Sigma receptors modulate nociception, offering a potential therapeutic target to treat pain, but relatively little is known regarding the role of sigma-2 receptors (S2R) in nociception. The purpose of this study was to investigate the in vivo analgesic and anti-allodynic activity and liabilities of a novel S2R selective ligand, 1-[4-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolin-2-yl)butyl]-3-methyl-1,3-dihydro-1,3-benzimidazol-2-one (CM-398). The inhibition of thermal, induced chemical, or inflammatory pain as well as the allodynia resulting from chronic nerve constriction injury (CCI) model of neuropathic pain were assessed in male mice. CM-398 dose-dependently (10–45 mg/kg i.p.) reduced mechanical allodynia in the CCI neuropathic pain model, equivalent at the higher dose to the effect of the control analgesic gabapentin (50 mg/kg i.p.). Likewise, pretreatment (i.p.) with CM-398 dose-dependently produced antinociception in the acetic acid writhing test (ED₅₀ (and 95% C.I.) = 14.7 (10.6–20) mg/kg, i.p.) and the formalin assay (ED₅₀ (and 95% C.I.) = 0.86 (0.44–1.81) mg/kg, i.p.) but was without effect in the 55 °C warm-water tail-withdrawal assay. A high dose of CM-398 (45 mg/kg, i.p.) exhibited modest locomotor impairment in a rotarod assay and conditioned place aversion, potentially complicating the interpretation of nociceptive testing. However, in an operant pain model resistant to these confounds, mice experiencing CCI and treated with CM-398 demonstrated robust conditioned place preference. Overall, these results demonstrate the S2R selective antagonist CM-398 produces antinociception and anti-allodynia with fewer liabilities than established therapeutics, adding to emerging data suggesting possible mediation of nociception by S2R, and the development of S2R ligands as potential treatments for chronic pain.

The macrocyclic tetrapeptide cyclo[Phe-d-Pro-Phe-Trp] (CJ-15,208) and its stereoisomer cyclo[Phe-d-Pro-Phe-d-Trp] exhibit different opioid activity profiles in vivo. The present study evaluated the influence of the Phe residues’ stereochemistry on the peptides’ opioid activity. Five stereoisomers were synthesized by a combination of solid-phase peptide synthesis and cyclization in solution. The analogs were evaluated in vitro for opioid receptor affinity in radioligand competition binding assays, and for opioid activity and selectivity in vivo in the mouse 55 °C warm-water tail-withdrawal assay. Potential liabilities of locomotor impairment, respiratory depression, acute tolerance development, and place conditioning were also assessed in vivo. All of the stereoisomers exhibited antinociception following either intracerebroventricular or oral administration differentially mediated by multiple opioid receptors, with kappa opioid receptor (KOR) activity contributing for all of the peptides. However, unlike the parent peptides, KOR antagonism was exhibited by only one stereoisomer, while another isomer produced DOR antagonism. The stereoisomers of CJ-15,208 lacked significant respiratory effects, while the [d-Trp]CJ-15,208 stereoisomers did not elicit antinociceptive tolerance. Two isomers, cyclo[d-Phe-d-Pro-d-Phe-Trp] (3) and cyclo[Phe-d-Pro-d-Phe-d-Trp] (5), did not elicit either preference or aversion in a conditioned place preference assay. Collectively, these stereoisomers represent new lead compounds for further investigation in the development of safer opioid analgesics.

Neuropathic pain is a significant problem with few effective treatments lacking adverse effects. The sigma-1 receptor (S1R) is a potential therapeutic target for neuropathic pain, as antagonists for this receptor effectively ameliorate pain in both preclinical and clinical studies. The current research examines the antinociceptive and anti-allodynic efficacy of SI 1/28, a recently reported benzylpiperazine derivative and analog of the S1R antagonist SI 1/13, that was 423-fold more selective for S1R over the sigma-2 receptor (S2R). In addition, possible liabilities of respiration, sedation, and drug reinforcement caused by SI 1/28 have been evaluated. Inflammatory and chemical nociception, chronic nerve constriction injury (CCI) induced mechanical allodynia, and adverse effects of sedation in a rotarod assay, conditioned place preference (CPP), and changes in breath rate and locomotor activity were assessed after i.p. administration of SI 1/28. Pretreatment with SI 1/28 produced dose-dependent antinociception in the formalin test, with an ED₅₀ (and 95% C.I.) value of 13.2 (7.42–28.3) mg/kg, i.p. Likewise, SI 1/28 produced dose-dependent antinociception against visceral nociception and anti-allodynia against CCI-induced neuropathic pain. SI 1/28 demonstrated no impairment of locomotor activity, conditioned place preference, or respiratory depression. In summary, SI 1/28 proved efficacious in the treatment of acute inflammatory pain and chronic neuropathy without liabilities at therapeutic doses, supporting the development of S1R antagonists as therapeutics for chronic pain.

New strategies facilitate the design of cyclic peptides which can penetrate the brain. We have designed a bicyclic peptide, OL-CTOP, composed of the sequences of a selective μ-opioid receptor antagonist, CTOP (f-cyclo(CYwOTX)T) (X = penicillamine, Pen; O = ornithine) and odorranalectin, OL (YASPK-cyclo(CFRYPNGVLAC)T), optimized its solid-phase synthesis and demonstrated its ability for nose-to-brain delivery and in vivo activity. The differences in reactivity of Cys and Pen thiol groups protected with trityl and/or acetamidomethyl protecting groups toward I₂ in different solvents were exploited for selective disulfide bond formation on the solid phase. Both the single step and the sequential strategy applied to macrocyclization reactions generated the desired OL-CTOP, with the sequential strategy yielding a large quantity and better purity of crude OL-CTOP. Importantly, intranasally (i.n.s.) administered OL-CTOP dose-dependently antagonized the analgesic effect of morphine administered to mice through the intracerebroventricular route and prevented morphine-induced respiratory depression. In summary, the results demonstrate the feasibility of our solid-phase synthetic strategy for the preparation of the OL-CTOP bicyclic peptide containing two disulfide bonds and reveal the potential of odorranalectin for further modifications and the targeted delivery to the brain.

Peptide-based opioid ligands are important candidates for the development of novel, safer, and more effective analgesics to treat pain. To develop peptide-based safer analgesics, we synthesized a mixture-based cyclic pentapeptide library containing a total of 24,624 pentapeptides and screened the mixture-based library samples using a 55 °C warm water tail-withdrawal assay. Using this phenotypic screening approach, we deconvoluted the mixture-based samples to identify a novel cyclic peptide Tyr-[D-Lys-Dap(Ant)-Thr-Gly] (CycloAnt), which produced dose- and time-dependent antinociception with an ED₅₀ (and 95% confidence interval) of 0.70 (0.52–0.97) mg/kg i.p. mediated by the mu-opioid receptor (MOR). Additionally, higher doses (≥3 mg/kg, i.p.) of CycloAnt antagonized delta-opioid receptors (DOR) for at least 3 h. Pharmacological characterization of CycloAnt showed the cyclic peptide did not reduce breathing rate in mice at doses up to 15 times the analgesic ED₅₀ value, and produced dramatically less hyperlocomotion than the MOR agonist, morphine. While chronic administration of CycloAnt resulted in antinociceptive tolerance, it was without opioid-induced hyperalgesia and with significantly reduced signs of naloxone-precipitated withdrawal, which suggested reduced physical dependence compared to morphine. Collectively, the results suggest this dual MOR/DOR multifunctional ligand is an excellent lead for the development of peptide-based safer analgesics.

The macrocyclic tetrapeptide CJ-15,208 (cyclo[Phe-D-Pro-Phe-Trp]) and its D-Trp isomer exhibit kappa opioid receptor (KOR) antagonism which prevents stress-induced reinstatement of extinguished cocaine-conditioned place preference. Here, we evaluated the effects of substitution of Trp and D-Trp on the peptides’ opioid activity, antinociceptive tolerance, and the ability to prevent relapse to extinguished drug-CPP. Six analogs were synthesized using a combination of solid-phase peptide synthesis and cyclization in solution. The analogs were evaluated in vitro for opioid receptor affinity in radioligand competition binding assays, efficacy in the [³⁵S]GTPγS assay, metabolic stability in mouse liver microsomes, and for opioid activity and selectivity in vivo in the mouse 55 °C warm-water tail-withdrawal assay. Potential liabilities of locomotor impairment, respiratory depression, acute tolerance, and conditioned place preference (CPP) were also assessed in vivo, and the ameliorating effect of analogs on the reinstatement of extinguished cocaine-place preference was assessed. Substitutions of other D-amino acids for D-Trp did not affect (or in one case increased) KOR affinity, while two of the three substitutions of an L-amino acid for Trp decreased KOR affinity. In contrast, all but one substitution increased mu opioid receptor (MOR) affinity in vitro. The metabolic stabilities of the analogs were similar to those of their respective parent peptides, with analogs containing a D-amino acid being much more rapidly metabolized than those containing an L-amino acid in this position. In vivo, CJ-15,208 analogs demonstrated antinociception, although potencies varied over an 80-fold range and the mediating opioid receptors differed by substitution. KOR antagonism was lost for all but the D-benzothienylalanine analog, and the 2′-naphthylalanine analog instead demonstrated significant delta opioid receptor (DOR) antagonism. Introduction of DOR antagonism coincided with reduced acute opioid antinociceptive tolerance and prevented stress-induced reinstatement of extinguished cocaine-CPP.