisting of 5% CO2 and 95% air at 37°C. Cell Membrane Preparation. Cells were washed twice and harvested in Versene solution (0.54 mM EDTA, 140 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.46 mM KH2PO4, and 1 mM glucose) and centrifuged at 500g for 3 min. The cell pellet was suspended in lysis buffer (5 mM Tris, pH 7.4, 5 mM EDTA, 5 mM EGTA, and 0.1 mM phenylmethylsulfonyl fluoride), passed through a 26 threeeighths-gauge needle 10 times, and then centrifuged at 46,000g for 30 min. The pellet was resuspended in lysis buffer and centrifuged again. The membrane pellet was resuspended in 50 mM Tris-HCl buffer (50 mM Tris, pH 7.4, and 1 mM EGTA), aliquoted and frozen in dry ice/ethanol, and stored at 80°C. All procedures were per-