HU-308: a specific agonist for CB(2), a peripheral cannabinoid receptor

Abstract

Two cannabinoid receptors have been identified: CB(1), present in the central nervous system (CNS) and to a lesser extent in other tissues, and CB(2), present outside the CNS, in peripheral organs. There is evidence for the presence of CB(2)-like receptors in peripheral nerve terminals. We report now that we have synthesized a CB(2)-specific agonist, code-named HU-308. This cannabinoid does not bind to CB(1) (K(i) > 10 microM), but does so efficiently to CB(2) (K(i) = 22.7 +/- 3.9 nM); it inhibits forskolin-stimulated cyclic AMP production in CB(2)-transfected cells, but does so much less in CB(1)-transfected cells. HU-308 shows no activity in mice in a tetrad of behavioral tests, which together have been shown to be specific for tetrahydrocannabinol (THC)-type activity in the CNS mediated by CB(1). However, HU-308 reduces blood pressure, blocks defecation, and elicits anti-inflammatory and peripheral analgesic activity. The hypotension, the inhibition of defecation, the anti-inflammatory and peripheral analgesic effects produced by HU-308 are blocked (or partially blocked) by the CB(2) antagonist SR-144528, but not by the CB(1) antagonist SR-141716A. These results demonstrate the feasibility of discovering novel nonpsychotropic cannabinoids that may lead to new therapies for hypertension, inflammation, and pain.

Scheme 1

Synthesis of HU-308. Step a, dry p-toluenesulfonic acid in methylene chloride; step b, potassium carbonate, methanol; step c, lithium aluminum hydride.

Figure 1

Binding of HU-308 to the CB₂ cannabinoid receptor, measured by competitive inhibition of [³H]HU-243 binding in COS-7 cells transfected with plasmids containing the CB₂ receptor gene (4).

Figure 2

Absence of psychoactive cannabinoid effect of HU-308. Mice (female C57/BL6) were tested 2.5 hr after i.p. injection of HU-308 (40 mg/kg), in the tetrad of tests for cannabinoid activity: Ambulation (a) and rearing (b) in open field; immobility on a ring (“catalepsy”) (c); hypothermia (d); and antinociception on a hot plate (e). It should be noted that no histopathological damage was observed when mice were kept for up to 60 s on a 59°C hot plate (44). Open bars, vehicle-treated; shaded bars, HU-308.

Figure 3

Intestinal immotility after HU-308 administration with or without SR-144528. Mice (female Sabra, 9–10 weeks old) were randomly divided into five groups of five. Each mouse was injected (i.p.) twice with a 45-min interval. The data presented reflect the number of fecal pellets voided over 105 min, after the second injection. In addition, 50 and 100 mg/kg of HU-308 caused an effect comparable to that recorded with 20 mg/kg (data not shown). *, Significantly less than controls (P < 0.05).

Figure 4

Hypotensive effects of HU-308. Anesthetized rats were cannulated. Baseline blood pressure was recorded before HU-308 was injected i.v. (30 mg/kg, n = 4) Lower doses did not have significant effects. In experiments with antagonists, SR-141716A (3 mg/kg, n = 3) or SR-144528 (1 mg/kg, n = 4), was injected 5 min before HU-308 (see text). The data represent the peak effect, occurring between 2.5 and 5 min. **, Significantly different (P < 0.01) from baseline (124 ± 5.2 mm/Hg). ***, Significantly different (P < 0.001) from baseline.

Figure 5

Effects of HU-308 and indomethacin on arachidonic acid (A′A)-induced swelling of the ear. The results are presented as the difference between the A′A-treated ear and the EtOH-treated ear, n = 5 for each treatment group. Mice (female Sabra) were treated with 4.5 mg of A′A (in 5 μl of EtOH), dispersed on the inner surface of one of the ears. The other ear was treated with 5 μl of EtOH. Ear swelling was assessed by measuring ear thickness with a dial thickness gauge, just before treatment, thence, every 15 min after application, for 90 min. (a) Time curve illustrating that peak swelling of the ear was achieved at about 30 min after A′A application. Vehicle, HU-308 (50 mg/kg), or indomethacin (20 mg/kg) was injected i.p. 60 min before A′A. Injection of HU-308 at 30 or 90 min before A′A yielded similar results (data not shown). (b) Effects of CB₁ and CB₂ receptor antagonists on the anti-inflammatory effect of HU-308. The CB₁ antagonist (SR1 = SR-141716A, 5 mg/kg) or the CB₂ receptor antagonist (SR2 = SR-144528, 1 mg/kg) was injected i.p., and followed 60 min afterward by HU-308 administration i.p. (50 mg/kg). A′A was administered 50 min after HU-308. *, Significantly different from vehicle-treated mice (P < 0.05); **, significantly different from vehicle-treated mice (P < 0.01); ***, significantly different from vehicle-treated mice (P < 0.001).

Figure 6

Effects of HU-308 with or without SR-144528 on formalin-induced peripheral pain. Vehicle or SR-144528 (0.5 mg/kg) was injected, i.p. 15 min before an injection of vehicle or HU-308 (50 mg/kg). Ninety min later, formalin was injected s.c. into one hind paw. Pain was assessed as the total number of licks of the injected paw, recorded every 5 min for 1 hr. Early (5 min) and late (25–60 min) phase pain were observed as described (21). However, HU-308-induced effects were observed only during the late phase. Data collected at 30 min after formalin injection are presented here. *, Significantly different from non-formalin-treated mice (P < 0.05); #, significantly different from vehicle-treated mice that had also received formalin (P < 0.05).