The macrocyclic tetrapeptide [D-Trp]CJ-15,208 produces short-acting κ opioid receptor antagonism in the CNS after oral administration.

BACKGROUND AND PURPOSE: Cyclic peptides are resistant to proteolytic cleavage, therefore potentially exhibiting activity after systemic administration. We hypothesized that the macrocyclic κ opioid receptor (KOR)-selective antagonist [D-Trp]CJ-15,208 would demonstrate antagonist activity after systemic, that is, s.c. and oral (per os, p. o.), administration. EXPERIMENTAL APPROACH: C57BL/6J mice were pretreated with [D-Trp]CJ-15,208 s.c. or p.o. before administration of the KOR-selective agonist U50,488 and the determination of antinociception in the warm-water tail-withdrawal assay. The locomotor activity of mice treated with [D-Trp]CJ-15,208 was determined by rotorod testing. Additional mice demonstrating cocaine conditioned place preference and subsequent extinction were pretreated daily with vehicle or [D-Trp]CJ-15,208 and then exposed to repeated forced swim stress or a single additional session of cocaine place conditioning before redetermining place preference. KEY RESULTS: Pretreatment with [D-Trp]CJ-15,208 administered s.c. or p.o. dose-dependently antagonized the antinociception induced by i.p. administration of U50,488 in mice tested in the warm-water tail-withdrawal assay for less than 12 and 6 h respectively. [D-Trp]CJ-15,208 also produced limited (<25%), short-duration antinociception mediated through KOR agonism. Orally administered [D-Trp]CJ-15,208 dose-dependently antagonized centrally administered U50,488-induced antinociception, and prevented stress-, but not cocaine-induced, reinstatement of extinguished cocaine-seeking behaviour, consistent with its KOR antagonist activity, without affecting locomotor activity. CONCLUSIONS AND IMPLICATIONS: The macrocyclic tetrapeptide [D-Trp]CJ-15,208 is a short-duration KOR antagonist with weak KOR agonist activity that is active after oral administration and demonstrates blood-brain barrier permeability. These data validate the use of systemically active peptides such as [D-Trp]CJ-15,208 as potentially useful therapeutics.

Discovery of novel antinociceptive α-conotoxin analogues from the direct in vivo screening of a synthetic mixture-based combinatorial library.

Marine cone snail venoms consist of large, naturally occurring combinatorial libraries of disulfide-constrained peptide neurotoxins known as conotoxins, which have profound potential in the development of analgesics. In this study, we report a synthetic combinatorial strategy that probes the hypervariable regions of conotoxin frameworks to discover novel analgesic agents by utilizing high diversity mixture-based positional-scanning synthetic combinatorial libraries (PS-SCLs). We hypothesized that the direct in vivo testing of these mixture-based combinatorial library samples during the discovery phase would facilitate the identification of novel individual compounds with desirable antinociceptive profiles while simultaneously eliminating many compounds with poor activity or liabilities of locomotion and respiration. A PS-SCL was designed based on the α-conotoxin RgIA-ΔR n-loop region and consisted of 10,648 compounds systematically arranged into 66 mixture samples. Mixtures were directly screened in vivo using the mouse 55 °C warm-water tail-withdrawal assay, which allowed deconvolution of amino acid residues at each position that confer antinociceptive activity. A second generation library of 36 individual α-conotoxin analogues was synthesized using systematic combinations of amino acids identified from PS-SCL deconvolution and further screened for antinociceptive activity. Six individual analogues exhibited comparable antinociceptive activity to that of the recognized analgesic α-conotoxin RgIA-ΔR, and were selected for further examination of antinociceptive, respiratory, and locomotor effects. Three lead compounds were identified that produced dose-dependent antinociception without significant respiratory depression or decreased locomotor activity. Our results represent a unique approach for rapidly developing novel lead α-conotoxin analogues as low-liability analgesics with promising therapeutic potential.

Central administration of angiotensin IV rapidly enhances novel object recognition among mice.

Angiotensin IV (Val(1)-Tyr(2)-Ile(3)-His(4)-Pro(5)-Phe(6)) has demonstrated potential cognitive-enhancing effects. The present investigation assessed and characterized: (1) dose-dependency of angiotensin IV's cognitive enhancement in a C57BL/6J mouse model of novel object recognition, (2) the time-course for these effects, (3) the identity of residues in the hexapeptide important to these effects and (4) the necessity of actions at angiotensin IV receptors for procognitive activity. Assessment of C57BL/6J mice in a novel object recognition task demonstrated that prior administration of angiotensin IV (0.1, 1.0, or 10.0, but not 0.01 nmol, i.c.v.) significantly enhanced novel object recognition in a dose-dependent manner. These effects were time dependent, with improved novel object recognition observed when angiotensin IV (0.1 nmol, i.c.v.) was administered 10 or 20, but not 30 min prior to the onset of the novel object recognition testing. An alanine scan of the angiotensin IV peptide revealed that replacement of the Val(1), Ile(3), His(4), or Phe(6) residues with Ala attenuated peptide-induced improvements in novel object recognition, whereas Tyr(2) or Pro(5) replacement did not significantly affect performance. Administration of the angiotensin IV receptor antagonist, divalinal-Ang IV (20 nmol, i.c.v.), reduced (but did not abolish) novel object recognition; however, this antagonist completely blocked the procognitive effects of angiotensin IV (0.1 nmol, i.c.v.) in this task. Rotorod testing demonstrated no locomotor effects with any angiotensin IV or divalinal-Ang IV dose tested. These data demonstrate that angiotensin IV produces a rapid enhancement of associative learning and memory performance in a mouse model that was dependent on the angiotensin IV receptor.

Inhibition of Gßγ-subunit signaling potentiates morphine-induced antinociception but not respiratory depression, constipation, locomotion, and reward.

Inhibition of Gßγ-subunit signaling to phospholipase C ß3 has been shown to potentiate morphine-mediated antinociception while attenuating the development of tolerance and dependence in mice. The objective of this study was to determine the effect of Gßγ-subunit inhibition on antinociception and other pharmacological effects, such as respiratory depression, constipation, and hyperlocomotion, mediated by the µ-opioid receptor. The Gßγ-subunit inhibitor, gallein, was administered to C57BL/6J mice by intraperitoneal injection before morphine, and data were compared with mice treated with vehicle, morphine, or gallein alone. Morphine-induced antinociception was measured using the 55°C warm-water tail-withdrawal test. Pretreatment with gallein produced a dose-dependent potentiation of morphine-mediated antinociception, producing up to a 10-fold leftward shift in the morphine dose-response curve and extending the duration of antinociception induced by a single dose of morphine. Gallein pretreatment also prevented acute antinociceptive tolerance induced by morphine. In contrast, the dose-dependent respiratory depression and hyperlocomotion induced by morphine were not potentiated by gallein pretreatment. Similarly, gallein pretreatment did not potentiate morphine-conditioned place preference responses or morphine-induced constipation, as measured as a reduction in excreta. These results suggest that selectively inhibiting Gßγ-mediated signaling may selectively increase µ-opioid receptor-mediated antinociception without matching increases in adverse physiological effects.

Novel opioid cyclic tetrapeptides: Trp isomers of CJ-15,208 exhibit distinct opioid receptor agonism and short-acting κ opioid receptor antagonism.

BACKGROUND AND PURPOSE: The κ opioid receptor antagonists demonstrate potential for maintaining abstinence from psychostimulant abuse, but existing non-peptide κ-receptor selective antagonists show exceptionally long activity. We hypothesized that the L- and D-Trp isomers of CJ-15,208, a natural cyclic tetrapeptide reported to be a κ-receptor antagonist in vitro, would demonstrate short-acting, dose-dependent antagonism in vivo, preventing reinstatement of cocaine-seeking behaviour. EXPERIMENTAL APPROACH: Affinity, selectivity and efficacy of the L-Trp and D-Trp isomers for opioid receptors were assessed in vitro in radioligand and GTPγS binding assays. Opioid receptor agonist and antagonist activities were characterized in vivo following i.c.v. administration with the 55°C warm water tail-withdrawal assay. The D-Trp isomer, which demonstrated primarily κ-receptor selective antagonist activity, was further evaluated for its prevention of stress- and drug-induced reinstatement of extinguished cocaine conditioned place preference (CPP). KEY RESULTS: The two isomers showed similar affinity and selectivity for κ receptors (K(i) 30-35 nM) as well as κ receptor antagonism in vitro. As expected, the D-Trp cyclic tetrapeptide exhibited minimal agonist activity and induced dose-dependent κ-receptor selective antagonism lasting less than 18 h in vivo. Pretreatment with this peptide prevented stress-, but not cocaine-induced, reinstatement of extinguished cocaine CPP. In contrast, the L-Trp cyclic tetrapeptide unexpectedly demonstrated mixed opioid agonist/antagonist activity. CONCLUSIONS AND IMPLICATIONS: The L-Trp and the D-Trp isomers of CJ-15,208 demonstrate stereospecific opioid activity in vivo. The relatively brief κ opioid receptor antagonism, coupled with the prevention of stress-induced reinstatement of extinguished cocaine-seeking behaviour, suggests the D-Trp isomer could be used therapeutically to maintain abstinence from psychostimulant abuse.

Unexpected opioid activity profiles of analogues of the novel peptide kappa opioid receptor ligand CJ-15,208.

An alanine scan was performed on the novel κ opioid receptor (KOR) peptide ligand CJ-15,208 to determine which residues contribute to the potent in vivo agonist activity observed for the parent peptide. These cyclic tetrapeptides were synthesized by a combination of solid-phase peptide synthesis of the linear precursors, followed by cyclization in solution. Like the parent peptide, each of the analogues exhibited agonist activity and KOR antagonist activity in an antinociceptive assay in vivo. Unlike the parent peptide, the agonist activity of the potent analogues was mediated predominantly, if not exclusively, by µâ€…opioid receptors (MOR). Thus analogues 2 and 4, in which one of the phenylalanine residues was replaced by alanine, exhibited both potent MOR agonist activity and KOR antagonist activity in vivo. These peptides represent novel lead compounds for the development of peptide-based opioid analgesics.

Kappa Opioid Receptor-Mediated Disruption of Novel Object Recognition: Relevance for Psychostimulant Treatment.

Kappa opioid receptor (KOR) agonists are potentially valuable as therapeutics for the treatment of psychostimulant reward as they suppress dopamine signaling in reward circuitry to repress drug seeking behavior. However, KOR agonists are also associated with sedation and cognitive dysfunction. The extent to which learning and memory disruption or hypolocomotion underlie KOR agonists' role in counteracting the rewarding effects of psychostimulants is of interest. C57BL/6J mice were pretreated with vehicle (saline, 0.9%), the KOR agonist (trans)-3,4-dichloro-N-methyl-N-[2-(1- pyrrolidinyl)-cyclohexyl] benzeneacetamide (U50,488), or the peripherally-restricted agonist D-Phe-D-Phe-D-lle-D-Arg- NH(2) (ffir-NH(2)), through central (i.c.v.) or peripheral (i.p.) routes of administration. Locomotor activity was assessed via activity monitoring chambers and rotorod. Cognitive performance was assessed in a novel object recognition task. Prolonged hypolocomotion was observed following administration of 1.0 and 10.0, but not 0.3 mg/kg U50,488. Central, but not peripheral, administration of ffir-NH(2) (a KOR agonist that does not cross the blood-brain barrier) also reduced motor behavior. Systemic pretreatment with the low dose of U50,488 (0.3 mg/kg, i.p.) significantly impaired performance in the novel object recognition task. Likewise, ffir-NH(2) significantly reduced novel object recognition after central (i.c.v.), but not peripheral (i.p.), administration. U50,488- and ffir-NH(2)-mediated deficits in novel object recognition were prevented by pretreatment with KOR antagonists. Cocaine-induced conditioned place preference was subsequently assessed and was reduced by pretreatment with U50,488 (0.3 mg/kg, i.p.). Together, these results suggest that the activation of centrally-located kappa opioid receptors may induce cognitive and mnemonic disruption independent of hypolocomotor effects which may contribute to the KOR-mediated suppression of psychostimulant reward.

Identification of two novel, potent, low-liability antinociceptive compounds from the direct in vivo screening of a large mixture-based combinatorial library.

Synthetic combinatorial methods now make it practical to readily produce hundreds of thousands of individual compounds, but it is clearly impractical to screen each separately in vivo. We theorized that the direct in vivo testing of mixture-based combinatorial libraries during the discovery phase would enable the identification of novel individual compounds with desirable antinociceptive profiles while simultaneously eliminating many compounds with poor absorption, distribution, metabolism, or pharmacokinetic properties. The TPI 1346 small-molecule combinatorial library is grouped in 120 mixtures derived from 26 functionalities at the first three positions and 42 functionalities at the fourth position of a pyrrolidine bis-cyclic guanidine core scaffold, totaling 738,192 compounds. These 120 mixtures were screened in vivo using the mouse 55 degrees C warm water tail-withdrawal assay to identify mixtures producing antinociception. From these data, two fully defined individual compounds (TPI 1818-101 and TPI 1818-109) were synthesized. These were examined for antinociceptive, respiratory, locomotor, and conditioned place preference effects. The tail-withdrawal assay consistently demonstrated distinctly active mixtures with analgesic activity that was blocked by pretreatment with the non-selective opioid antagonist, naloxone. Based on these results, synthesis and testing of TPI 1818-101 and 1818-109 demonstrated a dose-dependent antinociceptive effect three to five times greater than morphine that was antagonized by mu- or mu- and kappa-opioid receptor selective antagonists, respectively. Neither 1818-101 nor 1818-109 produced significant respiratory depression, hyperlocomotion, or conditioned place preference. Large, highly diverse mixture-based libraries can be screened directly in vivo to identify individual compounds, potentially accelerating the development of promising therapeutics.