Developing Burrowing As A Non-Evoked Readout Assessment For Novel Analgesic Drug Efficacy

Traditional drug development efforts to diminish the problem of chronic pain rely heavily on pain-evoked assays that can yield false positives, leading to confounding assessments of novel drugs. To avoid this, we are looking at burrowing as a novel readout assessment of analgesic efficacy. Preliminary data shows that induction of post-surgical pain reduces this innate behavior by about 35%. In attempt to increase this number, we tested a variety of additional parameters. Blue pads, placed at the bottom of each chamber in order to reduce the sound made when gravel is displaced, increased baseline amounts roughly 40%. Conversely, neither sex of the animal, nor time of day the experiment was performed was found to have an affect on the amount burrowed. Further optimizations of this assay look at habituation and test session length. We found that a 5-day habituation regiment could not be shortened, but a test session length of 15min produced comparable results to the original 60min session. Current studies are aimed at using HD webcams to quantify additional behaviors such as time spent burrowing and time spent in the tube. Ultimately, these studies could lead to the development of a beneficial assay for use in assessing novel analgesic drugs.https://dune.une.edu/neuro_studpost/1000/thumbnail.jp

Identification of a Novel “Almost Neutral” Mu Opioid Receptor Antagonist in CHO Cells Expressing the Cloned Human Mu Opioid Receptor

The basal (constitutive) activity of G protein-coupled receptors allows for the measurement of inverse agonist activity. Some competitive antagonists turn into inverse agonists under conditions where receptors are constitutively active. In contrast, neutral antagonists have no inverse agonist activity, and they block both agonist and inverse agonist activity. The mu opioid receptor (MOR) demonstrates detectable constitutive activity only after a state of dependence is produced by chronic treatment with a MOR agonist. We therefore sought to identify novel MOR inverse agonists, and novel neutral MOR antagonists in both untreated and agonist-treated MOR cells. CHO cells expressing the cloned human mu receptor (hMOR-CHO cells) were incubated for 20 hr with medium (control) or 10 μM (2S,4aR,6aR,7R,9S,10aS,10bR)-9-(benzoyloxy)-2-(3-furanyl)dodecahydro-6a,10b-dimethyl-4,10-dioxo-2H-naphtho-[2,1-c]pyran-7-carboxylic acid methyl ester (herkinorin, HERK). HERK-treatment generates a high degree of basal signaling and enhances the ability to detect inverse agonists. [35S]-GTP-γ-S assays were conducted using established methods. We screened 21 MOR “antagonists” using membranes prepared from HERK-treated hMOR-CHO cells. All antagonists, including CTAP and 6β-naltrexol, were inverse agonists. However, LTC-2 7 4 ( (-)-3-cyclopropylmethyl-2,3,4,4aα,5,6,7,7aα-octahydro-1H-benzofuro[3,2-e]isoquinolin-9-ol)) showed the lowest efficacy as an inverse agonist, and, at concentrations less than 5 nM, had minimal effects on basal [35S]-GTP-γ-S binding. Other efforts in this study identified KC-2-009 ((+)-3-((1R,5S)-2-((Z)-3-Phenylallyl)-2-azabicyclo[3.3.1]nonan-5-yl)phenol hydrochloride) as an inverse agonist at untreated MOR cells. In HERK-treated cells, KC-2-009 had the highest efficacy as an inverse agonist. In summary, we identified a novel and selective MOR inverse agonist (KC-2-009), and a novel MOR antagonist (LTC-274) that shows the least inverse agonist activity among 21 MOR antagonists. LTC-274 is a promising lead compound for developing a true MOR neutral antagonist

Behavioral pharmacology of the mixed-action delta-selective opioid receptor agonist BBI-11008: studies on acute, inflammatory and neuropathic pain, respiration, and drug self-administration

BBI-11008 had a 78-fold greater affinity for the delta opioid receptor than the mu receptor, and there was no binding to the kappa opioid receptor. BBI-11008 (3.2-100; 10-32 mg kg-1, i.v.) and morphine (1-10; 1-3.2 mg kg-1, i.v.) produced antinociceptive and anti-allodynic effects in assays of acute thermal nociception and complete Freund's adjuvant (CFA)-induced inflammatory pain, with BBI-11008 being less potent than morphine in both assays. BBI-11008 (1-18 mg kg-1, i.v.) had similar efficacy to gabapentin (10-56 mg kg-1, i.v.) in a spinal nerve ligation (SNL) model of neuropathic pain. In the respiration assay, with increasing %CO2 exposure, BBI-11008 produced an initial increase (32 mg kg-1, s.c.) and then decrease (56 mg kg-1, s.c.) in minute volume (MV) whereas morphine (3.2-32 mg kg-1, s.c.) produced dose-dependent decreases in MV. In the drug self-administration procedure, BBI-11008 did not maintain self-administration at any dose tested.12 month embargo; published online: 7 January 2020This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Can Amphipathic Helices Influence the CNS Antinociceptive Activity of Glycopeptides Related to β‑Endorphin?

Glycosylated β-endorphin analogues of various amphipathicity were studied in vitro and in vivo in mice. Opioid binding affinities of the O-linked glycopeptides (mono- or disaccharides) and unglycosylated peptide controls were measured in human receptors expressed in CHO cells. All were pan-agonists, binding to μ-, δ-, or κ-opioid receptors in the low nanomolar range (2.2–35 nM <i>K</i>_i’s). The glycoside moiety was required for intravenous (i.v.) but not for intracerebroventricular (i.c.v.) activity. Circular dichroism and NMR indicated the degree of helicity in H₂O, aqueous trifluoroethanol, or micelles. Glycosylation was essential for activity after i.v. administration. It was possible to manipulate the degree of helicity by the alteration of only two amino acid residues in the helical <i>address</i> region of the β-endorphin analogues without destroying μ-, δ-, or κ-agonism, but the antinociceptive activity after i.v. administration could not be directly correlated to the degree of helicity in micelles

Structural Requirements for CNS Active Opioid Glycopeptides

Glycopeptides related to β-endorphin penetrate the blood–brain barrier (BBB) of mice to produce antinociception. Two series of glycopeptides were assessed for opioid receptor binding affinity. Attempts to alter the mu-selectivity of [d-Ala²,<i>N</i>-MePhe⁴,Gly-ol⁵]­enkephalin (DAMGO)-related glycopeptides by altering the charged residues of the amphipathic helical address were unsuccessful. A series of pan-agonists was evaluated for antinociceptive activity (55 °C tail flick) in mice. A flexible linker was required to maintain antinociceptive activity. Circular dichroism (CD) in H₂O, trifluoroethanol (TFE), and SDS micelles confirmed the importance of the amphipathic helices (<b>11s</b> → <b>11sG</b> → <b>11</b>) for antinociception. The glycosylated analogues showed only nascent helices and random coil conformations in H₂O. Chemical shift indices (CSI) and nuclear Overhauser effects (NOE) with 600 MHz NMR and CD confirmed helical structures in micelles, which were rationalized by molecular dynamics calculations. Antinociceptive studies with mice confirm that these glycosylated endorphin analogues are potential drug candidates that penetrate the BBB to produce potent central effects