The Behavioral Effects of the Antidepressant Tianeptine Require the Mu-Opioid Receptor.

Depression is a debilitating chronic illness that affects around 350 million people worldwide. Current treatments, such as selective serotonin reuptake inhibitors, are not ideal because only a fraction of patients achieve remission. Tianeptine is an effective antidepressant with a previously unknown mechanism of action. We recently reported that tianeptine is a full agonist at the mu opioid receptor (MOR). Here we demonstrate that the acute and chronic antidepressant-like behavioral effects of tianeptine in mice require MOR. Interestingly, while tianeptine also produces many opiate-like behavioral effects such as analgesia and reward, it does not lead to tolerance or withdrawal. Furthermore, the primary metabolite of tianeptine (MC5), which has a longer half-life, mimics the behavioral effects of tianeptine in a MOR-dependent fashion. These results point to the possibility that MOR and its downstream signaling cascades may be novel targets for antidepressant drug development.

Preclinical findings predicting efficacy and side-effect profile of LY2940094, an antagonist of nociceptin receptors.

Nociceptin/Orphanin FQ (N/OFQ) is a 17 amino acid peptide whose receptor is designated ORL1 or nociceptin receptor (NOP). We utilized a potent, selective, and orally bioavailable antagonist with documented engagement with NOP receptors in vivo to assess antidepressant- and anxiolytic-related pharmacological effects of NOP receptor blockade along with measures of cognitive and motor impingement. LY2940094 ([2-[4-[(2-chloro-4,4-difluoro-spiro[5H-thieno[2,3-c]pyran-7,4'-piperidine]-1'-yl)methyl]-3-methyl-pyrazol-1-yl]-3-pyridyl]methanol) displayed antidepressant-like behavioral effects in the forced-swim test in mice, an effect absent in NOP -/- mice. LY2940094 also augmented the behavioral effect of fluoxetine without changing target occupancies (NOP and serotonin reuptake transporter [SERT]). LY2940094 did not have effects under a differential-reinforcement of low rate schedule. Although anxiolytic-like effects were not observed in some animal models (conditioned suppression, 4-plate test, novelty-suppressed feeding), LY2940094 had effects like that of anxiolytic drugs in three assays: fear-conditioned freezing in mice, stress-induced increases in cerebellar cGMP in mice, and stress-induced hyperthermia in rats. These are the first reports of anxiolytic-like activity with a systemically viable NOP receptor antagonist. LY2940094 did not disrupt performance in either a 5-choice serial reaction time or delayed matching-to-position assay. LY2940094 was also not an activator or suppressor of locomotion in rodents nor did it induce failures of rotarod performance. These data suggest that LY2940094 has unique antidepressant- and anxiolytic-related pharmacological effects in rodents. Clinical proof of concept data on this molecule in depressed patients have been reported elsewhere.

Consequences of opioid receptor mutation on actions of univalent and bivalent kappa and delta ligands.

INTRODUCTION: During the past decade, substantial evidence has documented that opioid receptor heterodimers form in cell lines expressing one or more opioid receptors. More recent studies have begun to investigate whether heterodimer formation also occurs in vivo. OBJECTIVES: We have used opioid receptor knockout mice to determine whether the in vivo intrathecal (i.t.) pharmacological potency of delta, kappa, and bivalent kappa/delta ligands is altered in the absence of the KOR-1 and/or DOR-1 genes. RESULTS: We observe that both NorBNI (a kappa antagonist) and KDN-21 (a kappa/delta bivalent antagonist) specifically inhibit DPDPE but not deltorphin II i.t potency in wild-type mice but that following mutation of KOR-1, the ability of either compound to reduce DPDPE potency is lost. In contrast, knockout of KOR-1 unexpectedly slightly reduces the potency of deltorphin II (delta2) but not DPDPE (delta1). Finally, two compounds with kappa agonist activity, 6'-GNTI (a putative kappa/delta heterodimer selective agonist) and KDAN-18 (kappa agonist/delta antagonist bivalent ligand) show reduced potency in DOR-1 KO mice. CONCLUSIONS: These results show, genetically, that bivalent ligands with kappa agonist activity require delta receptors for maximal potency in vivo, which is consistent with the presence of opioid heterodimer/oligomer complexes in vivo, and also highlight the complexity of delta drug action even when complementary pharmacologic and genetic approaches are used.

Kappa2 opioid receptor subtype binding requires the presence of the DOR-1 gene.

Over the past several years substantial evidence has documented that opioid receptor homo- and heterodimers form in cell lines expressing one or more of the opioid receptors. We used opioid receptor knockout mice to determine whether in vivo pharmacological characteristics of kappa1 and kappa2 opioid receptors changed following knockout of specific opioid receptors. Using displacement of the general opioid ligand diprenorphine, we observed that occupancy or knockout of the DOR-1 gene increases the binding density of kappa1 receptors and eliminates kappa2 receptors in crude membrane preparations while the total density of kappa opioid binding sites is unchanged. Further, the analgesic potency of U69,593 in cumulative dose response curves is enhanced in mice lacking the DOR-1 gene. These results demonstrate that the DOR-1 gene is required for the expression of the kappa2 opioid receptor subtype and are consistent with the possibility that a KOR-1/DOR-1 heterodimer mediates kappa2 pharmacology.

The tail pigmentation pattern of C57BL/6J mice affects nociception/pain quantification in the tail flick test.

The heat radiant tail flick test is commonly used to quantify nociception and pain levels. Likewise, the C57BL/6J strain of mice is frequently used in pain-related studies as transgenic mice are often backcrossed onto this background. C57BL/6J mice naturally develop non-pigmented patches of variable length on the distal part of the tail that could conceivably modify the response latency in tail flick assays. Here we find that these non-pigmented regions, in a position-independent manner, significantly increase the response latency in the heat radiant tail flick assay, but not the warm water immersion test. This finding demonstrates that the extent of pigmentation, and not other potential variables between pigmented and non-pigmented skin, affects radiant heat tail flick latency, and should be considered in the design of pain-related studies using mice with variable tail pigmentation.

Nociceptin/orphanin FQ (N/OFQ)-evoked bradycardia, hypotension, and diuresis are absent in N/OFQ peptide (NOP) receptor knockout mice.

Intracerebroventricular administration of the opioid-like peptide nociceptin/orphanin FQ (N/OFQ) produces bradycardia, hypotension, and diuresis in mice. We hypothesized that these responses are solely caused by selective activation of central N/OFQ peptide (NOP) receptors. To test this premise, we first examined whether i.c.v. N/OFQ produced dose-dependent diuretic and cardiovascular depressor responses in commercially available C57BL/6 mice. Next, using doses established in these studies, we examined the renal excretory and cardiovascular responses to i.c.v. N/OFQ in conscious transgenic NOP receptor knockout mice (NOP(-/-)). In metabolic studies, i.c.v. N/OFQ, but not saline vehicle, dose-dependently increased urine output (V) in NOP(+/+); this response was significant at 3 nmol (N/OFQ, V = 0.39 +/- 0.10 ml/2 h; saline, 0.08 +/- 0.05 ml/2 h). The N/OFQ-evoked diuresis was absent in littermate NOP(-/-) (N/OFQ, V = 0.06 +/- 0.06 ml/2 h; saline, 0.03 +/- 0.03 ml/2 h). There were no significant changes in urinary sodium or potassium excretion or free water clearance in either group. In telemetry studies, i.c.v. N/OFQ dose dependently lowered heart rate (HR) and mean arterial pressure (MAP). At 3 nmol N/OFQ, both HR and MAP were reduced in NOP(+/+) (peak DeltaHR = -217 +/- 31 bpm; peak DeltaMAP =-47 +/- 7 mm Hg) compared with saline (peak DeltaHR =-14 +/- 5 bpm; peak DeltaMAP = 2 +/- 3 mm Hg). These N/OFQ-evoked bradycardic and hypotensive responses were absent in NOP(-/-) (peak DeltaHR =-13 +/- 17 bpm; peak DeltaMAP =-2 +/- 4 mm Hg, respectively). Basal 24-h cardiovascular and renal excretory function were not different between NOP(-/-) and NOP(+/+) mice. These results establish that the bradycardia, hypotension and diuresis produced by centrally administered N/OFQ are mediated by selective activation of NOP receptors.

Antinociceptive and hypothermic effects of Salvinorin A are abolished in a novel strain of kappa-opioid receptor-1 knockout mice.

Salvia divinorum is a natural occurring hallucinogen that is traditionally used by the Mazatec Indians of central Mexico. The diterpene salvinorin A was identified as an active component of S. divinorum over 20 years ago, but only recently has biochemical screening indicated that a molecular target of salvinorin A in vitro is the kappa-opioid receptor. We have examined whether salvinorin A, the C2-substituted derivative salvinorinyl-2-propionate, and salvinorin B can act as kappa-opioid receptor agonists in vivo. We found that following intracerebroventricular injection over a dose range of 1 to 30 microg of both salvinorin A and salvinorinyl-2-propionate produces antinociception in wild-type mice but not in a novel strain of kappa-opioid receptor knockout mice. Moreover, both salvinorin A and salvinorinyl-2-propionate reduce rectal body temperature, similar to conventional kappa-opioid receptor agonists, in a genotype-dependent manner. In addition, we determined that salvinorin A has high affinity for kappa 1- but not kappa 2-opioid receptors, demonstrating selectivity for this receptor subclass. Finally, treatment over the same dose range with salvinorin B, which is inactive in vitro, produced neither antinociceptive nor hypothermic effects in wild-type mice. These data demonstrate that salvinorin A is the active component of S. divinorum, selective for kappa(1)-opioid receptors, and that salvinorin A and specific structurally related analogs produce behavioral effects that require the kappa-opioid receptor.