Rank Order Of Potency Research Articles

Oxycodone-like discriminative stimulus effects of fentanyl-related emerging drugs of abuse in mice

BackgroundFentanyl and its structurally related compounds have emerged as the most significant contributors to opioid overdose fatalities in recent years. While there is abundant information about the pharmacological effects of fentanyl, far less is known of its more recently abused analogs. The objective of this study was to determine whether fentanyl and several fentanyl-related substances would engender oxycodone-like responding in a mouse model of oxycodone discrimination. Oxycodone was selected as the training drug due to its high selectivity for mu opioid receptors. Compounds that elicited oxycodone-like responding in this procedure would likely evoke overlapping subjective experiences. MethodsAdult male C57BL/6 mice were trained to discriminate 1.3 mg/kg oxycodone from vehicle in a food-reinforced, two-lever choice procedure. Generalization tests were conducted with fentanyl and the following fentanyl-related compounds: ocfentanil, 3-furanyl fentanyl, crotonylfentanyl, and valerylfentanyl. ResultsFentanyl and each of its analogs completely generalized to the 1.3 mg/kg oxycodone discriminative stimulus and naltrexone pretreatment significantly decreased oxycodone-like responding for each compound. Rank order potency for engendering oxycodone-appropriate responding was ocfentanil > fentanyl > 3-furanyl fentanyl ≈ crotonylfentanyl > oxycodone > valerylfentanyl. Drug doses that evoked full substitution also significantly suppressed response rates compared to vehicle. ConclusionsThese results indicate that the discriminative stimulus, and by extension, the interoceptive and subjective effects of the tested fentanyl analogs, overlap with those of oxycodone. These observations consequentially support the prediction that they would also engender the likelihood for abuse similar to oxycodone.This article is part of the Special Issue entitled ‘New Vistas in Opioid Pharmacology’.

Naphthenic Acid Mixtures and Acid-Extractable Organics from Oil Sands Process-Affected Water Impair Embryonic Development of Silurana (Xenopus) tropicalis.

Naphthenic acids (NAs) are carboxylic acids naturally occurring in crude oils and bitumen and are suspected to be the primary toxic substances in wastewaters associated with oil refineries and mining of oil sands. Oil sands process-affected water (OSPW) generated by the extraction of bitumen from oil sands are a major source of NAs and are currently stored in tailings ponds. We report on the acute lethality and teratogenic effects of aquatic exposure of Silurana (Xenopus) tropicalis embryos to commercial NA extracts and from the acid extractable organics (AEOs) fraction of a Canadian OSPW. Using electrospray ionization-high resolution mass spectrometry, we determined that the O2 species proportion were 98.8, 98.9 and 58.6% for commercial mixtures Sigma 1 (S1M) and Sigma 2 (S2M), and AEOs, respectively. The 96h LC50 estimates were 10.4, 11.7, and 52.3 mg/L for S1M, S2M, and the AEOs, respectively. The 96h EC50 estimates based on frequencies of developmental abnormalities were 2.1, 2.6, and 14.2 mg/L for S1M, S2M, and the AEOs, respectively. The main effects observed were reduced body size, edema, and cranial, heart, gut and ocular abnormalities. Increasing concentrations of the mixtures resulted in increased severity and frequency of abnormalities ( p < 0.05). The rank-order potency was S1M > S2M > AEO based on LC50 and EC50 estimates. These data provide insight into the effects NAs in amphibian embryos and can contribute to the development of environmental guidelines for the management of OSPW.

Serotonin enhances oxybuprocaine- and proxymetacaine-induced cutaneous analgesia in rats

The aim of the study was to investigate the analgesic effects of adding serotonin to oxybuprocaine or proxymetacaine preparations. We employed a rat model of the cutaneous trunci muscle reflex (CTMR) to conduct the dose-response curves and duration of drugs (oxybuprocaine, proxymetacaine, or serotonin) as an infiltrative anesthetic. The use of isobolographic methods to analyze the drug-drug interactions. We showed that oxybuprocaine and proxymetacaine, as well as serotonin produced dose-dependent skin antinociception. On the basis of 50% effective dose (ED50), the rank order of drug potency was serotonin [7.22 (6.45–8.09) μmol/kg] < oxybuprocaine [1.03 (0.93–1.15) μmol/kg] < proxymetacaine [0.59 (0.53–0.66) μmol/kg] (P < 0.01 for each comparison). The sensory block duration of serotonin was longer (P < 0.01) than that of oxybuprocaine or proxymetacaine at the equipotent doses (ED25, ED50, and ED75). The mixture of serotonin with oxybuprocaine or proxymetacaine produced a better analgesic effect than the drug itself. We have concluded that oxybuprocaine, proxymetacaine, or serotonin displays dose-related cutaneous analgesia. Oxybuprocaine or proxymetacaine is more potent and has a shorter duration of cutaneous analgesia than serotonin. Serotonin produces a synergistic antinociceptive interaction with oxybuprocaine or proxymetacaine.

Spinal sensory and motor blockade by intrathecal doxylamine and triprolidine in rats.

The aim of this experiment was mainly to examine the effects of intrathecally injected doxylamine and triprolidine, two antihistamine drugs spinal motor and sensory functions. After intrathecally injecting the rats with five different doses, the dose-response curves of spinal sensory and motor block with doxylamine and triprolidine were constructed. In comparison with the local anaesthetic mepivacaine, the quality and duration of spinal anaesthesia with doxylamine or triprolidine were conducted. Doxylamine, mepivacaine and triprolidine elicited spinal motor and sensory (nociception and proprioception) blockades in a dose-dependent fashion. On the ED50 (50% effective dose) basis, the rank order of drug potency was triprolidine>mepivacaine>doxylamine (P<0.05) at provoking spinal motor, proprioceptive and nociceptive blockades. On the equianaesthetic doses (ED25 , ED50 and ED75 ), the duration of spinal anaesthesia with doxylamine was longer (P<0.01) than that with mepivacaine or triprolidine. Moreover, doxylamine or triprolidine displayed greater potency (ED50 ) (P<0.05) and duration (P<0.05) of sensory block over motor block. Doxylamine or triprolidine produces a dose-dependent effect of spinal motor and sensory block. Triprolidine with a better nociception-selective actionover motor block has a better potency than mepivacaine or doxylamine. Doxylamine and triprolidine produce longer durations than mepivacaine.

Lanosterol Synthase Regulates Human Rhinovirus Replication in Human Bronchial Epithelial Cells.

Human rhinovirus (RV) infections are a significant risk factor for exacerbations of asthma and chronic obstructive pulmonary disease. Thus, approaches to prevent RV infection in such patients would give significant benefit. Through RNA interference library screening, we identified lanosterol synthase (LSS), a component of the cholesterol biosynthetic pathway, as a novel regulator of RV replication in primary normal human bronchial epithelial cells. Selective knock down of LSS mRNA with short interfering RNA inhibited RV2 replication in normal human bronchial epithelial cells. Small molecule inhibitors of LSS mimicked the effect of LSS mRNA knockdown in a concentration-dependent manner. We further demonstrated that the antiviral effect is not dependent on a reduction in total cellular cholesterol but requires a 24-hour preincubation with the LSS inhibitor. The rank order of antiviral potency of the LSS inhibitors used was consistent with LSS inhibition potency; however, all compounds showed remarkably higher potency against RV compared with the LSS enzyme potency. We showed that LSS inhibition led to an induction of 24(S),25 epoxycholesterol, an important regulator of the sterol pathway. We also demonstrated that LSS inhibition led to a profound increase in expression of the innate antiviral defense protein, IFN-β. We found LSS to be a novel regulator of RV replication and innate antiviral immunity and identified a potential molecular mechanism for this effect, via induction of 24(S),25 epoxycholesterol. Inhibition of LSS could therefore be a novel therapeutic target for prevention of RV-induced exacerbations.

NOP receptor pharmacological profile - A dynamic mass redistribution study.

The Nociceptin/Orphanin FQ (N/OFQ) peptide NOP receptor is coupled to pertussis toxin (PTX)-sensitive G proteins (Gi/o) whose activation leads to the inhibition of both cAMP production and calcium channel activity, and to the stimulation of potassium currents. The label free dynamic mass redistribution (DMR) approach has been demonstrated useful for investigating the pharmacological profile of G protein-coupled receptors. Herein, we employ DMR technology to systematically characterize the pharmacology of a large panel of NOP receptor ligands. These are of peptide and non-peptide nature and display varying degrees of receptor efficacy, ranging from full agonism to pure antagonism. Using Chinese hamster ovary (CHO) cells expressing the human NOP receptor we provide rank orders of potency for full and partial agonists as well as apparent affinities for selective antagonists. We find the pharmacological profile of NOP receptor ligands to be similar but not identical to values reported in the literature using canonical assays for Gi/o-coupled receptors. Our data demonstrate that holistic label-free DMR detection can be successfully used to investigate the pharmacology of the NOP receptor and to characterize the cellular effects of novel NOP receptor ligands.

GABA beyond the synapse: defining the subtype-specific pharmacodynamics of non-synaptic GABAA receptors.

Physiologically relevant combinations of recombinant GABAA receptor (GABAR) subunits were expressed in HEK293 cells. Using whole-cell voltage clamp and rapid drug application, we measured the GABAR-subtype-specific properties to convey either synaptic or extrasynaptic signalling in a range of physiological contexts. α4βδ GABARs are optimally tuned to submicromolar tonic GABA and transient surges of micromolar GABA concentrations. α5β2γ2l GABARs are better suited to higher tonic GABA levels, but also convey robust responses to brief synaptic and perisynaptic GABA fluctuations. α1β2/3δ GABARs function well at prolonged, micromolar (>2μm) GABA levels, but not to low tonic (<1μm GABA) or synaptic/transient GABAergic signalling. These results help illuminate the context- and isoform-specific modes of GABAergic signalling in the brain. GABAA receptors (GABARs) mediate a remarkable diversity of signalling modalities in vivo. Yet most published work characterizing responses to GABA has focused on the properties needed to convey fast, phasic synaptic inhibition. We therefore aimed to characterize the most prevalent (α4βδ, α5β3γ2L) and least prevalent (α1β2δ) non-synaptic GABAR currents, using whole-cell voltage clamp recordings of recombinant GABAR expressed in HEK293 cells and drug application protocols to recapitulate the GABA concentration profiles occurring during both fast synaptic and slow extrasynaptic signalling. We found that α4βδ GABARs were very sensitive to submicromolar GABA, with a rank order potency of α4β2δ ≥ α4β1δ ≈ α4β3δ GABARs. In comparison, the GABA EC50 was up to 20 times higher for α1β2γ2L GABARs, with α1β2δ and α5β3γ2L GABARs having intermediate GABA potency. Both α4βδ and α5β3γ2L GABAR currents exhibited slow, but substantial, desensitization as well as prolonged rates of deactivation. These GABAR current properties defined distinct 'dynamic ranges' of responsiveness to changing GABA for α4β2δ (0.1-1μm), α5β3γ2L (0.5-7μm) and α1β2γ2L (0.6-9μm) GABARs. Finally, α1β2δ GABARs were notable for their relative lack of desensitization and extremely quick deactivation. In summary, our results help delineate the roles that specific GABARs may play in mediating non-synaptic GABA signals. Since ambient GABA levels may be altered during development as well as by drugs and disease states, these findings may help future efforts to understand disrupted inhibition underlying a variety of neurological illnesses, such as epilepsy.

Real-Time Ligand Binding of Fluorescent VEGF-A Isoforms that Discriminate between VEGFR2 and NRP1 in Living Cells

SummaryFluorescent VEGF-A isoforms have been evaluated for their ability to discriminate between VEGFR2 and NRP1 in real-time ligand binding studies in live cells using BRET. To enable this, we synthesized single-site (N-terminal cysteine) labeled versions of VEGF165a, VEGF165b, and VEGF121a. These were used in combination with N-terminal NanoLuc-tagged VEGFR2 or NRP1 to evaluate the selectivity of VEGF isoforms for these two membrane proteins. All fluorescent VEGF-A isoforms displayed high affinity for VEGFR2. Only VEGF165a-TMR bound to NanoLuc-NRP1 with a similar high affinity (4.4 nM). Competition NRP1 binding experiments yielded a rank order of potency of VEGF165a > VEGF189a > VEGF145a. VEGF165b, VEGF-Ax, VEGF121a, and VEGF111a were unable to bind to NRP1. There were marked differences in the kinetic binding profiles of VEGF165a-TMR for NRP1 and VEGFR2. These data emphasize the importance of the kinetic aspects of ligand binding to VEGFR2 and its co-receptors in the dynamics of VEGF signaling.

Abstract 4877: Allosteric inhibition of SHP2 variants containing cancer-associated activating mutations

Abstract SHP2 (PTPN11) is a non-receptor protein tyrosine phosphatase and scaffold protein that functions in multiple signal transduction pathways. Genetic and pharmacologic evidence supports a role for SHP2 in driving the proliferation of cancer cells dependent upon a range of activated RTKs, certain RAS and BRAF mutations, and NF1 loss-of-function mutations. In addition, dominant activating mutations in PTPN11 fuel pathogenic RAS/MAPK signaling and underlie certain human RASopathies (40% of Noonan syndrome and 80% of LEOPARD syndrome) and cancers (35% of JMML and up to 5% of many other cancers). These mutants destabilize an autoinhibited conformation of SHP2, which can also be activated by binding to diphosphotyrosine motifs in vivo or to synthetic diphosphopeptides in vitro. Multiple selective, allosteric inhibitors that stabilize this autoinhibited state and potently inhibit wild-type (WT) SHP2 have recently been reported, but the impact of these inhibitors on mutant forms of the protein is less well established. Here we investigate the activity of a diverse set of over fifty allosteric SHP2 inhibitors on multiple cancer-associated activating mutants of SHP2, both in the presence and absence of an activating diphosphopeptide. The rank order of potencies of the inhibitors remains identical for all mutants, although a decrease in potency relative to WT is seen in some mutants, which is magnified further in the presence of activating peptide. These results conform to a simple equilibrium model, where inhibitors show a reduction in potency against activated mutants that is proportional to the energetic magnitude of the activating mutation. Surprisingly, some common activating mutations of SHP2 (e.g., D61G) have a relatively modest energetic effect, and exhibit similar sensitivity to allosteric inhibitors as WT SHP2. We follow up these biochemical results with cellular inhibition studies in HEK293 cells stably transfected with the respective SHP2 mutants. Collectively, these results suggest that potent allosteric SHP2 inhibitors will be effective at inhibiting the growth of cancers driven by a subset of activating mutations in SHP2. Citation Format: David Wildes, Naing Aay, Andreas Buckl, Daphne Hsieh, Ashutosh S. Jogalekar, Gert Kiss, Elena S. Koltun, Abby Marquez, Kevin T. Mellem, Jason Romero, Mae Saldajeno-Concar, Christopher J. Schulze, Chris M. Semko, Walter Won, Robert J. Nichols, Carlos Stahlhut, Christos Tzitzilonis, Adrian L. Gill, Jacqueline A. Smith. Allosteric inhibition of SHP2 variants containing cancer-associated activating mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4877.

In vivo profiling of four centrally administered opioids for antinociception, constipation and respiratory depression: Between-colony differences in Sprague Dawley rats.

Outbred rodent stocks including Sprague Dawley rats, are known for their genetic diversity and so they are often used to develop animal models of human disease. Although between-colony differences in pharmaco-behavioural studies have been published previously, a direct head-to-head comparison study, whereby all research was performed in the same laboratory by the same experimenter utilising the supraspinal route of drug administration in the same strain of rat, is lacking. Herein, we report our head-to-head comparison study, involving assessment of antinociception, constipation and respiratory depression evoked by single bolus intracerebroventricular (ICV) doses of morphine, buprenorphine, DPDPE and U69,593 using male Sprague Dawley rats sourced from a different breeding colony (BC2) from that (BC1) used by us previously. Our data show that there are marked differences in the potency rank order for morphine and buprenorphine between rats sourced from BC2 and BC1. Although ICV morphine evoked a bell-shaped dose-response curve in the constipation test for rats from both colonies, this occurred at higher doses for rats from BC2. In conclusion, our head-to-head comparison shows considerable between-colony differences for the same rat strain, in the potency rank order of two clinically important strong opioid analgesics given by the ICV route.

ApoE isoforms and carboxyl-terminal-truncated apoE4 forms affect neuronal BACE1 levels and Aβ production independently of their cholesterol efflux capacity.

The β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) initiates the production of amyloid-β peptide (Aβ), which is central to the pathogenesis of Alzheimer's disease (AD). Changes in brain cholesterol homeostasis have been suggested to affect Aβ metabolism. Cholesterol homeostasis is maintained in the brain by apolipoprotein E (apoE). The apoE4 isoform constitutes the major risk factor for AD. Here, we investigated the effect of apoE forms on Aβ generation and on BACE1 levels. We also examined the potential involvement in these processes of cholesterol transporters ABCG1 and ABCG4 or the lipoprotein receptor SR-BI, which are implicated in cholesterol efflux to apoE. It was found that reconstituted lipoprotein-associated apoE isoforms promoted the increase of Aβ production and oligomerization and of BACE1 levels in human neuroblastoma SK-N-SH cells, with an apoE4 ≥ apoE3 > apoE2 potency rank order. Progressive carboxyl-terminal apoE4 deletions between residues 230-299 decreased the protein's ability to increase BACE1, while further truncations up to residue 166 prevented apoE4 from increasing BACE1 and Aβ levels in SK-N-SH and primary mouse neuronal cells. ABCG1, but not ABCG4 or SR-BI, moderately increased Aβ production and BACE1 levels in SK-N-SH cells. All apoE forms affected Aβ production/oligomerization and BACE1 levels in a pattern that did not follow that of their capacity to promote ABCG1, ABCG4 or SR-BI-mediated cholesterol efflux. Overall, our data indicate that apoE-containing lipoprotein particles can have a direct effect on BACE1 levels and Aβ secretion and possibly contribute to AD pathogenetic processes, independently of their capacity to promote cholesterol efflux.

Adding Dopamine to Proxymetacaine or Oxybuprocaine Solutions Potentiates and Prolongs the Cutaneous Antinociception in Rats.

We evaluated the interaction of dopamine-proxymetacaine and dopamine- oxybuprocaine antinociception using isobolograms. This experiment uses subcutaneous drug (proxymetacaine, oxybuprocaine, and dopamine) injections under the skin of the rat's back, thus simulating infiltration blocks. The dose-related antinociceptive curves of proxymetacaine and oxybuprocaine alone and in combination with dopamine were constructed, and then the antinociceptive interactions between the local anesthetic and dopamine were analyzed using isobolograms. Subcutaneous proxymetacaine, oxybuprocaine, and dopamine produced a sensory block to local skin pinpricks in a dose-dependent fashion. The rank order of potency was proxymetacaine (0.57 [0.52-0.63] μmol/kg) > oxybuprocaine (1.05 [0.96-1.15] μmol/kg) > dopamine (165 [154-177] μmol/kg; P < .01 for each comparison) based on the 50% effective dose values. On the equianesthetic basis (25% effective dose, 50% effective dose, and 75% effective dose), the nociceptive block duration of proxymetacaine or oxybuprocaine was shorter than that of dopamine (P < .01). Oxybuprocaine or proxymetacaine coinjected with dopamine elicited a synergistic antinociceptive effect and extended the duration of action. Oxybuprocaine and proxymetacaine had a higher potency and provoked a shorter duration of sensory block compared with dopamine. The use of dopamine increased the quality and duration of skin antinociception caused by oxybuprocaine and proxymetacaine.

Saccharomyces cerevisiae as a platform for assessing sphingolipid lipid kinase inhibitors.

Successful medicinal chemistry campaigns to discover and optimize sphingosine kinase inhibitors require a robust assay for screening chemical libraries and for determining rank order potencies. Existing assays for these enzymes are laborious, expensive and/or low throughput. The toxicity of excessive levels of phosphorylated sphingoid bases for the budding yeast, Saccharomyces cerevisiae, affords an assay wherein inhibitors added to the culture media rescue growth in a dose-dependent fashion. Herein, we describe our adaptation of a simple, inexpensive, and high throughput assay for assessing inhibitors of sphingosine kinase types 1 and 2 as well as ceramide kinase and for testing enzymatic activity of sphingosine kinase type 2 mutants. The assay was validated using recombinant enzymes and generally agrees with the rank order of potencies of existing inhibitors.

Drimane Sesquiterpenoids Noncompetitively Inhibit Human α4β2 Nicotinic Acetylcholine Receptors with Higher Potency Compared to Human α3β4 and α7 Subtypes.

The drimane sesquiterpenoids drimenin, cinnamolide, dendocarbin A, and polygodial were purified from the Canelo tree ( Drimys winteri) and chemically characterized by spectroscopic methods. The pharmacological activity of these natural compounds were determined on hα4β2, hα3β4, and hα7 nicotinic acetylcholine receptors (AChRs) by Ca2+ influx measurements. The results established that drimane sesquiterpenoids inhibit AChRs with the following selectivity: hα4β2 > hα3β4 > hα7. In the case of hα4β2 AChRs, the following potency rank order was determined (IC50's in μM): drimenin (0.97 ± 0.35) > cinnamolide (1.57 ± 0.36) > polygodial (62.5 ± 19.9) ≫ dendocarbin A (no activity). To determine putative structural features underlying the differences in inhibitory potency at hα4β2 AChRs, additional structure-activity relationship and molecular docking experiments were performed. The Ca2+ influx and structural results supported a noncompetitive mechanism of inhibition, where drimenin interacted with luminal and nonluminal (TMD-β2 intrasubunit) sites. The structure-activity relationship results, i.e., the lower the ligand polarity, the higher the inhibitory potency, supported the nonluminal interaction. Ligand binding to both sites might inhibit the hα4β2 AChR by a cooperative mechanism, as shown experimentally ( nH > 1). Drimenin could be used as a molecular scaffold for the development of more potent inhibitors with higher selectivity for the hα4β2 AChR.

Clinical testing of three novel transient receptor potential cation channel subfamily V member 1 antagonists in a pharmacodynamic intradermal capsaicin model.

The transient receptor potential cation channel subfamily V 1 (TRPV1) is involved in nociception and has thus been of interest for drug developers, as a target for novel analgesics. However, several oral TRPV1 antagonists have failed in development, and novel approaches to target TRPV1 with innovative chemistry are needed. This work describes an intradermal microdosing approach in humans for pharmacodynamic deductions and pharmacological profiling of compounds. First, a human capsaicin model was developed, to generate pharmacodynamic translational data (Study Part A, n=24). Then, three small molecule TRPV1 antagonists (AZ11760788, AZ12048189 and AZ12099548) were investigated in healthy volunteers (Study Part B, n=36), applying the established model. Pain and flare were assessed by Visual Analogue Score and laser Doppler, respectively. The developed model proved useful for pharmacologic deductions; all compounds caused a dose-dependent inhibition of capsaicin-induced pain and flare responses, with a rank order potency of AZ11760788>AZ12048189≫AZ12099548. In addition, the dose-response data showed that the minimal antagonist concentrations needed to inhibit TRPV1 was ≥6-7 times the equilibrium dissociation constant for each compound. With careful design of a pharmacodynamic translational human pain model, it was possible to rank order TRPV1 efficacy among three investigational TRPV1 antagonists, and to estimate human efficacious concentrations. This fast and cost-effective translational approach allows for generation of human target engagement information early in drug development. This could be of value for other development programmes where pharmacological targets are expressed in peripheral sensory nerves.

Effects of Urocortins on Intestinal Ion Secretion in the Mouse Colon

Background and AimsUrocortins (Ucn1, Ucn2, and Ucn3) belong to the corticotropin‐releasing factor (CRF)‐like peptide family and bind to two subtypes of CRF receptors (CRF1 and CRF2) to exert their biological effects. Ucn1 binds to both CRF1 and CRF2 receptors with equal high affinity, whereas Ucn2 and Ucn3 bind exclusively to CRF2. CRF and urocortins are expressed in the gastrointestinal tract. CRF has been implicated in stress‐stimulated intestinal ion secretion and diarrhea. However, the effects of urocortins on intestinal ion secretion have remain uninvestigated. The aim of the present study was to investigate the role of urocortins in the regulation of intestinal ion secretion.MethodsMucosa/submucosa preparations from mouse colon were mounted in Ussing flux chambers for measurement of short‐circuit current (Isc) as an indicator of mucosal ion secretion.ResultsApplication of CRF, Ucn1, Ucn2, and Ucn3 to the basolateral side of the preparation increased baseline Isc in a concentration‐dependent manner with an EC50 (in M) of 1.28×10−6, 6.04×10−7, 2.89×10−7, or 2.98×10−7, respectively, with the rank order of potency: Ucn2≈Ucn3&gt;Ucn1&gt;CRF. At 1 μM, Ucn3 caused a greater rise in Isc (ΔIsc: 47.01±8.94 μA/cm2), compared with Ucn2 (17.71±2.63 μA/cm2), Ucn1 (7.06±4.33 μA/cm2), or CRF (8.92±4.11 μA/cm2). The CRF1 receptor antagonist NBI 27914 significantly suppressed Ucn1‐evoked ΔIsc, but had no effect on Ucn2‐ or Ucn3‐evoked ΔIsc. The CRF2 receptor antagonist antisauvagine‐30 significantly suppressed Ucn1‐evoked ΔIsc, and completely blocked Ucn2‐ or Ucn3‐evoked ΔIsc. The neuronal blocker tetrodotoxin had no effect on Ucn1‐, Ucn2‐, or Ucn3‐evoked DIsc.ConclusionsThe results suggest that urocortins stimulate colonic ion secretion independent the submucosal plexus of the enteric nervous system. Both CRF1 and CRF2 receptors are involved in Ucn1‐stimulated ion secretion, while Ucn2‐ and Unc3‐stimulated ion secretion is mediated by the CRF2 receptors. Ucn2 and Ucn3 are more potent than CRF in stimulation of colonic ion secretion and may play a dominant role in stress‐induced hyper secretion and diarrhea.Support or Funding InformationNIH R15 DK097460‐01A1 (SL) and UWL graduate research service and educational leadership grant (AK).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Reinforcing Potency and Effectiveness of Synthetic Cathinones: Potency versus Selectivity for DAT

Synthetic cathinones found in “bath salts” preparations interact with monoamine transporters and function as either cocaine‐like monoamine uptake inhibitors or amphetamine‐like substrates. Abuse of these synthetic cathinones has resulted in regulation of some cathinones internationally. Modifications to the chemical structure of regulated cathinones (e.g., MDPV) produce a series of new cathinones (e.g., α‐PVP, α‐PPP, MDPBP, and MDPPP) which may not be subject to existing regulations. Although MDPV is a more potent and effective reinforcer than cocaine, little is known about the reinforcing effects of other synthetic cathinones. Thus, this study examined intravenous self‐administration of cocaine, MDPV, α‐PVP, α‐PPP, MDPBP, and MDPPP to test the hypotheses that 1) stimulants with greater potency to inhibit the dopamine transporter (DAT) are more potent reinforcers, and 2) stimulants with greater selectivity for DAT relative to the serotonin transporter (SERT) are more effective reinforcers. Potencies to inhibit monoamine uptake at DAT and SERT were determined using synaptosomes prepared from rat brains. Separate groups of male Sprague‐Dawley rats (n=12 per group) were used to generate full dose‐response curves under a progressive ratio (PR) schedule of reinforcement for α‐PVP, α‐PPP, MDPBP, and MDPPP; full dose‐response curves for MDPV and cocaine were also generated in each rat (n=48). Rank order potency to inhibit DAT was MDPV &gt; α‐PVP ≈ MDPBP &gt; MDPPP ≈ α‐PPP ≈ cocaine, while rank order DAT/SERT selectivity was α‐PVP &gt; α‐PPP &gt; MDPV &gt; MDPBP &gt; MDPPP &gt; cocaine. The potency to inhibit DAT correlated with the potency of each drug to maintain responding (MDPV &gt; α‐PVP ≈ MDPBP &gt; MDPPP ≈ α‐PPP ≈ cocaine). Relative reinforcing effectiveness as determined by comparing the maximum number of infusions obtained for each drug under a PR schedule of reinforcement was α‐PVP &gt; α‐PPP ≈ MDPV &gt; MDPBP ≈ MDPPP &gt; cocaine, which correlated with DAT/SERT selectivity. These data suggest that (1) potency at DAT is an important determinant of the potency of each drug to function as a reinforcer, (2) each of these synthetic cathinones functions as a more effective reinforcer than cocaine, and (3) DAT/SERT selectivity is related to reinforcing effectiveness and may predict the abuse‐related effects of novel synthetic cathinones in humans.Support or Funding InformationThese experiments were supported by NIH grants R01DA039146 (GTC) and T32DA031115 (BMG) and the Intramural Research Programs of NIDA (MHB, KCR) and NIAAA (KCR).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Potency of polycyclic aromatic hydrocarbons in chicken and Japanese quail embryos.

Birds are receptors of concern for polycyclic aromatic hydrocarbons (PAHs), yet limited data describing the relative potency of PAH congeners are available for avian species. In the present study, we determined embryonic median lethal dose (LD50) values for 5 PAH congeners in chicken (Gallus gallus) and one PAH congener in Japanese quail (Coturnix japonica). Graded concentrations of each test compound were injected into the air cell of chicken or quail eggs before incubation. Embryos were monitored through development (quail) or hatching (chicken). All PAHs tested caused dose-dependent increases in embryo mortality, but few other effects (e.g., weight changes, deformities) were observed. In chicken, windows of developmental sensitivity were identified between embryonic days 4 and 9 and between embryonic days 20 and 22. The rank order potency of benzo[k]fluoranthene (BkF; 76 μg/kg) ≈ dibenz[ah]anthracene (83 μg/kg) > indeno[1,2,3-cd]pyrene (325 μg/kg) > benzo[a]pyrene (461 μg/kg) > benz[a]anthracene (529 μg/kg) corresponded well with previous in vitro estimates in birds. Previously published ethoxyresorufin-O-deethylase median effect concentrations from cultured chicken embryo hepatocytes were highly predictive of our LD50s (p < 0.001, r2 = 0.99). To explore differences in sensitivity between species, Japanese quail eggs were injected with BkF, the most potent PAH. We found that chicken and quail were nearly equally sensitive to BkF. The present results contribute to our developing understanding of variability in responses to PAHs among congeners and species. Environ Toxicol Chem 2018;37:1556-1564. © 2018 SETAC.

Identification and Complete Stereochemical Assignments of the New Resolvin Conjugates in Tissue Regeneration in Human Tissues that Stimulate Proresolving Phagocyte Functions and Tissue Regeneration

Resolvin conjugates in tissue regeneration (RCTRs) are new chemical signals that accelerate resolution of inflammation, infection, and tissue regeneration. Herein, using liquid chromatography-tandem mass spectrometry-based metabololipidomics, we identified RCTRs in human spleen, lymph node, bone marrow, and brain. In human spleen incubated with Staphylococcus aureus, endogenous RCTRs were increased along with conversion of deuterium-labeled docosahexaenoic acid, conferring pathway activation. Physical and biological properties of endogenous RCTRs were matched with those prepared by total organic synthesis. The complete stereochemical assignment of bioactive RCTR1 is 8R-glutathionyl-7S,17S-dihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid, RCTR2 is 8R-cysteinylglycinyl-7S,17S-dihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid, and RCTR3 is 8R-cysteinyl-7S,17S-dihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid. These stereochemically defined RCTRs stimulated human macrophage phagocytosis, efferocytosis, and planaria tissue generation. Proteome profiling demonstrated that RCTRs regulated both proinflammatory and anti-inflammatory cytokines with human macrophages. In microfluidic chambers, the three RCTRs limited human polymorphonuclear cell migration. In hind-limb ischemia-reperfusion-initiated organ injury, both RCTR2 and RCTR3 reduced polymorphonuclear cell infiltration into lungs. In infectious peritonitis, RCTR1 shortened the resolution intervals. Each RCTR (1 nmol/L) accelerated planaria tissue regeneration by approximately 0.5 days, with direct comparison to both maresin and protectin CTRs. Together, these results identify a new bioactive RCTR (ie, RCTR3) in human tissues and establish the complete stereochemistry and rank-order potencies of three RCTRs invivo. Moreover, RCTR1, RCTR2, and RCTR3 each exert potent anti-inflammatory and proresolving actions with human leukocytes.

Receptor binding profiles and behavioral pharmacology of ring-substituted N,N-diallyltryptamine analogs

Substantial effort has been devoted toward understanding the psychopharmacological effects of tryptamine hallucinogens, which are thought to be mediated by activation of 5-HT2A and 5-HT1A receptors. Recently, several psychoactive tryptamines based on the N,N-diallyltryptamine (DALT) scaffold have been encountered as recreational drugs. Despite the apparent widespread use of DALT derivatives in humans, little is known about their pharmacological properties. We compared the binding affinities of DALT and its 2-phenyl-, 4-acetoxy-, 4-hydroxy-, 5-methoxy-, 5-methoxy-2-methyl-, 5-fluoro-, 5-fluoro-2-methyl-, 5-bromo-, and 7-ethyl-derivatives at 45 receptor and transporter binding sites. Additionally, studies in C57BL/6 J mice examined whether these substances induce the head twitch response (HTR), a 5-HT2A receptor-mediated response that is widely used as a behavioral proxy for hallucinogen effects in humans. Most of the test drugs bound to serotonin receptors, σ sites, α2-adrenoceptors, dopaminergic D3 receptors, histaminergic H1 receptors, and the serotonin transporter. DALT and several of the ring-substituted derivatives were active in the HTR assay with the following rank order of potency: 4-acetoxy-DALT > 5-fluoro-DALT > 5-methoxy-DALT > 4-hydroxy-DALT > DALT > 5-bromo-DALT. 2-Phenyl-DALT, 5-methoxy-2-methyl-DALT, 5-fluoro-2-methyl-DALT, and 7-ethyl-DALT did not induce the HTR. HTR potency was not correlated with either 5-HT1A or 5-HT2A receptor binding affinity, but a multiple regression analysis indicated that 5-HT2A and 5-HT1A receptors make positive and negative contributions, respectively, to HTR potency (R2 = 0.8729). In addition to supporting the established role of 5-HT2A receptors in the HTR, these findings are consistent with evidence that 5-HT1A activation by tryptamine hallucinogens buffers their effects on HTR.This article is part of the Special Issue entitled ‘Psychedelics: New Doors, Altered Perceptions’.