Lack of tolerance to anxiolysis and withdrawal symptoms in mice repeatedly treated with AC-5216, a selective TSPO ligand.

AC-5216, a ligand for the translocator protein (18 kDa) (TSPO), produces anxiolytic-like effects in animal models of anxiety without causing the side effects normally associated with conventional benzodiazepines. This study aimed to investigate whether repeated administration of AC-5216 induces tolerance to anxiolytic-like effects of AC-5216 and produces withdrawal on abrupt cessation, and compare the results with those of diazepam. In the tolerance experiment, AC-5216 (0.1 mg/kg, p.o.) produced significant anxiolytic-like effects in both groups of mice pretreated with the vehicle and AC-5216 twice daily for 14 days. Diazepam (0.1 mg/kg, p.o.) also retained its anxiolytic effects in mice repeatedly treated with diazepam. In the withdrawal experiment, mice were orally treated with either AC-5216 (0.1, 1 or 10 mg/kg; twice daily) or diazepam (0.1, 1 or 10 mg/kg; twice daily) for 14 days, and examined, during a treatment withdrawal period, for anxiogenic-like effects in the social interaction test, and for body weight loss as indices of emotional and somatic withdrawal symptoms, respectively. In AC-5216-treated groups, neither anxiogenic-like effects nor body weight loss was observed upon treatment withdrawal at any of the doses tested. In contrast, in diazepam 1 mg/kg- and 10 mg/kg-treated groups, treatment withdrawal not only induced anxiogenic-like effects on the second day of the withdrawal period, but also decreased body weight gain and brought about body weight loss in mice. These findings indicate that AC-5216 when repeatedly administered does not induce tolerance to its anxiolytic-like effects or withdrawal symptoms.

Involvement of neurosteroids in the anxiolytic-like effects of AC-5216 in mice.

AC-5216, a ligand for the translocator protein (18 kDa) (TSPO), previously called the peripheral benzodiazepine receptor (PBR), produces anxiolytic-like effects mediated by TSPO in animal models of anxiety. Since stimulation of TSPO is considered to promote the synthesis of neurosteroids, we investigated the possible role of endogenous neurosteroids that positively act on the GABA(A) receptor in the anxiolytic-like effects of AC-5216. In our experiments, the effects of trilostane and finasteride, two inhibitors of steroidogenic enzymes, and picrotoxin, a GABA(A) receptor-gated Cl(-) channel blocker, on the anxiolytic-like effects of AC-5216 were examined in the social interaction test in mice. Also, the anxiolytic-like effects of allopregnanolone and progesterone were examined. The anxiolytic-like effects of AC-5216 (0.1 mg/kg, p.o.) were inhibited by trilostane (10-30 mg/kg, s.c.), finasteride (10-30 mg/kg, s.c.), and picrotoxin (0.03-0.3 mg/kg, s.c.), while those of diazepam (0.1 mg/kg, p.o.) were inhibited by picrotoxin only. The anxiolytic-like effects of progesterone (1-3 mg/kg, s.c.) were inhibited by finasteride (3-30 mg/kg) and picrotoxin (0.1-0.3 mg/kg), although those of allopregnanolone (10 mg/kg, s.c.) were inhibited by picrotoxin only. These results demonstrate that the anxiolytic-like effects of AC-5216 are due to newly synthesized neurosteroids that enhance GABA(A) receptor function.

Antianxiety and antidepressant-like effects of AC-5216, a novel mitochondrial benzodiazepine receptor ligand.

We investigated the ability of N-benzyl-N-ethyl-2-(7,8-dihydro-7-methyl-8-oxo-2-phenyl-9H-purin-9-yl)acetamide (AC-5216), a novel mitochondrial benzodiazepine receptor (MBR) ligand, to produce anti-anxiety and antidepressant-like effects in various animal models. AC-5216 showed high affinity for MBRs prepared from rat whole brain (Ki 0.297 nm), rat glioma cells (IC50 3.04 nm) and human glioma cells (IC50 2.73 nm), but only negligible affinity for the other main receptors including central benzodiazepine receptors. AC-5216 produced anti-anxiety effects in the Vogel-type conflict test in rats, and in the light/dark box and social interaction tests in mice at 0.1-3, 0.003-0.01 and 0.01-0.3 mg kg(-1), p.o., respectively. These effects of AC-5216 were antagonized by PK11195, an MBR antagonist. In the forced swimming test in rats, AC-5216 (3-30 mg kg(-1), p.o.) reduced the immobility time, and this effect was blocked by PK11195. AC-5216 had no myorelaxant effects, did not affect the memory or prolong hexobarbitone-induced sleep in mice, even at doses as high as 1000 mg kg(-1), p.o. Although it did slightly prolong the ethanol-induced sleep time at 1000 mg kg(-1), AC-5216 (1-100 mg kg(-1), p.o.) produced no distinct change in the rat electroencephalogram. These results indicate that AC-5216 produces anti-anxiety and antidepressant-like effects that are mediated by MBR, but does not cause the side effects normally associated with conventional benzodiazepines. Hence, AC-5216 shows potential for the treatment of stress-related disorders including anxiety and depression.

Development of potent bifunctional endomorphin-2 analogues with mixed mu-/delta-opioid agonist and delta-opioid antagonist properties.

The C terminus of endomorphin-2 (EM-2) analogues (Tyr-Pro-Phe-NH-X) was modified with aromatic, heteroaromatic, or aliphatic groups (X = phenethyl,benzyl, phenyl, naphthyl, pyridyl, quinolyl, isoquinolyl, tert-butyl, cyclohexyl, or adamantyl; 3-18) to study their effect on opioid activity. Only 9 (1-naphthyl), 11 (5-quinolyl), 16 (cyclohexyl), and 18 (2-adamantyl) exhibited mu-opioid receptor affinity in the nanomolar range (K(i) = 2.41-6.59 nM), which, however, was 3- to 10-fold less than the parent peptide. Replacement of Tyr(1) by Dmt (2',6'-dimethyl-l-tyrosine) (19-32) exerted profound effects: (i) acquisition of high mu-opioid receptor affinity (K(i) = 0.11-0.52 nM) except 23 (Ph); (ii) presence of potent functional mu-opioid receptor agonism (IC(50) < 1 nM) for 19 ([Dmt(1)]EM-2), 27 (1-naphthyl), 29 (5-quinolyl), and 32 (5-isolquinolyl); (iii) association of weak delta-opioid antagonist activity (pA(2) = 5.41-7.18) except 19 ([Dmt(1)]EM-2), 20 (H), 27 (1-naphthyl), and in particular 29 (5-quinolyl) with its potent delta-agonism (IC(50) = 0.62 nM, pA(2) = 5.88); (iv) production of antinociception after ic administration of 32 (5-isoquinolyl) in mice, a bioactivity absent in the corresponding Tyr(1) analogue (14); and (v) preferential cis orientation (cis/trans = 3:2 to 7:3) at the Dmt-Pro amide bond, in contrast to the Tyr-Pro amide trans orientation (cis/trans = 1:2 to 1:3). Thus, [Dmt(1)]EM-2 analogues with hydrophobic C-terminal extensions provide model compounds with potent mu-opioid receptor bioactivity and dual functional agonism.