Rank Order Of Potency Research Articles

An in vitro bioassay for evaluating the effect of inhaled bronchodilators on airway smooth muscle

PurposeThe development of inhaled drug products is expensive and involves time-consuming pharmacokinetic (PK) and pharmacodynamic (PD) studies. There are few in vitro cell-based assays to evaluate the disposition and action of orally inhaled drugs to guide early product development and minimise risk. The aim of the present study was to develop a co-culture bioassay, combining an airway epithelial cell line (Calu-3) with cultured human primary airway smooth muscle cells (ASM), integrated with apparatus to deliver pharmaceutical aerosols. MethodsAn assay for measuring cyclic adenosine monophosphate (cAMP) in ASM derived from healthy donors was adapted to provide a biochemical surrogate for ASM relaxation. Concentration-response curves for cAMP were established for three drugs that elicit ASM relaxation: isoprenaline (ISO), forskolin (FOR) and salbutamol sulphate. The ASM bioassay was incorporated into a co-culture model in which air-interfaced Calu-3 cell layers, representing the permeability barrier of the airway epithelium, were grown on transwell inserts above ASM cells cultured in the well of the base-plate. The sensitivity of this bioassay to salbutamol delivered using different formulations and aerosol products was evaluated. ResultsASM responded with concentration dependent increases in cAMP when exposed to 10-9 to 10-5 M ISO, FOR or salbutamol sulphate solutions for 15 or 30 min. Salbutamol formulated with different counter ions elicited differential cAMP responses in ASM (xinafoate > base = sulphate) suggesting that this bioassay could discriminate between formulations with different potency. A similar rank order of potency was observed for the different salbutamol salts when applied as aerosols to the co-culture model. DiscussionWe have developed a novel bioassay using human ASM in co-culture with human respiratory epithelial cells to better mimic various elements that contribute to the rate and extent of local drug availability in the lungs following topical administration. The bioassay offers an opportunity to investigate the factors determining the activity of inhaled bronchodilator drugs in a more biologically relevant system than that has previously been described and with further development and validation, this novel bioassay could provide a method to guide the more efficient development of inhaled bronchodilators, reducing the current reliance on in vivo studies.

Pharmacological characterisation of small molecule C5aR1 inhibitors in human cells reveals biased activities for signalling and function

The complement fragment C5a is a core effector of complement activation. C5a, acting through its major receptor C5aR1, exerts powerful pro-inflammatory and immunomodulatory functions. Dysregulation of the C5a-C5aR1 axis has been implicated in numerous immune disorders, and the therapeutic inhibition of this axis is therefore imperative for the treatment of these diseases. A myriad of small-molecule C5aR1 inhibitors have been developed and independently characterised over the past two decades, however the pharmacological properties of these compounds has been difficult to directly compare due to the wide discrepancies in the model, read-out, ligand dose and instrumentation implemented across individual studies. Here, we performed a systematic characterisation of the most commonly reported and clinically advanced small-molecule C5aR1 inhibitors (peptidic: PMX53, PMX205 and JPE1375; non-peptide: W545011, NDT9513727, DF2593A and CCX168). Through signalling assays measuring C5aR1-mediated cAMP and ERK1/2 signalling, and β-arrestin 2 recruitment, this study highlighted the signalling-pathway dependence of the rank order of potencies of the C5aR1 inhibitors. Functional experiments performed in primary human macrophages demonstrated the high insurmountable antagonistic potencies for the peptidic inhibitors as compared to the non-peptide compounds. Finally, wash-out studies provided novel insights into the duration of inhibition of the C5aR1 inhibitors, and confirmed the long-lasting antagonistic properties of PMX53 and CCX168. Overall, this study revealed the potent and prolonged antagonistic activities of selected peptidic C5aR1 inhibitors and the unique pharmacological profile of CCX168, which thus represent ideal candidates to fulfil diverse C5aR1 research and clinical therapeutic needs.

High-Throughput Fluorescence-Based Activity Assay for Arginase-1.

Arginase-1, which converts the amino acid L-arginine into L-ornithine and urea, is a promising new drug target for cancer immunotherapy, as it has a role in the regulation of T-cell immunity in the tumor microenvironment. To enable the discovery of small-molecule Arginase-1 inhibitors by high-throughput screening, we developed a novel homogeneous (mix-and-measure) fluorescence-based activity assay. The assay measures the conversion of L-arginine into L-ornithine by a decrease in fluorescent signal due to quenching of a fluorescent probe, Arginase Gold. This way, inhibition of Arginase-1 results in a gain of signal when compared with the uninhibited enzyme. Side-by-side profiling of reference inhibitors in the fluorescence-based assay and a colorimetric urea formation assay revealed similar potencies and the same potency rank order among the two assay formats. The fluorescence-based assay was successfully automated for high-throughput screening of a small-molecule library in 384-well format with a good Z'-factor and hit confirmation rate. Finally, we show that the assay can be used to study the binding kinetics of inhibitors.

Inhibition of Neuromuscular Contractions of Human and Rat Colon by Bergamot Essential Oil and Linalool: Evidence to Support a Therapeutic Action.

Bergamot essential oil (BEO) added to food and drink promotes a citrus flavour. Folklore suggests benefits on gastrointestinal functions but with little supporting evidence. BEO and major constituents (linalool, limonene, linalyl acetate) were therefore examined for any ability to influence neuromuscular contractions of human and rat colon. Circular muscle strips (macroscopically-normal human colon obtained following ethical approval at cancer surgery; Sprague–Dawley rats) were suspended in baths (Krebs solution; 37 °C; 5% CO2 in O2) for measurement of neuronally-mediated contractions (prevented by tetrodotoxin or atropine) evoked by electrical field stimulation (5 Hz, 0.5 ms pulse width, 10s/minute, maximally-effective voltage), or contractions evoked by KCl (submaximally-effective concentrations). BEO and each constituent concentration dependently inhibited neuronally-mediated and KCl-induced contractions. In human: apparent pIC50 for BEO (volume/volume Krebs), respectively, 3.8 ± 0.3 and 4.4 ± 0.3; Imax 55.8% ± 4.2% and 37.5% ± 4.2%. For the constituents, the rank order of potency differed in human (linalool > limonene >> linalyl-acetate) and rat colon (linalyl-acetate > limonene = linalool), but rank order of efficacy was similar (linalool >> (BEO) = linalyl-acetate >> limonene). Thus, linalool had high efficacy but greater potency in human colon (Imax 76.8% ± 6.9%; pIC50 6.7 ± 0.2; n = 4) compared with rat colon (Imax 75.3% ± 1.9%; pIC50 5.8 ± 0.1; n = 4). The ability of BEO and linalool to inhibit human colon neuromuscular contractility provides a mechanism for use as complementary treatments of intestinal disorders.

Differential activation of G protein-mediated signaling by synthetic cannabinoid receptor agonists.

Synthetic cannabinoid receptor agonists (SCRAs) are new psychoactive substances associated with acute intoxication and even death. However, the molecular mechanisms through which SCRAs may exert their toxic effects remain unclear—including the potential differential activation of G protein subtypes by cannabinoid receptor type 1 (CB1), a major target of SCRA. We measured CB1‐mediated activation of Gαs and Gαi/o proteins by SCRAs by examining stimulation (pertussis toxin, PTX treated) as well as inhibition (non‐PTX treated) of forskolin (FSK)‐induced cyclic adenosine monophosphate (cAMP) accumulation in human embryonic kidney (HEK) cells stably expressing CB1. Real‐time measurements of stimulation and inhibition of cAMP levels were made using a BRET biosensor. We found that the maximum concentration of SCRAs tested (10 µmol L−1), increased cAMP levels 12%‐45% above that produced by FSK alone, while the phytocannabinoid THC did not significantly alter cAMP levels in PTX‐treated HEK‐CB1 cells. All SCRAs had greater potency to inhibit FSK‐induced cAMP levels than to stimulate cAMP levels. The rank order of potencies for SCRA stimulation of cAMP (Gαs) was PB‐22 > 5F‐MDMB‐PICA > JWH‐018 ≈ AB‐FUBINACA > XLR‐11. By contrast, the potency of SCRAs for inhibition of cAMP (Gαi/o) was 5F‐MDMB‐PICA > AB‐FUBINACA > PB‐22 > JWH‐018 > XLR‐11. The different rank order of potency and EMax of the SCRAs to stimulate Gαs‐like signaling compared to Gαi/o signaling suggests differences in G protein preference between SCRAs. Understanding the apparent differences among these drugs may contribute to unravelling their complex effects in humans.

Dependence and Cytotoxicity of Components of Cannabis

Cannabis use among the younger population in Japan has been steadily increasing. The aim of the present review is to highlight recent knowledge regarding the molecular mechanisms of action and health risks associated with cannabis and synthetic cannabinoid consumption. We investigated the effects of Δ9-tetrahydrocannabinol (THC) and synthetic cannabinoids on place conditioning in ICR mice. Both Δ9-THC and synthetic cannabinoids produce a significant conditioned place preference. These rewarding effects were completely suppressed by the cannabinoid CB1 receptor type antagonist AM251. The cytotoxicological effects of Δ9-THC and synthetic cannabinoids were also characterized in the limbic forebrain of mice in primary culture in vitro. Δ9-THC and synthetic cannabinoids caused cell death in a dose-dependent manner. The rank order of cytotoxicological potency was synthetic cannabinoids>Δ9-THC and related to the agonistic activities of the CB1 receptor. A recent review on the harmful effects of cannabis use in humans reported that behavioral impairments, especially in terms of attention, memory, and complex information-processing ability, can last for many weeks after cessation of cannabis use among heavy users. In addition, cannabis use could be a risk factor for drug dependence and later psychosis among adolescents. The results of animal and human studies suggest that CB1 receptors play an important role in the expression of harmful effects of cannabis and synthetic cannabinoid use. Moreover, concern regarding increasing concentrations of Δ9-THC in cannabis in many countries has been noted, because more potent cannabis may be associated with worse adverse effects.

Involvement of NO in Antinociception of NSAIDS in Murine Formalin Hind Paw Assay.

There are different animal models to evaluate pain among them the formalin hind paw assay which is widely used since some of its events appear to be similar to the clinical pain of humans. The assay in which a dilute solution of formalin is injected into the dorsal hindpaw of a murine produces two 'phases' of pain behavior separated by a inactive period. The early phase (Phase I) is probably due to direct activation of nociceptors and the second phase (Phase II) is due to ongoing inflammatory input and central sensitization. Mice were used to determine the potency antinociceptive of piroxicam (1,3,10,and 30 mg/kg), parecoxib (0.3, 1,3,10 and 30 mg/kg), dexketoprofen (3,10,30 and 100 mg/kg) and ketoprofen (3,10,30 and 100 mg/kg). Dose-response for each NSAIDs were created before and after 5 mg/kg of L-NAME i.p. or 5 mg/kg i.p. of 7-nitroindazole. A least-squares linear regression analysis of the log dose-response curves allowed the calculation of the dose that produced 50% of antinociception (ED50) for each drug. The ED50 demonstrated the following rank order of potency, in the phase I: piroxicam > dexketoprofen > ketoprofen > parecoxib and in the phase II: piroxicam > ketoprofen > parecoxib > dexketoprofen. Pretreatment of the mice with L-NAME or 7-nitroindazol induced a significant increase of the analgesic power of the NSAIDs, with a significant reduction of the ED50. It is suggested that NO may be involved in both phases of the trial, which means that nitric oxide regulates the bioactivity of NSAIDs.

In vitro profiling of opioid ligands using the cAMP formation inhibition assay and the β-arrestin2 recruitment assay: No two ligands have the same profile

Previously, we showed that no two of seven opioids administered by the intracerebroventricular route had the same potency rank order for evoking antinociception, constipation and respiratory depression in rats. To gain insight at the cellular level, this study was designed to systematically investigate the activity profiles of six commonly used opioid ligands using the forskolin-stimulated cAMP assay and a β-arrestin2 recruitment assay in cultured HEK-293 cells transfected with MOP(μ), DOP(δ) or KOP(κ) receptors(-r).Morphine was a potent agonist at the MOP-r in the cAMP assay whereas it was a weak agonist at the KOP-r and DOP-r. Oxycodone had moderate efficacy and low potency at the MOP-r. Buprenorphine was a potent MOP-r and DOP-r agonist; its efficacy rank order was DOP > MOP > KOP. Fentanyl was a potent agonist at the MOP-r; its efficacy rank order was MOP > DOP > KOP. For DPDPE, its agonist efficacy was confined to the DOP-r, whereas for U69593, its efficacy rank order was KOP>> MOP.For the β-arrestin2 assay, fentanyl had full efficacy at the MOP-r whereas morphine and oxycodone were weak with insignificant efficacy at DOP and KOP receptors. Buprenorphine did not recruit β-arrestin2 at all three opioid-receptors. DPDPE and U69593 had full efficacy for β-arrestin2 recruitment to the DOP-r and KOP-r respectively.Despite the low efficacy and potency of morphine, oxycodone and buprenorphine in recruiting β-arrestin2 to the MOP-r herein, these opioids all evoked respiratory depression and constipation in rats. Together, our findings discount a key role for β-arrestin2 recruitment at the MOP-r in evoking opioid-related side-effects.

Caenorhabditis elegans muscle Cys-loop receptors as novel targets of terpenoids with potential anthelmintic activity.

The anthelmintic treatment of nematode infections remains the pillar of worm control in both human and veterinary medicine. Since control is threatened by the appearance of drug resistant nematodes, there is a need to develop novel compounds, among which phytochemicals constitute potential anthelmintic agents. Caenorhabditis elegans has been pivotal in anthelmintic drug discovery and in revealing mechanisms of drug action and resistance. By using C. elegans, we here revealed the anthelmintic actions of three plant terpenoids -thymol, carvacrol and eugenol- at the behavioral level. Terpenoids produce a rapid paralysis of worms with a potency rank order carvacrol > thymol > eugenol. In addition to their paralyzing activity, they also inhibit egg hatching, which would, in turn, lead to a broader anthelmintic spectrum of activity. To identify drug targets, we performed an in vivo screening of selected strains carrying mutations in receptors involved in worm locomotion for determining resistance to the paralyzing effect of terpenoids. The assays revealed that two Cys-loop receptors with key roles in worm locomotion -Levamisole sensitive nicotinic receptor (L-AChR) and GABA(A) (UNC-49) receptor- are involved in the paralyzing effects of terpenoids. To decipher the mechanism by which terpenoids affect these receptors, we performed electrophysiological studies using a primary culture of C. elegans L1 muscle cells. Whole cell recordings from L1 cells demonstrated that terpenoids decrease macroscopic responses of L-AChR and UNC-49 receptor to their endogenous agonists, thus acting as inhibitors. Single-channel recordings from L-AChR revealed that terpenoids decrease the frequency of opening events, probably by acting as negative allosteric modulators. The fact that terpenoids act at different receptors may have important advantages regarding efficacy and development of resistance. Thus, our findings give support to the use of terpenoids as either an alternative or a complementary anthelmintic strategy to overcome the ever-increasing resistance of parasites to classical anthelmintic drugs.

Evaluation of antioxidant activity of bee products of different bee races in Turkey

Bee products with high bioactive properties have a very important place in human nutrition. Biochemical properties of these products such as honey, pollen, and propolis vary according to flora, season, bee race, and production methods. In this study, the biological activities of honey, bee pollen, and propolis samples from four bee races in Turkey (Apis mellifera caucasica, Apis mellifera anatoliaca, Apis mellifera syriaca, and Apis mellifera carnica) were examined. Besides the determining of total polyphenol and total flavonoid contents, the antioxidant capacity was investigated by three different assays: DPPH (1,1-diphenyl-2-picrylhydrazyl) radical-scavenging activities, CUPRAC (cupric reducing antioxidant capacity) and FRAP (ferric reducing antioxidant power). The rank order of antioxidant potencies was as follows in the four bee races: propolis > pollen > honey. The polyphenol content of the products was evaluated and Apis mellifera syriaca for honey, Apis mellifera anatoliaca for pollen, and Apis mellifera carnica for propolis were found higher than the others. When bee races were compared, it was seen that flora is a more effective factor than bee race for antioxidant capacity.

Differential Signaling Mediated by ApoE2, ApoE3, and ApoE4 in Human Neurons Parallels Alzheimer's Disease Risk.

In blood, apolipoprotein E (ApoE) is a component of circulating lipoproteins and mediates the clearance of these lipoproteins from blood by binding to ApoE receptors. Humans express three genetic ApoE variants, ApoE2, ApoE3, and ApoE4, which exhibit distinct ApoE receptor-binding properties and differentially affect Alzheimer's disease (AD), such that ApoE2 protects against, and ApoE4 predisposes to AD. In brain, ApoE-containing lipoproteins are secreted by activated astrocytes and microglia, but their functions and role in AD pathogenesis are largely unknown. Ample evidence suggests that ApoE4 induces microglial dysregulation and impedes Aβ clearance in AD, but the direct neuronal effects of ApoE variants are poorly studied. Extending previous studies, we here demonstrate that the three ApoE variants differentially activate multiple neuronal signaling pathways and regulate synaptogenesis. Specifically, using human neurons (male embryonic stem cell-derived) cultured in the absence of glia to exclude indirect glial mechanisms, we show that ApoE broadly stimulates signal transduction cascades. Among others, such stimulation enhances APP synthesis and synapse formation with an ApoE4>ApoE3>ApoE2 potency rank order, paralleling the relative risk for AD conferred by these ApoE variants. Unlike the previously described induction of APP transcription, however, ApoE-induced synaptogenesis involves CREB activation rather than cFos activation. We thus propose that in brain, ApoE acts as a glia-secreted signal that activates neuronal signaling pathways. The parallel potency rank order of ApoE4>ApoE3>ApoE2 in AD risk and neuronal signaling suggests that ApoE4 may in an apparent paradox promote AD pathogenesis by causing a chronic increase in signaling, possibly via enhancing APP expression.SIGNIFICANCE STATEMENT Humans express three genetic variants of apolipoprotein E (ApoE), ApoE2, ApoE3, and ApoE4. ApoE4 constitutes the most important genetic risk factor for Alzheimer's disease (AD), whereas ApoE2 protects against AD. Significant evidence suggests that ApoE4 impairs microglial function and impedes astrocytic Aβ clearance in brain, but the direct neuronal effects of ApoE are poorly understood, and the differences between ApoE variants in these effects are unclear. Here, we report that ApoE acts on neurons as a glia-secreted signaling molecule that, among others, enhances synapse formation. In activating neuronal signaling, the three ApoE variants exhibit a differential potency of ApoE4>ApoE3>ApoE2, which mirrors their relative effects on AD risk, suggesting that differential signaling by ApoE variants may contribute to AD pathogenesis.

Differential inhibitory effects of resveratrol on excitotoxicity and synaptic plasticity: involvement of NMDA receptor subtypes

ABSTRACT Objectives: The neuroprotective effects of resveratrol against excitatory neurotoxicity have been associated with N-methyl-D-aspartate receptor (NMDAR) inhibition. This study examined the differential inhibitory effects of resveratrol on NMDAR-mediated responses in neuronal cells with different NMDAR subtype composition. Methods: The effects of resveratrol on NMDA-induced cell death and calcium influx in immature and mature rat primary cortical neurons were determined and compared. Moreover, the potencies and efficacies of resveratrol to inhibit NR1/NR2A, NR1/NR2B, NR1/NR2C, and NR1/NR2D NMDAR expressed in HEK 293 cells were evaluated. Results: Resveratrol significantly attenuated NMDA-induced cell death in mature neurons, but not in immature neurons. Resveratrol also concentration-dependently reduced NMDA-induced calcium influx among all NMDAR subtypes, but displayed NR2 subunit selectivity, with a potency rank order of NR2B = NR2D > NR2A = NR2C and an efficacy rank order of NR2B = NR2C > NR2A = NR2D. Data show the stronger inhibitory effects of resveratrol on NR1/NR2B than other subtypes. Moreover, resveratrol did not affect hippocampal long-term potentiation (LTP), but impaired long-term depression (LTD). Discussion: These findings reveal the specific NMDAR modulating profile of resveratrol, providing further insight into potential mechanisms underlying the protective effects of resveratrol on neurological disorders.

Effect fingerprints of antipsychotic drugs on neural networks in vitro.

We compared the acute effect of typical (haloperidol) and atypical (aripiprazole, clozapine, olanzapine) antipsychotic drugs (APDs) on spontaneous electrophysiological activity of in vitro neuronal networks cultured on microelectrode arrays (MEAs). Network burst analysis revealed a "regularizing" effect of all APDs at therapeutic concentrations, i.e., an increase of network-wide temporal synchronization. At supratherapeutic concentrations, all APDs but olanzapine mediated a decrease of burst and spike rates, burst duration, number of spikes in bursts, and network synchrony. The rank order of potency of APDs was: haloperidol > aripiprazole > clozapine > olanzapine (no suppression). Disruption of network function was not due to enhanced cell death as assessed by trypan blue staining. APDs promoted distinct concentration-dependent alterations yielding acute effect fingerprints of the tested compounds. These effects were rather characteristic for individual compounds than distinctive for typical vs. atypical APDs. Thus, this dichotomy may be of value in distinguishing clinical features but has no apparent basis on the network or local circuitry level.

Exploring the overlapping binding sites of ifenprodil and EVT-101 in GluN2B-containing NMDA receptors using novel chicken embryo forebrain cultures and molecular modeling.

N‐methyl‐d‐aspartate receptors (NMDAR) are widely expressed in the brain. GluN2B subunit‐containing NMDARs has recently attracted significant attention as potential pharmacological targets, with emphasis on the functional properties of allosteric antagonists. We used primary cultures from chicken embryo forebrain (E10), expressing native GluN2B‐containing NMDA receptors as a novel model system. Comparing the inhibition of calcium influx by well‐known GluN2B subunit‐specific allosteric antagonists, the following rank order of potency was found: EVT‐101 (EC 50 22 ± 8 nmol/L) > Ro 25‐6981 (EC 50 60 ± 30 nmol/L) > ifenprodil (EC 50 100 ± 40 nmol/L) > eliprodil (EC 50 1300 ± 700 nmol/L), similar to previous observations in rat cortical cultures and cell lines overexpressing chimeric receptors. The less explored Ro 04‐5595 had an EC 50 of 186 ± 32 nmol/L. Venturing to explain the differences in potency, binding properties were further studied by in silico docking and molecular dynamics simulations using x‐ray crystal structures of GluN1/GluN2B amino terminal domain. We found that Ro 04‐5595 was predicted to bind the recently discovered EVT‐101 binding site, not the ifenprodil‐binding site. The EVT‐101 binding pocket appears to accommodate more structurally different ligands than the ifenprodil‐binding site, and contains residues essential in ligand interactions necessary for calcium influx inhibition. For the ifenprodil site, the less effective antagonist (eliprodil) fails to interact with key residues, while in the EVT‐101 pocket, difference in potency might be explained by differences in ligand‐receptor interaction patterns.

Changes in neuronal activity in rat primary cortical cultures induced by illicit drugs and new psychoactive substances (NPS) following prolonged exposure and washout to mimic human exposure scenarios

The use of new psychoactive substances (NPS) is increasing despite associated health risks and limited pharmacological and toxicological knowledge. Information is available mainly for acute effects on specific targets like monoamine transporters and receptors. Recently, we have shown the ability of several NPS and illicit drugs to modulate neuronal activity during acute exposure. While these acute measurements provide valuable information regarding the potency and possible structure-activity relationships, an exposure scenario more representative of human exposure would increase insight and aid translation to the human situation.Therefore, we investigated the effects on neuronal activity after acute (30 min) and prolonged (5 h) exposure to amphetamine-type stimulants, cathinones, hallucinogens, piperazines and cocaine using rat primary cortical cultures grown on multi-well microelectrode arrays. To investigate the reversibility of effects, activity was also measured after a washout period of 19 h.During acute exposure, all compounds concentration-dependently decreased neuronal activity. Compared to acute exposure, prolonged exposure did not further decrease neuronal activity. Following washout, effects of 3 out of 11 drugs (methamphetamine, cocaine, and benzylpiperazine) were fully reversible, whereas effects induced by MDMA, PMMA and α-PVP were partially reversible. Neuronal activity did not recover after 19 h washout following exposure to the highest concentration of MDPV, 2C-B, 25B-NBOMe, and TFMPP. On the contrary, exposure to low concentrations of methylone, and to some extent of 2C-B, increased neuronal activity after the washout period.Hazard characterization of emerging NPS should include at least an acute exposure to determine a potency rank order. Supplementing the (acute and prolonged) exposure scenario with a washout period allows investigation of the reversibility of effects. The possibility of a neuronal network to regain activity after drug exposure appears independent of drug class or IC50 values for acute and prolonged exposure. Even though neuronal activity (partly) recovers after washout following exposure to most drugs, it is perturbing that complete recovery of neuronal activity is observed only for a minority of the tested drugs.

AMB‐FUBINACA (FUB‐AMB) and related "spice" compounds induce gene‐dose effects in mice lacking cannabinoid‐1 receptor (CB1R) alleles

Synthetic cannabinoids (SCs), commonly known as ‘K2' or 'spice’, are a constantly evolving class of new psychoactive substances that pose public health risks. Little information is available about novel SCs when they first emerge into the recreational drug marketplace. In this study, we investigated the pharmacology of AMB‐FUBINACA (FUB‐AMB) and 5F‐AMB, two recently encountered indazole‐based SCs responsible for mass intoxications and fatalities. The effects of known cannabinoid‐1 receptor (CB1R) agonists AM‐2201 and Δ9‐THC were examined for comparison. Groups of genetically‐engineered mice lacking CB1R alleles (CB1R −/− and CB1R +/−), and their C57Bl/6J wild‐type (WT) counterparts, received surgically‐implanted subcutaneous (s.c.) temperature transponders under isoflurane anesthesia. Beginning one week after surgery, mice received s.c. injection of either SCs or vehicle and were subjected to a “triad” test battery consisting of sequential assessment of: 1] body temperature, 2] catalepsy bar latency and 3] hot‐plate analgesia latency. All SCs induced significant dose‐ and time‐dependent hypothermia, catalepsy and analgesia in WT mice, which lasted up to 120 min. The rank order of potency was FUB‐AMB>5F‐AMB=AM‐2201>Δ9‐THC. Importantly, ED50 values in the triad test battery for FUB‐AMB (0.09–0.22 mg/kg, s.c.) were at least 50‐fold more potent than those associated with Δ9‐THC (12.72–30.37 mg/kg, s.c.), and SCs were more efficacious in their ability to produce hypothermia. Homozygous CB1R −/− mice were not affected by any dose of the compounds tested, whereas heterozygous CB1R +/− mice displayed responses intermediate between homozygous and WT. CB1R binding assays in WT mouse brain revealed that IC50 values for inhibition of [3H]SR141716 binding were 1.2, 2.2, 12.6 and 50.6 nM for FUB‐AMB, AM‐2201, 5F‐AMB and Δ9‐THC, respectively. Our findings demonstrate that newer indazole‐based SCs exert their effects via potent agonist actions at CB1R, and do not support the notion that SCs induce in vivo effects by non‐CB receptor mechanisms. Given the widespread abuse of synthetic cannabinoids, CB1R antagonists should be made available for overdose rescue, and medical marijuana could be deployed as an agonist treatment for dependence on these substances.Support or Funding InformationThis research was generously supported by IRP, NIDA, NIH (DA00523)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Comprehensive Study of the Chemosensitizing Effects of AZD‐1775, WEE‐1 Inhibitor in Combination with DNA Damaging Treatments in the Jurkat Leukemia Cell Model

Current treatment for leukemia focuses on DNA damaging treatments (DNAdt), including chemo (cytarabine, vincristine, doxorubicin, etc.) and/or radiotherapy. However, Acute Lymphoblastic Leukemia (ALL) mechanisms of resistance limit the value of these treatments. Recent studies evaluated the G2‐M gatekeeper Wee1 inhibition as a possible alternative treatment for other types of cancer, such as: breast, prostate, pancreatic, lung, leukemia, glioblastoma, among others. AZD‐1775, a well‐documented Wee1 (G2‐M‐Gatekeeper) inhibitor, has shown chemosensitizing, synergistic effects, and currently is undergoing clinical trials. Therefore, our working hypothesis is that: AZD‐1775 sensitizes Leukemia cell lines to DNA‐damaging drugs, via a mitotic catastrophe process. This study evaluates the cytotoxic and chemo‐sensitization effect of AZD‐1775 on an ALL T‐cell model (Jurkat). Even more, this study innovates the understanding of AZD‐1775 by carefully crafting a panel of drugs with different mechanisms that induce DNA damage. Cell viability assays (resazurin‐based; high throughput 384 well plates) were done to determine the inhibitory effects of AZD‐1775 and a series of DNAdt drugs alone and in combinations. The corresponding IC50 value for all curves obtained were calculated using GraphPAD Prism (Vers.7). The nature of the interactions (synergy, additivity, antagonism) between AZD‐1775 and the different DNAdt drugs was determined with Combenefit and the CompuSyn Software. The Combenefit freeware permitted fitting and analysis of the data to various synergy models (Loewe‐additivity, Bliss‐independence and Highest‐single Agent); while, GraphPad Prism and CompuSyn allowed estimates of drug combination indexes (CI) and dose‐reduction indexes (DRI). The individual agents exhibited the following rank order of inhibitory potencies (IC50±SEM): cytarabine (160nM±6nM SEM) ≥ AZD‐1775 (370nM±9nM SEM) >> etoposide (2μM±60nM SEM) = cisplatin (3μM±127nM SEM) > 5′‐fluorouracil (34μM±2.7μM SEM). The results demonstrate a significant synergistic effect of AZD‐1775 with cytarabine, 5FU, cisplatin and etoposide. Conversely, most of these drugs were also capable of potentiating the effect of AZD‐1775 in a synergistic manner. In all models of synergy analysis, statistically significant synergistic interactions were confined to specific concentration ranges of drug‐combinations. For example, the most significant synergistic interactions were obtained with AZD‐1775 concentrations of 58nM to 270nM, and cytarabine concentrations of 8nM to 125nM, with the Bliss‐independence model. In conclusion, our findings strongly suggest that AZD‐1775 can be used as a chemosensitizing agent in ALL, capable of potentiating the effect and susceptibility to DNAdt; whilst, allowing the patient to require lower doses of these chemotherapies to decrease adverse effects.Support or Funding InformationPartially supported by NIGMS/INBRE award P20GM103475. The content is solely the responsibility of the author and does not necessarily represent the official views of any of the supporting agencies.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Relative Contribution of Dopamine, Norepinephrine, and Serotonin to the Discriminative Stimulus Effects of MDPV

The abuse of synthetic cathinones (“bath salts”) has increased worldwide over the past decade. Bath salt preparations often contain multiple synthetic cathinones in addition to other stimulants such as cocaine or caffeine. Synthetic cathinones are known to interact with dopamine, norepinephrine, and serotonin transporters (DAT, NET, and SERT, respectively). Although recent studies suggest that interactions with DAT are crucial for the reinforcing effects of synthetic cathinones, actions at SERT were found to negatively modulate reinforcing effectiveness. The goal of the current study was to characterize the relative contributions of dopamine, norepinephrine, and serotonin to the discriminative stimulus effects of MDPV by evaluating the substitution profiles of 1) a series of structurally related cathinones (MDPBP, MDPPP, α‐PPP, and α‐PVP) and cocaine, and 2) direct‐acting dopamine (quinpirole [D2/3], SKF 82958 [D1/5], and apomorphine [D1‐5]), and norepinephrine (phenylephrine [α1], and clonidine [α2]) receptor agonists in male Sprague‐Dawley rats (n=8) trained to discriminate 1.0 mg/kg MDPV from saline under a fixed ratio 10 schedule of sucrose pellet delivery. The mu‐opioid receptor agonist fentanyl served as a negative control. As expected, each of the cathinones and cocaine produced dose‐dependent increases in MDPV‐appropriate responding, whereas fentanyl failed to increase MDPV‐appropriate responding at doses smaller than those that suppressed responding. The rank order potency of the synthetic cathinones to increase MDPV‐appropriate responding is positively correlated with their potency to inhibit DAT and NET, but not SERT, and is consistent with the rank order to maintain self‐administration. The D2/3 receptor‐preferring agonist quinpirole dose‐dependently increased MDPV‐appropriate responding whilst SKF 82958 [D1/5], apomorphine [D1‐5], phenylephrine [α1], and clonidine [α2] failed to increase MDPV‐appropriate responding at doses smaller than those that suppressed responding. Together, these data provide evidence that the discriminative stimulus effects of MDPV are primarily mediated by its capacity to inhibit DAT, and the subsequent activation of dopamine D2 and/or D3 receptors. Moreover, these studies suggest do not support a role for serotonergic or adrenergic systems in mediating/modulating the discriminative stimulus effects of MDPV. Future studies will utilize antagonists of dopamine, adrenergic, and serotoninergic receptors to better characterize their relative contributions to the discriminative stimulus effects of MDPV.Support or Funding InformationThis work was supported by NIH/NIDA grant (R01 DA039146; GTC) and NIDA‐ and NIAAA‐IRPs (KCR)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification of a novel scaffold for a small molecule GPR139 receptor agonist

GPR139 is an orphan G protein-coupled receptor (GPCR) that is primarily expressed in the brain in regions known to regulate motor control and metabolism. Here, we screened a diverse 4,000 compound library in order to identify GPR139 agonists. We identified 11 initial hits in a calcium mobilization screen, including one compound, AC4, which contains a different chemical scaffold to what has previously been described for GPR139 agonists. Our mutagenesis data shows that AC4 interacts with the same hotspots in the binding site of GPR139 as those reported to interact with the reference agonists 1a and 7c. We additionally tested and validated 160 analogs in a calcium mobilization assay and found 5 compounds with improved potency compared to AC4. In total, we identified 36 GPR139 agonists with potencies in the nanomolar range (90–990 nM). The most potent compounds were confirmed as GPR139 agonists using an orthogonal ERK phosphorylation assay where they displayed a similar rank order of potency. Accordingly, we herein introduce multiple novel GPR139 agonists, including one with a novel chemical scaffold, which can be used as tools for future pharmacological and medicinal chemistry exploration of GPR139.

Discriminative stimulus effects of mecamylamine and nicotine in rhesus monkeys: Central and peripheral mechanisms

Mecamylamine is a non-competitive nicotinic acetylcholine receptor (nAChR) antagonist that has been prescribed for hypertension and as an off-label smoking cessation aid. Here, we examined pharmacological mechanisms underlying the interoceptive effects (i.e., discriminative stimulus effects) of mecamylamine (5.6 mg/kg s.c.) and compared the effects of nAChR antagonists in this discrimination assay to their capacity to block a nicotine discriminative stimulus (1.78 mg/kg s.c.) in rhesus monkeys. Central (pempidine) and peripherally restricted nAChR antagonists (pentolinium and chlorisondamine) dose-dependently substituted for the mecamylamine discriminative stimulus in the following rank order potency (pentolinium > pempidine > chlorisondamine > mecamylamine). In contrast, at equi-effective doses based on substitution for mecamylamine, only mecamylamine antagonized the discriminative stimulus effects of nicotine, i.e., pentolinium, chlorisondamine, and pempidine did not. NMDA receptor antagonists produced dose-dependent substitution for mecamylamine with the following rank order potency (MK-801 > phencyclidine > ketamine). In contrast, behaviorally active doses of smoking cessation aids including nAChR agonists (nicotine, varenicline, and cytisine), the smoking cessation aid and antidepressant bupropion, and the benzodiazepine midazolam did not substitute for the discriminative stimulus effects of mecamylamine. These data suggest that peripheral nAChRs and NMDA receptors may contribute to the interoceptive stimulus effects produced by mecamylamine. Based on the current results, the therapeutic use of mecamylamine (i.e., for smoking or to alleviate green tobacco sickness) should be weighed against the potential for mecamylamine to produce interoceptive effects that overlap with another class of abused drugs (i.e., NMDA receptor agonists).