Modulation of gastrointestinal function by MuDelta,a mixed μ opioid receptor agonist/ μ opioid receptor antagonist

BACKGROUND & PURPOSE

Loperamide is a selective µ opioid receptor agonist acting locally in the gastrointestinal (GI) tract as an effective anti-diarrhoeal but can cause constipation. We tested whether modulating µ opioid receptor agonism with δ opioid receptor antagonism, by combining reference compounds or using a novel compound (‘MuDelta’), could normalize GI motility without constipation.

EXPERIMENTAL APPROACH

MuDelta was characterized in vitro as a potent µ opioid receptor agonist and high-affinity δ opioid receptor antagonist. Reference compounds, MuDelta and loperamide were assessed in the following ex vivo and in vivo experiments: guinea pig intestinal smooth muscle contractility, mouse intestinal epithelial ion transport and upper GI tract transit, entire GI transit or faecal output in novel environment stressed mice, or four weeks after intracolonic mustard oil (post-inflammatory). Colonic δ opioid receptor immunoreactivity was quantified.

KEY RESULTS

δ Opioid receptor antagonism opposed µ opioid receptor agonist inhibition of intestinal contractility and motility. MuDelta reduced intestinal contractility and inhibited neurogenically-mediated secretion. Very low plasma levels of MuDelta were detected after oral administration. Stress up-regulated δ opioid receptor expression in colonic epithelial cells. In stressed mice, MuDelta normalized GI transit and faecal output to control levels over a wide dose range, whereas loperamide had a narrow dose range. MuDelta and loperamide reduced upper GI transit in the post-inflammatory model.

CONCLUSIONS AND IMPLICATIONS

MuDelta normalizes, but does not prevent, perturbed GI transit over a wide dose-range in mice. These data support the subsequent assessment of MuDelta in a clinical phase II trial in patients with diarrhoea-predominant irritable bowel syndrome.     

κ-opioid receptors are not necessary for the antidepressant treatment of neuropathic pain

Background and Purpose

Tricyclic antidepressants are used clinically as first-line treatments for neuropathic pain. Opioid receptors participate in this pain-relieving action, and preclinical studies in receptor-deficient mice have highlighted a critical role for δ-, but not μ-opioid receptors. In this study, we investigated whether κ-opioid (KOP) receptors have a role in the antiallodynic action of tricyclic antidepressants.

Experimental Approach

We used a model of neuropathic pain induced by unilateral sciatic nerve cuffing. In this model, the mechanical allodynia was evaluated using von Frey filaments. Experiments were conducted in C57BL/6J mice, and in KOP receptor-deficient mice and their wild-type littermates. The tricyclic antidepressant nortriptyline (5 mg·kg⁻¹) was delivered twice a day for over 2 weeks. Agonists and antagonists of opioid receptors were used to test the selectivity of the KOP receptor antagonist norbinaltorphimine (nor-BNI) in mice with neuropathic pain.

Key Results

After 12 days of treatment, nortriptyline relieved neuropathic allodynia in both wild-type and KOP receptor-deficient mice. Surprisingly, acute nor-BNI reversed the effect of nortriptyline in both wild-type and KOP receptor-deficient mice. Further experiments showed that nor-BNI action was selective for KOP receptors at a late time-point after its administration (8 h), but not at an early time-point, when it may also interact with δ-opioid (DOP) receptors.

Conclusions and Implications

KOP receptors are not necessary for the effect of a tricyclic antidepressant against neuropathic allodynia. These findings together with previous data indicate that the DOP receptor is the only opioid receptor that is necessary for the antiallodynic action of antidepressants.     

Pharmacological mechanisms underlying the antinociceptive and tolerance effects of the 6,14-bridged oripavine compound 030418

Aim:

To investigate possible pharmacological mechanisms underlying the antinociceptive effect of and tolerance to N-methyl-7α-[(R)-1-hydroxy-1-methyl-3-(thien-3-yl)-propyl]-6,14-endo-ethanotetrahydronororipavine (030418), a derivative of thienorphine.

Methods:

The binding affinity and efficacy of 030418 were determined using receptor binding and guanosine 5′-O-(3-[³⁵S]thio)triphosphate ([³⁵S]GTPγS) assays in CHO-μ, CHO-κ, CHO-δ, and CHO-ORL1 cell membranes. The analgesic activity of and tolerance to 030418 were evaluated in thermal nociceptive tests in mice. The effects of 030418 on opioid receptors were further investigated using in vivo pharmacological antagonist blockade and in vitro tissue preparations.

Results:

The compound 030418 displayed high binding affinity to all subtypes of opioid receptors with K_i values in the nanomolar range. In [³⁵S]GTPγS binding assay, the maximal stimulation of 030418 to μ-, κ-, δ-receptors and the ORL1 receptor was 89%, 86%, 67% and 91%, respectively. In hot-plate test, the antinociceptive effect of 030418 was more potent and longer than morphine. The nonselective opioid receptor antagonist naloxone could completely block 030418-induced antinociception, while both the μ-opioid receptor antagonist β-FNA and the κ-opioid receptor antagonist nor-BNI attenuated 030418-induced antinociception. In contrast, the ORL1 receptor antagonist J-113397 enhanced the antinociceptive effect of 030418. Additionally, chronic treatment with 030418 resulted in a dramatic development of tolerance that could not be effectively prevented by J-113397. In guinea pig ileum preparation, the existing action of 030418 could be removed with difficulty after prolonged washing.

Conclusion:

The compound 030418 is a novel agonist of opioid receptors with high efficiency, long-lasting effect and liability to tolerance, which may be closely correlated with the methyl group at the N₁₇ position and the high hydrophobicity of the C₇-thiophene group in its chemical structure.     

Quantitative pharmacological analysis of antagonist binding kinetics at CRF1 receptors in vitro and in vivo

BACKGROUND AND PURPOSE

A series of novel non-peptide corticotropin releasing factor type-1 receptor (CRF₁) antagonists were found to display varying degrees of insurmountable and non-competitive behaviour in functional in vitro assays. We describe how we attempted to relate this behaviour to ligand receptor-binding kinetics in a quantitative manner and how this resulted in the development and implementation of an efficient pharmacological screening method based on principles described by Motulsky and Mahan.

EXPERIMENTAL APPROACH

A non-equilibrium binding kinetic assay was developed to determine the receptor binding kinetics of non-peptide CRF₁ antagonists. Nonlinear, mixed-effects modelling was used to obtain estimates of the compounds association and dissociation rates. We present an integrated pharmacokinetic–pharmacodynamic (PKPD) approach, whereby the time course of in vivo CRF₁ receptor binding of novel compounds can be predicted on the basis of in vitro assays.

KEY RESULTS

The non-competitive antagonist behaviour appeared to be correlated to the CRF₁ receptor off-rate kinetics. The integrated PKPD model suggested that, at least in a qualitative manner, the in vitro assay can be used to triage and select compounds for further in vivo investigations.

CONCLUSIONS AND IMPLICATIONS

This study provides evidence for a link between ligand offset kinetics and insurmountable/non-competitive antagonism at the CRF₁ receptor. The exact molecular pharmacological nature of this association remains to be determined. In addition, we have developed a quantitative framework to study and integrate in vitro and in vivo receptor binding kinetic behaviour of CRF₁ receptor antagonists in an efficient manner in a drug discovery setting.     

Pharmacological characterization of LPS and opioid interactions at the toll-like receptor 4

Background and Purpose

Previous work in our laboratory showed opioid agents inhibit cytokine expression in astrocytes. Recently, Watkins and colleagues hypothesized that opioid agonists activate toll-like receptor 4 (TLR4) signalling, which leads to neuroinflammation. To test this hypothesis, we characterized LPS and opioid effects on TLR4 signalling in reporter cells.

Experimental Approach

NF-κB reporter cells expressing high levels of TLR4 were used to compare LPS and opioid effects on NF-κB activation, a pathway activated by TLR4 stimulation.

Key Results

LPS increased TLR4 signalling in a concentration-dependent manner and was antagonized by LPS antagonist (LPS-RS, from Rhodobacter sphaeroides). A concentration ratio analysis showed that LPS-RS was a competitive antagonist. The opioid agonists, morphine and fentanyl, produced minor activation of TLR4 signalling when given alone. When tested following LPS stimulation, opioid agonists inhibited NF-κB activation but this inhibition was not blocked by the general opioid antagonist, naloxone, nor by the selective μ opioid receptor antagonist, β-FNA. Indeed, both naloxone and β-FNA also inhibited NF-κB activation in reporter cells. Further examination of fentanyl and β-FNA effects revealed that both opioid agents inhibited LPS signalling in a non-competitive fashion.

Conclusions and Implications

These results show that LPS-RS is a competitive antagonist at the TLR4 complex, and that both opioid agonists and antagonists inhibit LPS signalling in a non-competitive fashion through a non-GPCR, opioid site(s) in the TLR4 signalling pathway. If confirmed, existing opioid agents or other drug molecules more selective at this novel site may provide a new therapeutic approach to the treatment of neuroinflammation.     

Determining Pharmacological Selectivity of the Kappa Opioid Receptor Antagonist LY2456302 Using Pupillometry as a Translational Biomarker in Rat and Human

Background:

Selective kappa opioid receptor antagonism is a promising experimental strategy for the treatment of depression. The kappa opioid receptor antagonist, LY2456302, exhibits ~30-fold higher affinity for kappa opioid receptors over mu opioid receptors, which is the next closest identified pharmacology.

Methods:

Here, we determined kappa opioid receptor pharmacological selectivity of LY2456302 by assessing mu opioid receptor antagonism using translational pupillometry in rats and humans.

Results:

In rats, morphine-induced mydriasis was completely blocked by the nonselective opioid receptor antagonist naloxone (3mg/kg, which produced 90% mu opioid receptor occupancy), while 100 and 300mg/kg LY2456302 (which produced 56% and 87% mu opioid receptor occupancy, respectively) only partially blocked morphine-induced mydriasis. In humans, fentanyl-induced miosis was completely blocked by 50mg naltrexone, and LY2456302 dose-dependently blocked miosis at 25 and 60mg (minimal-to-no blockade at 4–10mg).

Conclusions:

We demonstrate, for the first time, the use of translational pupillometry in the context of receptor occupancy to identify a clinical dose of LY2456302 achieving maximal kappa opioid receptor occupancy without evidence of significant mu receptor antagonism.     

In vivo effects of the pure aryl hydrocarbon receptor antagonist GNF-351 after oral administration are limited to the gastrointestinal tract

Background and Purpose

GNF-351 is a potent aryl hydrocarbon receptor (AHR) antagonist that inhibits dioxin response element-dependent and independent activities. Here, the absorption, metabolism and in vivo AHR antagonist activity of GNF-351 were investigated.

Experimental Approach

LC-MS metabolomics was used to analyse GNF-351 metabolism in vitro and in vivo. Recombinant drug-metabolizing enzymes were employed to determine the enzymes involved in GNF-351 metabolism. Analysis of target AHR genes was performed to investigate the inhibitory effects of GNF-351 towards AHR activation.

Key Results

Several phase I metabolites were generated after GNF-351 was incubated with microsomes from human or mouse liver and intestine, including two oxidized GNF-351 and one tri-demethylated GNF-351. Poor absorption from the intestine resulted in no detectable levels of GNF-351 in mouse serum (0–6 h) and urine (24 h) and almost all GNF-351 was found in the faeces after 24 h. Analysis of faeces further revealed all the in vitro phase I metabolites. Novel metabolites were detected, including one di-oxidized GNF-351, two oxidized and tri-demethylated GNF-351, one dehydrogenated product of oxidized GNF-351, and one sulfation product of di-oxidized GNF-351. Cytochromes P450 were demonstrated to be the major enzymes involved in metabolism of GNF-351. After oral administration to mice, GNF-351 readily inhibited β-naphthoflavone-induced AHR activation in ileum and colon, but not that in the liver.

Conclusion and Implications

While poor absorption and extensive metabolism after oral administration limited the in vivo effects of the pure AHR antagonist GNF-351 in liver, it could be used to inhibit AHR activation in intestine and colon.     

The cognition-enhancing activity of E1R,a novel positive allosteric modulator of sigma-1 receptors

Background and Purpose

Here, we describe the in vitro and in vivo effects of (4R,5S)-2-(5-methyl-2-oxo-4-phenyl-pyrrolidin-1-yl)-acetamide (E1R), a novel positive allosteric modulator of sigma-1 receptors.

Experimental Approach

E1R was tested for sigma receptor binding activity in a [³H](+)-pentazocine assay, in bradykinin (BK)-induced intracellular Ca²⁺ concentration ([Ca²⁺]_i) assays and in an electrically stimulated rat vas deferens model. E1R''s effects on cognitive function were tested using passive avoidance (PA) and Y-maze tests in mice. A selective sigma-1 receptor antagonist (NE-100), was used to study the involvement of the sigma-1 receptor in the effects of E1R. The open-field test was used to detect the effects of E1R on locomotion.

Key Results

Pretreatment with E1R enhanced the selective sigma-1 receptor agonist PRE-084''s stimulating effect during a model study employing electrically stimulated rat vasa deferentia and an assay measuring the BK-induced [Ca²⁺]_i increase. Pretreatment with E1R facilitated PA retention in a dose-related manner. Furthermore, E1R alleviated the scopolamine-induced cognitive impairment during the PA and Y-maze tests in mice. The in vivo and in vitro effects of E1R were blocked by treatment with the selective sigma-1 receptor antagonist NE-100. E1R did not affect locomotor activity.

Conclusion and Implications

E1R is a novel 4,5-disubstituted derivative of piracetam that enhances cognition and demonstrates efficacy against scopolamine-induced cholinergic dysfunction in mice. These effects are attributed to its positive modulatory action on the sigma-1 receptor and this activity may be relevant when developing new drugs for treating cognitive symptoms related to neurodegenerative diseases.     

Development of β-amino-carbonyl compounds as androgen receptor antagonists

Aim： Androgen receptor （AR） antagonists have proven to be useful in the early control of prostate cancer. The aim of this study was to identify and characterize a novel β-amino-carbonyl-based androgen receptor antagonist. Methods Different isomers of the β-amino-carbonyl compounds were obtained by chiral separation. The bioactivities of the isomers were evaluated by AR nuclear translocation, mammalian two-hybrid, competitive receptor binding and cell proliferation assays. The expression of genes downstream of AR was analyzed with real-time PCR. The therapeutic effects on tumor growth in vivo were observed in male SClD mice bearing LNCaP xenografts. Results： Compound 21 was previously identified as an AR modulator by the high-throughput screening of a diverse compound library. In the present study, the two isomers of compound 21, termed compounds 21-1 and 2.1.-2, were characterized as partial AR agonists in terms of androgen-induced AR nuclear translocation, prostate-specific antigen expression and cell proliferation. Further structural modifications led to the discovery of a androgen receptor antagonist （compound 6012）, which blocked androgen receptor nuclear translocation, androgen-responsive gene expression and androgen-dependent LNCaP cell proliferation. Four stereoisomers of compound 6012 were isolated, and their bioactivities were assessed. The pharmacological effects of 6012, including AR binding, androgen-induced AR translocation, NH2- and COOH-terminal interaction, growth inhibition of LNCaP cells in vitro and LNCaP xenograft growth in nude mice, were mainly restricted to isomer 6012-4 （1R, 3S）. Conclusion： Compound 6012-4 was determined to be a novel androgen receptor antagonist with prostate cancer inhibitory activities comparable to bicalutamide both in vitro and in vivo.     

Characterization of RO4583298 as a novel potent, dual antagonist with in vivo activity at tachykinin NK₁ and NK₃ receptors

BACKGROUND AND PURPOSE

Clinical results of osanetant and talnetant (selective-NK₃ antagonists) indicate that blocking the NK₃ receptor could be beneficial for the treatment of schizophrenia. The objective of this study was to characterize the in vitro and in vivo properties of a novel dual NK₁/NK₃ antagonist, RO4583298 (2-phenyl-N-(pyridin-3-yl)-N-methylisobutyramide derivative).

EXPERIMENTAL APPROACH

RO4583298 in vitro pharmacology was investigated using radioligand binding ([³H]-SP, [³H]-osanetant, [³H]-senktide), [³H]-inositol-phosphate accumulation Schild analysis (SP- or [MePhe⁷]-NKB-induced) and electrophysiological studies in guinea-pig substantia nigra pars compacta (SNpc). The in vivo activity of RO4583298 was assessed using reversal of GR73632-induced foot tapping in gerbils (GFT; NK₁) and senktide-induced tail whips in mice (MTW; NK₃).

KEY RESULTS

RO4583298 has a high-affinity for NK₁ (human and gerbil) and NK₃ (human, cynomolgus monkey, gerbil and guinea-pig) receptors and behaves as a pseudo-irreversible antagonist. Unusually it binds with high-affinity to mouse and rat NK₃, yet with a partial non-competitive mode of antagonism. In guinea-pig SNpc, RO4583298 inhibited the senktide-induced potentiation of spontaneous activity of dopaminergic neurones with an apparent non-competitive mechanism of action. RO4583298 (p.o.) robustly blocked the GFT response, and inhibited the MTW.

CONCLUSIONS AND IMPLICATIONS

RO4583298 is a high-affinity, non-competitive, long-acting in vivo NK₁/NK₃ antagonist; hence providing a useful in vitro and in vivo pharmacological tool to investigate the roles of NK₁ and NK₃ receptors in psychiatric disorders.     

Pharmacology and anti-addiction effects of the novel κ opioid receptor agonist Mesyl Sal B,a potent and long-acting analogue of salvinorin A

BACKGROUND AND PURPOSE

Acute activation of κ opioid (KOP) receptors results in anticocaine-like effects, but adverse effects, such as dysphoria, aversion, sedation and depression, limit their clinical development. Salvinorin A, isolated from the plant Salvia divinorum, and its semi-synthetic analogues have been shown to have potent KOP receptor agonist activity and may induce a unique response with similar anticocaine addiction effects as the classic KOP receptor agonists, but with a different side effect profile.

EXPERIMENTAL APPROACH

We evaluated the duration of effects of Mesyl Sal B in vivo utilizing antinociception assays and screened for cocaine-prime induced cocaine-seeking behaviour in self-administering rats to predict anti-addiction effects. Cellular transporter uptake assays and in vitro voltammetry were used to assess modulation of dopamine transporter (DAT) function and to investigate transporter trafficking and kinase signalling pathways modulated by KOP receptor agonists.

KEY RESULTS

Mesyl Sal B had a longer duration of action than SalA, had anti-addiction properties and increased DAT function in vitro in a KOP receptor-dependent and Pertussis toxin-sensitive manner. These effects on DAT function required ERK1/2 activation. We identified differences between Mesyl Sal B and SalA, with Mesyl Sal B increasing the V_max of dopamine uptake without altering cell-surface expression of DAT.

CONCLUSIONS AND IMPLICATIONS

SalA analogues, such as Mesyl Sal B, have potential for development as anticocaine agents. Further tests are warranted to elucidate the mechanisms by which the novel salvinorin-based neoclerodane diterpene KOP receptor ligands produce both anti-addiction and adverse side effects.

LINKED ARTICLES

This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2     

In vitro and in vivo characterization of A-940894: a potent histamine H4 receptor antagonist with anti-inflammatory properties

Background and purpose:

The histamine H₄ receptor is widely expressed in cells of immune origin and has been shown to play a role in a variety of inflammatory processes mediated by histamine. In this report, we describe the in vitro and in vivo anti-inflammatory properties of a potent histamine H₄ receptor antagonist, A-940894 (4-piperazin-1-yl-6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-d]pyrimidin-2-ylamine).

Experimental approach:

We have analysed the pharmacological profile of A-940894 at mouse native, rat recombinant and human recombinant and native, histamine H₄ receptors by radioligand binding, calcium mobilization, mast cell shape change, eosinophil chemotaxis assays and in the mouse model of zymosan-induced peritonitis.

Key results:

A-940894 potently binds to both human and rat histamine H₄ receptors and exhibits considerably lower affinity for the human histamine H₁, H₂ or H₃ receptors. It potently blocked histamine-evoked calcium mobilization in the fluorometric imaging plate reader assays and inhibited histamine-induced shape change of mouse bone marrow-derived mast cells and chemotaxis of human eosinophils in vitro. In a mouse mast cell-dependent model of zymosan-induced peritonitis, A-940894 significantly blocked neutrophil influx and reduced intraperitoneal prostaglandin D₂ levels. Finally, A-940894 has good pharmacokinetic properties, including half-life and oral bioavailability in rats and mice.

Conclusions and Implications:

These data suggest that A-940894 is a potent and selective histamine H₄ receptor antagonist with pharmacokinetic properties suitable for long-term in vivo testing and could serve as a useful tool for the further characterization of histamine H₄ receptor pharmacology.     

Modulating human proteinase activated receptor 2 with a novel antagonist (GB88) and agonist (GB110)

BACKGROUND AND PURPOSE

Many cells express proteinase activated receptor 2 (PAR2) on their plasma membrane. PAR2 is activated by proteolytic enzymes, such as trypsin and tryptase that cleave the receptor N-terminus, inititating signalling to intracellular G proteins. Studies on PAR2 have relied heavily upon activating effects of proteases and peptide agonists that lack stability and bioavailability in vivo.

EXPERIMENTAL APPROACH

A novel small molecule agonist GB110 and an antagonist GB88 were characterized in vitro against trypsin, peptide agonists, PAR2 antibody, PAR1 agonists and flow cytometry,in seven cell lines using intracellular Ca²⁺ mobilization and examined in vivo against PAR2- and PAR1-induced rat paw oedema.

KEY RESULTS

GB110 is a potent non-peptidic agonist activating PAR2-mediated Ca²⁺ release in HT29 cells (EC₅₀∼200 nM) and six other human cell lines, inducing PAR2 internalization. GB88 is a unique PAR2 antagonist, inhibiting PAR2 activated Ca²⁺ release (IC₅₀∼2 µM) induced by native (trypsin) or synthetic peptide and non-peptide agonists. GB88 was a competitive and surmountable antagonist of agonist 2f-LIGRLO-NH₂, a competitive but insurmountable antagonist of agonist GB110, and a non-competitive insurmountable antagonist of trypsin. GB88 was orally active and anti-inflammatory in vivo, inhibiting acute rat paw oedema elicited by agonist GB110 and proteolytic or peptide agonists of PAR2 but not by corresponding agonists of PAR1 or PAR4.

CONCLUSIONS AND IMPLICATIONS

The novel PAR2 agonist and antagonist modulate intracellular Ca²⁺ and rat paw oedema, providing novel molecular tools for examining PAR2-mediated diseases.     

A novel, orally active LPA1 receptor antagonist inhibits lung fibrosis in the mouse bleomycin model

Background and purpose:

The aim of this study was to assess the potential of an antagonist selective for the lysophosphatidic acid receptor, LPA₁, in treating lung fibrosis We evaluated the in vitro and in vivo pharmacological properties of the high affinity, selective, oral LPA₁-antagonist (4′-{4-[(R)-1-(2-chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (AM966).

Experimental approach:

The potency and selectivity of AM966 for LPA₁ receptors was determined in vitro by calcium flux and cell chemotaxis assays using recombinant and native cell cultures. The in vivo efficacy of AM966 to reduce tissue injury, vascular leakage, inflammation and fibrosis was assessed at several time points in the mouse bleomycin model.

Key results:

AM966 was a potent antagonist of LPA₁ receptors, with selectivity for this receptor over the other LPA receptors. In vitro, AM966 inhibited LPA-stimulated intracellular calcium release (IC₅₀ = 17 nM) from Chinese hamster ovary cells stably expressing human LPA₁ receptors and inhibited LPA-induced chemotaxis (IC₅₀ = 181 nM) of human IMR-90 lung fibroblasts expressing LPA₁ receptors. AM966 demonstrated a good pharmacokinetic profile following oral dosing in mice. In the mouse, AM966 reduced lung injury, vascular leakage, inflammation and fibrosis at multiple time points following intratracheal bleomycin instillation. AM966 also decreased lactate dehydrogenase activity and tissue inhibitor of metalloproteinase-1, transforming growth factor β1, hyaluronan and matrix metalloproteinase-7, in bronchoalveolar lavage fluid.

Conclusions and implications:

These findings demonstrate that AM966 is a potent, selective, orally bioavailable LPA₁ receptor antagonist that may be beneficial in treating lung injury and fibrosis, as well as other diseases that are characterized by pathological inflammation, oedema and fibrosis.     

Discrete cue-conditioned alcohol-seeking after protracted abstinence: pattern of neural activation and involvement of orexin₁ receptors

BACKGROUND AND PURPOSE

The enduring propensity for alcoholics to relapse even following years of abstinence presents a major hurdle for treatment. Here we report a model of relapse following protracted abstinence and investigate the pattern of neuronal activation following cue-induced reinstatement and administration of the orexin₁ receptor antagonist SB-334867 in inbred alcohol-preferring rats.

EXPERIMENTAL APPROACH

Rats were trained to self-administer alcohol under operant conditions and divided into two groups: immediate (reinstated immediately following extinction) and delayed (extinguished and then housed for 5 months before reinstatement). Prior to reinstatement, animals were treated with vehicle (immediate n= 11, delayed n= 11) or SB-334867 (20 mg·kg⁻¹ i.p.; immediate n= 6, delayed n= 11). Fos expression was compared between each group and to animals that underwent extinction only.

KEY RESULTS

SB-334867 significantly attenuated cue-induced reinstatement in both groups. Immediate reinstatement increased Fos expression in the nucleus accumbens (NAc), infra-limbic (IL), pre-limbic (PrL), orbitofrontal (OFC) and piriform cortices, the lateral and dorsomedial hypothalamus, central amygdala and basolateral amygdala (BLA), and the bed nucleus of the stria terminalis. Following delayed reinstatement, Fos expression was further elevated in cortical structures. Concurrent with preventing reinstatement, SB-334867 decreased Fos in NAc core, PrL and OFC following immediate reinstatement. Following protracted abstinence, SB-334867 treatment decreased reinstatement-induced Fos in the PrL, OFC and piriform cortices.

CONCLUSIONS AND IMPLICATIONS

Cue-induced alcohol seeking can be triggered following protracted abstinence in rats. The effects of SB-334867 on both behaviour and Fos expression suggest that the orexin system is implicated in cue-induced reinstatement, although some loci may shift following protracted abstinence.     

Positive allosteric modulation of the human cannabinoid (CB1) receptor by RTI-371, a selective inhibitor of the dopamine transporter

Background and purpose

In our search for an indirect dopamine agonist as therapy for cocaine addiction, several selective inhibitors of the dopamine transporter (DAT), which are 3-phenyltropane analogues, were assayed for their effect on locomotor activity in mice. Interestingly, several of the compounds showed a poor correlation between stimulation of locomotion and DAT inhibition. One of the compounds, 3β-(4-methylphenyl)-2β-[3-(4-chlorophenyl)isoxazol-5-yl]tropane (RTI-371), was shown to cross the blood-brain barrier, by binding studies in vivo, and block cocaine-induced locomotor stimulation. As poor pharmacokinetics could not explain the behavioural effects of RTI-371, this compound was screened through our functional assays for activity at other CNS receptors. Initial screening identified RTI-371 as a positive allosteric modulator of the human CB₁ (hCB₁) receptor.

Experimental approach

The effect of RTI-371 and other DAT-selective inhibitors on CP55940-stimulated calcium mobilization was characterized in a calcium mobilization-based functional assay for the hCB₁ receptor. Selected compounds were also characterized in a similar assay for human µ opioid receptor activation to assess the specificity of their effects.

Key results

RTI-371 and several other DAT-selective inhibitors with atypical actions on locomotor behaviour increased the efficacy of CP55940 in a concentration-dependent manner.

Conclusions and implications

These results suggest that the lack of correlation between the DAT-binding affinity and locomotor stimulation of RTI-371 could be due at least in part to its activity as a positive modulator of the hCB₁ receptor.     

Permeation of topically applied caffeine through human skin – a comparison of in vivo and in vitro data

AIMS

Due to ethical reasons, in vivo penetration studies are not applicable at all stages of development of new substances. Therefore, the development of appropriate in vitro methods is essential, as well as the comparison of the obtained in vivo and in vitro data, in order to identify their transferability. The aim of the present study was to investigate the follicular penetration of caffeine in vitro and to compare the data with the in vivo results determined previously under similar conditions.

METHODS

The Follicular Closing Technique (FCT) represents a method to investigate the follicular penetration selectively. In the present study, FCT was combined with the Franz diffusion cell in order to differentiate between follicular and intercellular penetration of caffeine into the receptor medium in vitro. Subsequently, the results were compared with the data obtained in an earlier study investigating follicular and intercellular penetration of caffeine in vivo.

RESULTS

The comparison of the data revealed that the in vitro experiments were valuable for the investigation of the follicular penetration pathway, which contributed in vivo as well as in vitro to approximately 50% of the total penetration, whereas the kinetics of caffeine penetration were shown to be significantly different.

CONCLUSIONS

The combination of FCT with the Franz diffusion cell represents a valuable method to investigate follicular penetration in vitro. Nevertheless, in vivo experiments should not be abandoned as in vitro, structural changes of skin occur and blood flow and metabolism are absent, probably accounting for reduced penetration rates in vitro.     

Zoledronate and pamidronate depress neutrophil functions and survival in mice

BACKGROUND AND PURPOSE

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) has been identified as a severe complication of patients previously treated with i.v. bisphosphonates. It has been noted that necrotic bone from BRONJ sites display signs of bacterial infection suggesting that an immune defect may play a role in the pathophysiology of BRONJ. Here, we have examined the effect of two potent bisphosphonates, zoledronate and pamidronate, on neutrophil function, differentiation and survival.

EXPERIMENTAL APPROACH

The effect of bisphosphonates on chemotaxis, NADPH oxidase activity and neutrophil survival were assessed in vitro using bone marrow-derived primary neutrophils or in vitro differentiated haematopoetic progenitors from mice. The same parameters and the number of circulating neutrophils were quantified in neutrophils isolated from mice treated in vivo with zoledronate. In vivo recruitment of neutrophils was assessed by sodium periodate-induced peritonitis.

KEY RESULTS

Zoledronate and pamidronate inhibited in vitro neutrophil chemotaxis and NADPH oxidase activity in a dose-dependent manner. In vivo recruitment of neutrophils was also suppressed. Zoledronate did not affect in vitro differentiation of neutrophils but shortened their life span in a granulocyte-colony stimulating factor-dependent manner. fMLP-induced activation of RhoA activity was decreased by zoledronate treatment.

CONCLUSIONS AND IMPLICATIONS

Our results show that bisphosphonate exposure leads to impaired neutrophil chemotaxis, neutrophil NADPH oxidase activity and reduced circulating neutrophil counts. This work suggests that bisphosphonates have the potential to depress the innate immune system for a prolonged time, possibly contributing to the pathogenesis of BRONJ.     

Peripheral interactions between cannabinoid and opioid systems contribute to the antinociceptive effect of crotalphine

Background and Purpose

Crotalphine is an antinociceptive peptide that, despite its opioid-like activity, does not induce some of the characteristic side effects of opioids, and its amino acid sequence has no homology to any known opioid peptide. Here, we evaluated the involvement of the peripheral cannabinoid system in the crotalphine effect and its interaction with the opioid system.

Experimental Approach

Hyperalgesia was evaluated using the rat paw pressure test. Involvement of the cannabinoid system was determined using a selective cannabinoid receptor antagonist. Cannabinoid and opioid receptor activation were evaluated in paw slices by immunofluorescence assays using conformation state-sensitive antibodies. The release of endogenous opioid peptides from skin tissue was measured using a commercial enzyme immunoassay (EIA).

Key Results

Both p.o. (0.008–1.0 μg·kg⁻¹) and intraplantar (0.0006 μg per paw) administration of crotalphine induced antinociception in PGE₂-induced hyperalgesia. Antinociception by p.o. crotalphine (1 μg·kg⁻¹) was blocked by AM630 (50 μg per paw), a CB₂ receptor antagonist, and by antiserum anti-dynorphin A (1 μg per paw). Immunoassay studies confirmed that crotalphine increased the activation of both κ-opioid (51.7%) and CB₂ (28.5%) receptors in paw tissue. The local release of dynorphin A from paw skin was confirmed by in vitro EIA and blocked by AM630.

Conclusions and Implications

Crotalphine-induced antinociception involves peripheral CB₂ cannabinoid receptors and local release of dynorphin A, which is dependent on CB₂ receptor activation. These results enhance our understanding of the mechanisms involved in the peripheral effect of crotalphine, as well as the interaction between the opioid and cannabinoid systems.     

Inhibitory activity of the novel CB2 receptor agonist, GW833972A, on guinea-pig and human sensory nerve function in the airways

Background and purpose:

Sensory nerves regulate central and local reflexes such as airway plasma protein leakage, bronchoconstriction and cough. Sensory nerve activity may be enhanced during inflammation such that these protective effects become exacerbated and deleterious. Cannabinoids are known to inhibit airway sensory nerve function. However, there is still controversy surrounding which receptor is involved in eliciting these effects.

Experimental approach:

We have adopted a pharmacological approach, including using a novel, more selective CB₂ receptor agonist, GW 833972A (1000-fold selective CB₂/CB₁), and receptor selective antagonists to investigate the inhibitory activity of cannabinoids on sensory nerve activity in vitro and in vivo in guinea-pig models of cough and plasma extravasation.

Key results:

GW 833972A inhibited capsaicin-induced depolarization of the human and guinea-pig and prostaglandin E₂ (PGE₂) and hypertonic saline-induced depolarization of the guinea-pig isolated vagus nerve in vitro. GW 833972A also inhibited citric acid-induced cough but not plasma extravasation in the guinea-pig and this effect was blocked by a CB₂ receptor antagonist.

Conclusions and implications:

This confirms and extends previous studies highlighting the role of CB₂ receptors in the modulation of sensory nerve activity elicited both by the exogenous ligands capsaicin and hypertonic saline but also by endogenous modulators such as PGE₂ and low pH stimuli. These data establish the CB₂ receptor as an interesting target for the treatment of chronic cough.