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Comparative Study
, 324 (3), 1073-83

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

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Comparative Study

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

Yulin Wang et al. J Pharmacol Exp Ther.

Abstract

Salvinorin (Sal) A is a naturally occurring, selective kappa 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 approximately 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-[(35)S]thio)triphosphate binding and was approximately 5- and approximately 7-fold more potent than U50,488H and Sal A, respectively. In Chinese hamster ovary cells stably expressing KOPR, all three kappa 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 approximately 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.

Figures

Fig. 1
Fig. 1
Chemical structures of Sal A, Sal B, and MOM-Sal B.
Fig. 2
Fig. 2
A, competitive inhibition by MOM-Sal B, Sal A, or U50,488H of [3H]diprenorphine binding to KOPR. Membranes were prepared from CHO-FLAG-hKOPR cells. Binding was carried out with ~0.4 nM [3H]di-prenorphine in the presence or absence of various concentrations of MOM-Sal B, Sal A, or U50,488H as described under Materials and Methods. Data were normalized to the percentage of specific binding. Each value represents the mean ± S.E.M. of at least three independent experiments performed in duplicate. Apparent Ki values are shown in Table 1. B, stimulation of [35S]GTPγS binding to membranes of CHO-FLAG-hKOPR cells by MOM-Sal B, Sal A, or U50,488H. [35S]GTPγS binding to membranes was performed with various concentrations of each compound as described under Materials and Methods. Nonspecific binding was determined in the presence of 10 μM cold GTPγS. Basal [35S] GTPγS binding in the absence of added compounds was ~2000 dpm. Data were normalized to percentage of the basal [35S]GTPγS binding. Each value represents the mean ± S.E.M. of at least three independent experiments performed in duplicate. EC50 values and maximal responses are shown in Table 1.
Fig. 3
Fig. 3
Dose-response relationship of MOM-Sal B-, Sal A-, and U50,488H-induced internalization of FLAG-hKOPR expressed in CHO cells. CHO-FLAG-hKOPR cells grown on 12-well plates were left untreated or treated with different concentrations of MOM-Sal B, Sal A, or U50,488H at 37°C for 30 min. Cells were then suspended and incubated with M1 anti-FLAG mouse monoclonal antibody followed by goat anti-mouse IgG (H+L) conjugated with Alexa Fluor 488 at 4°C. Cell surface immunofluorescence was measured by fluorescence flow cytometry on a FACScan, and internalized receptor was determined as described under Materials and Methods. Each value represents the mean ± S.E.M. of three independent experiments.
Fig. 4
Fig. 4
Dose-response relationship of MOM-Sal B-, Sal A-, and U50,488H-induced down-regulation of FLAG-hKOPR expressed in CHO cells. CHO-FLAG-HKOPR cells were treated with vehicle (VEH) or various concentrations of MOM-Sal B, Sal A, or U50,488H at 37°C for 4 h. Cells were harvested, solubilized with 2× Laemmli sample buffer, and subjected to SDS-polyacrylamide gel electrophoresis. The receptors were detected by sequential incubation with rabbit anti-FLAG antibodies and horseradish peroxidase-conjugated goat anti-rabbit antibody followed by enhanced chemiluminescence. A, one representative immunoblotting result is shown. B, immunoblot results were quantitated by ImageGauge software and plotted relative to the amount of receptors detected in control cells. Receptor down-regulation was determined as described under Materials and Methods. Each value represents mean ± S.E.M. of at least three independent experiments.
Fig. 5
Fig. 5
Effects of MOM-Sal B on exploratory activities of rats in a Y-maze. Rats were divided into four groups of six animals and administered MOM-Sal B (1, 2.5, and 5 mg/kg s.c.) or vehicle. The incidence of arm entries, vertical rears, and grooming episodes by the rats was counted for 3 min. *, P < 0.05; **, P < 0.01, compared with vehicle.
Fig. 6
Fig. 6
Dose-related antinociception effects of MOM-Sal B in the rat hot-plate test. Rats were divided into 10 groups of 6 to 11 animals, and basal response latencies were determined. Rats were administered MOM-Sal B (A; 0.5, 1, 2.5, or 5 mg/kg i.p.), Sal A (10 mg/kg i.p.), U50,488H (B; 5, 10, or 20 mg/kg i.p.), or an equivalent volume of vehicle. The vehicle for MOM-Sal B and Sal A was 50% dimethyl sulfoxide/saline, whereas that for U50,488H was sterile, pyrogen-free saline. Response latencies were measured at 30-min intervals for 2 h. The cut-off was 30 s. Data are expressed as the mean ± S.E.M. of the %MPA from baseline (time 0). *, P < 0.05, compared with vehicle. C, dose-related relationship of antinociception effects of MOM-Sal B and U50,488H 30 min after administration in the rat hot-plate test. Data are expressed as the mean ± S.E.M. of response latencies (seconds). *, P < 0.05 by Student’s t test, compared with response latency induced by U50,488H at 20 mg/kg.
Fig. 7
Fig. 7
Dose-related hypothermic effects of MOM-Sal B in rats. Rats were divided into 10 groups of six to nine animals, and body temperatures were measured. Rats were administered MOM-Sal B (A; 0.5, 1, 2.5, or 5 mg/kg i.p.), Sal A (10 mg/kg i.p.), U50,488H (B; 5, 10, or 20 mg/kg i.p.), or an equivalent volume of vehicle (50% dimethyl sulfoxide/saline for MOM-Sal B and Sal A and sterile, pyrogen-free saline for U50,488H). Rectal temperature was measured at 30-min intervals for 2 h. Data are expressed as the mean ± S.E.M. of the change in body temperature (ΔTb) from baseline (time 0). *, P < 0.05, compared with vehicle. C, dose-related relationship of MOM-Sal B- and U50,488H-induced hypothermic effects. Data are expressed as the mean ± S.E.M. of the ΔTb from baseline.
Fig. 8
Fig. 8
Effects of norBNI on the antinociception and hypothermia caused by MOM-Sal B. Twenty-four hours after receiving norBNI (10 mg/kg s.c.) or vehicle (VEH), rats were given MOM-Sal B (2.5 mg/kg i.p.) or VEH. Response latency in the hot-plate or body temperature tests was measured 30 min after MOM-Sal B injection. Data are expressed as the mean ± S.E.M. of the %MPA (A) or ΔTb (B) from baseline. n = 6 rats per group. *, P < 0.05 compared with VEH + VEH; +, P < 0.05 compared with VEH + MOM-Sal B.

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