Rank Order Of Potency Research Articles

Assessment of pharmacological effects and abuse potential of 5F-EDMB-PICA, CUMYL-PEGACLONE, and NM-2201 in mice.

The newly emerging synthetic cannabinoids (SCs) 5F-EDMB-PICA, CUMYL-PEGACLONE, and NM-2201 have been observed to produce effects by activating cannabinoid type 1 (CB1) receptors. Nevertheless, the pharmacological effects and potential for abuse of these three substances remain to be studied. These substances have yet to be regulated in many countries. We investigated the safety, pharmacological effects, rewarding effects, and cannabinoid withdrawal of 5F-EDMB-PICA, CUMYL-PEGACLONE, and NM-2201. This study evaluated the drug safety and the cannabinoid-specific pharmacological effects of the three substances through acute toxicity experiments (in which the LD50 of each substance was obtained) and tetrad experiments (comprising assessments of hypothermia, analgesia, locomotion inhibition, and catalepsy). Furthermore, the conditioned place preference (CPP) experiments and withdrawal experiments were conducted to evaluate the rewarding effect and cannabinoid withdrawal potential of the substances in question. The results demonstrated that all three drugs exhibited certain acute toxic effects and could potentially induce tetrad effects. The data were analyzed using non-linear regression, and the corresponding ED50 values and 95% confidence intervals (CI) were obtained. The rank order of potency was determined to be CUMYL-PEGACLONE > 5F-EDMB-PICA > NM-2201. In the CPP experiments, it was demonstrated that 5F-EDMB-PICA significantly induced an increase in CPP score at a dose of 0.3mg/kg, while NM-2201 caused an increase in CPP score and a significant aversion effect at a dose of 2 and 3mg/kg, respectively. It is noteworthy that all three types of SCs were observed to produce a significant biphasic effect, indicating that CPP scores were biphasic for all compounds. Following the administration of the CB1 receptor antagonist rimonabant, a notable increase in head twitches and paw tremors was observed, indicating that these three SCs induce cannabinoid withdrawal through the mediation of CB1 receptors. The results of this study indicated that these SCs possess cannabinoid-specific pharmacological effects and abuse potential, which provides substantial experimental data to support the future regulation of these substances.

Comparing CB1 receptor GIRK channel responses to receptor internalization using a kinetic imaging assay

The type 1 cannabinoid receptor (CB1R) mediates neurotransmitter release and synaptic plasticity in the central nervous system. Endogenous, plant-derived, synthetic cannabinoids bind to CB1R, initiating the inhibitory G-protein (Gi) and the β-arrestin signaling pathways. Within the Gi signaling pathway, CB1R activates G protein-gated, inwardly-rectifying potassium (GIRK) channels. The β-arrestin pathway reduces CB1R expression on the cell surface through receptor internalization. Because of their association with analgesia and drug tolerance, GIRK channels and receptor internalization are of interest to the development of pharmaceuticals. This research used immortalized mouse pituitary gland cells transduced with a pH-sensitive, fluorescently-tagged human CB1R (AtT20-SEPCB1) to measure GIRK channel activity and CB1R internalization. Cannabinoid-induced GIRK channel activity is measured by using a fluorescent membrane-potential sensitive dye. We developed a kinetic imaging assay that visualizes and measures CB1R internalization. All cannabinoids stimulated a GIRK channel response with a rank order potency of WIN55,212-2 > (±)CP55,940 > Δ9-THC > AEA. Efficacy was expressed relative to (±)CP55,940 with a rank order efficacy of (±)CP55,940 > WIN55, 212-2 > AEA > Δ9-THC. All cannabinoids stimulated CB1R internalization with a rank order potency of (±)CP55,940 > WIN55, 212-2 > AEA > Δ9-THC. Internalization efficacy was normalized to (±)CP55,940 with a rank order efficacy of WIN55,212-2 > AEA > (±)CP55,940 > Δ9-THC. (±)CP55,940 was significantly more potent and efficacious than AEA and Δ9-THC at stimulating a GIRK channel response; no significant differences between potency and efficacy were observed with CB1R internalization. No significant differences were found when comparing a cannabinoid’s GIRK channel and CB1R internalization response. In conclusion, AtT20-SEPCB1 cells can be used to assess cannabinoid-induced CB1R internalization. While cannabinoids display differential Gi signaling when compared to each other, this did not extend to CB1R internalization.

Pharmacological differences between human and mouse P2X4 receptor explored using old and new tools.

There is growing interest in the P2X4 receptor as a therapeutic target for several cardiovascular, inflammatory and neurological conditions. Key to exploring the physiological and pathophysiological roles of P2X4 is access to selective compounds to probe function in cells, tissues and animal models. There has been a recent growth in selective antagonists for P2X4, though agonist selectivity is less well studied. As there are some known pharmacological differences between P2X receptors from different species, it is important to understand these differences when designing a pharmacological strategy to probe P2X4 function in human tissue and mouse models. Here, we provide a systematic comparison of agonist and antagonist pharmacology in 1321N1 cells expressing either human or mouse P2X4 orthologues. We identify a rank order of agonist potency of ATP > 2-MeSATP > αβmeATP = BzATP > CTP = γ-[(propargyl)-imido]-ATP for human P2X4 and ATP > 2-MeSATP = CTP > ATPγS = γ-[(propargyl)-imido]-ATP = BzATP for mouse. Human P2X4 is not activated by ATPγS but can be activated by αβmeATP. We identify a rank order of antagonist potency of BAY-1797 = PSB-12062 = BX-430 > 5-BDBD > TNP-ATP = PPADS for human P2X4 and BAY-1797 > PSB-12062 = PPADS > TNP-ATP for mouse. Mouse P2X4 is not antagonised by 5-BDBD or BX-430. The study reveals key pharmacological differences between human and mouse P2X4, highlighting caution when selecting tools for comparative studies between human and mouse and ascribing cellular responses of some commonly used agonists to P2X4.

Efficiency of single pharmaceutical surfactants to mimic intestinal biorelevant media solubilization and dissolution of etravirine: Comparison of intrinsic and film dissolution models

We understand that quality control dissolution media may best anticipate in vivo product performance by mimicking in vivo media, but preferably involve at most a single pharmaceutical surfactant for routine laboratory use. The objective here was to estimate the concentrations of six pharmaceutical surfactants to mimic etravirine solubility and intrinsic dissolution rate, as well as dissolution rate from a film model, in each Fed State Simulated Intestinal Fluid Version 2 (FeSSIF-V2) and Fasted State Simulated Intestinal Fluid Version 2 (FaSSIF-V2). Solubility studies and colloid sizing measurements were conducted. Results indicate that all six surfactants were more efficient than FeSSIF-V2 or FaSSIF-V2 at solubilizing drug, and also exhibited higher micelle diffusivities than FeSSIF-V2 and FaSSIF-V2 mixed-micelles. The rank-order potency (on mM basis) of the six pharmaceutical surfactants to mimic etravirine solubility in each FeSSIF-V2 and FaSSIF-V2 was: polysorbate 80 (PS80) > polysorbate 20 (PS20) > polyoxyethylene(23) lauryl ether (POE23) > POE10 > hexadecyltrimethylammonium bromide (HEX) > sodium lauryl sulfate (SLS). This rank-order potency was almost the same to mimic drug dissolution rate into each FeSSIF-V2 and FaSSIF-V2, except POE10 > POE23. For the most potent surfactant, PS80, 0.461 mM and 0.140 mM PS80 was estimated to mimic etravirine's solubility and dissolution rate into FeSSIF-V2, respectively, using the intrinsic dissolution model. The low PS80 concentration to mimic dissolution rate reflects the relatively high diffusivity of PS80 micelles, compared to FeSSIF-V2 mixed-micelle diffusivity, which was the case for all six pharmaceutical surfactants. Results are also presented in terms of a film dissolution model for surfactant-mediated dissolution, where dissolution enhancement was less than that in the intrinsic dissolution model, and the film model required lower surfactant concentration than in intrinsic dissolution model to mimic FeSSIF-V2-enhanced dissolution. Findings have promised to identify single pharmaceutical surfactant concentrations that mimic key performance attributes of biorelevant media.

Comparison of the µ-opioid receptor antagonists methocinnamox (MCAM) and naloxone to reverse and prevent the ventilatory depressant effects of fentanyl, carfentanil, 3-methylfentanyl, and heroin in male rats.

The number of opioid overdose deaths has increased significantly over the past decade. The life-threatening effect of opioids is hypoventilation that can be reversed by the µ-opioid receptor (MOR) antagonist naloxone; however, because of the very short duration of action of naloxone, re-emergence of MOR agonist-induced hypoventilation can occur, requiring additional doses of naloxone. The MOR antagonist methocinnamox (MCAM) antagonizes hypoventilation by the non-morphinan fentanyl and the morphinan heroin in laboratory animals with an unusually long duration of action. Whole-body plethysmography was used to compare the potency and effectiveness of MCAM and naloxone for preventing and reversing hypoventilation by fentanyl, heroin, and the ultra-potent and longer-acting fentanyl analogs carfentanil and 3-methylfentanyl in male rats breathing normal air. Sessions comprised a 45-minute habituation period followed by intravenous (i.v.) administration of saline or an acute dose of MOR agonist. The rank order of potency to decrease ventilation was 3-methylfentanyl > carfentanil > fentanyl > heroin. MCAM (0.0001-0.1 mg/kg) and naloxone (0.0001-0.01 mg/kg) dose-dependently reversed hypoventilation by 3-methylfentanyl (0.01 mg/kg), carfentanil (0.01 mg/kg), fentanyl (0.1 mg/kg), or heroin (3.2 mg/kg). For prevention studies, MCAM, naloxone, or vehicle was administered i.v. 22, 46, or 70 hours prior to a MOR agonist. When administered 22 hours earlier, MCAM (0.1-1.0 mg/kg) but not naloxone (1.0 mg/kg) prevented hypoventilation by each MOR agonist. This study demonstrates the effectiveness of MCAM to reverse and prevent hypoventilation by MOR agonists including ultra-potent fentanyl analogs that have a long duration of action. Significance Statement The number of opioid overdose deaths increased over the past decade despite the availability of antagonists that can prevent and reverse the effects of opioids. This study demonstrates the effectiveness and long duration of action of the µ-opioid receptor (MOR) antagonist methocinnamox (MCAM) for reversing and preventing hypoventilation by MOR agonists including ultra-potent fentanyl analogs. These results provide support for the notion that MCAM has the potential to positively impact the ongoing opioid crisis by reversing and preventing opioid overdose.

Extracellular domain mutations of the EGF receptor differentially modulate high-affinity and low-affinity responses to EGF receptor ligands

The EGF receptor is mutated in a number of cancers. In most cases, the mutations occur in the intracellular tyrosine kinase domain. However, in glioblastomas, many of the mutations are in the extracellular ligand binding domain. To determine what changes in receptor function are induced by such extracellular domain mutations, we analyzed the binding and biological response to the seven different EGF receptor ligands in three common glioblastoma mutants--R84K, A265V, and G574V. Our data indicate that all three mutations significantly increase the binding affinity of all seven ligands. In addition, the mutations increase the potency of all ligands for stimulating receptor autophosphorylation, phospholipase Cγ, Akt, and MAP kinase activity. In all mutants, the rank order of ligand potency seen at the wild-type receptor was retained, suggesting that the receptors still discriminate among the different ligands. However, the low affinity ligands, EPR and EPG, did show larger than average enhancements of potency for stimulating Akt and MAPK, but not receptor autophosphorylation and phospholipase Cγ activation. Relative to the wild-type receptor, these changes lead to an increase in the responsiveness of these mutants to physiological concentrations of ligands and an alteration in the ratio of activation of the different pathways. This may contribute to their oncogenic potential. In the context of recent findings, our data also suggest that so-called ‘high’ affinity biological responses arise from activation by isolated receptor dimers, whereas ‘low’ affinity biological responses require clustering of receptors which occurs at higher concentrations of ligand.

Assessing the Toxicity of Benzotriazole Ultraviolet Stabilizers to Fishes: Insights into Aryl Hydrocarbon Receptor-Mediated Effects.

Benzotriazole ultraviolet stabilizers (BUVSs) are chemicals used to mitigate UV-induced damage to manufactured goods. Their presence in aquatic environments and biota raises concerns, as certain BUVSs activate the aryl hydrocarbon receptor (AhR), which is linked to adverse effects in fish. However, potencies of BUVSs as AhR agonists and species sensitivities to AhR activation are poorly understood. This study evaluated the toxicity of three BUVSs using embryotoxicity assays. Zebrafish (Danio rerio) embryos exposed to BUVSs by microinjection suffered dose-dependent increases in mortality, with LD50 values of 4772, 11 608, and 56 292 ng/g-egg for UV-P, UV-9, and UV-090, respectively. The potencies and species sensitivities to AhR2 activation by BUVSs were assessed using a luciferase reporter gene assay with COS-7 cells transfected with the AhR2 of zebrafish and eight other fishes. The rank order of potency for activation of the AhR2 from all nine species was UV-P > UV-9 > UV-090. However, AhR2s among species differed in sensitivities to activation by up to 100-fold. An approximate reversed rank order of species sensitivity was observed compared to the rank order of sensitivity to 2,3,7,8-tetrachlorodibenzo[p]dioxin, the prototypical AhR agonist. Despite this, a pre-existing quantitative adverse outcome pathway linking AhR activation to embryo lethality could predict embryotoxicities of BUVSs in zebrafish.

The Psychedelic N,N-Dipropyltryptamine Prevents Seizures in a Mouse Model of Fragile X Syndrome via a Mechanism that Appears Independent of Serotonin and Sigma1 Receptors.

The serotonergic psychedelic psilocybin shows efficacy in treating neuropsychiatric disorders, though the mechanism(s) underlying its therapeutic effects remain unclear. We show that a similar psychedelic tryptamine, N,N-dipropyltryptamine (DPT), completely prevents audiogenic seizures (AGS) in an Fmr1 knockout mouse model of fragile X syndrome at a 10 mg/kg dose but not at lower doses (3 or 5.6 mg/kg). Despite showing in vitro that DPT is a serotonin 5-HT2A, 5-HT1B, and 5-HT1A receptor agonist (with that rank order of functional potency, determined with TRUPATH Gα/βγ biosensors), pretreatment with selective inhibitors of 5-HT2A/2C, 5-HT1B, or 5-HT1A receptors did not block DPT's antiepileptic effects; a pan-serotonin receptor antagonist was also ineffective. Because 5-HT1A receptor activation blocks AGS in Fmr1 knockout mice, we performed a dose-response experiment to evaluate DPT's engagement of 5-HT1A receptors in vivo. DPT elicited 5-HT1A-dependent effects only at doses greater than 10 mg/kg, further supporting that DPT's antiepileptic effects were not 5-HT1A-mediated. We also observed that the selective sigma1 receptor antagonist, NE-100, did not impact DPT's antiepileptic effects, suggesting DPT engagement of sigma1 receptors was not a crucial mechanism. Separately, we observed that DPT and NE-100 at high doses caused convulsions on their own that were qualitatively distinct from AGS. In conclusion, DPT dose-dependently blocked AGS in Fmr1 knockout mice, but neither serotonin nor sigma1 receptor antagonists prevented this action. Thus, DPT might have neurotherapeutic effects independent of its serotonergic psychedelic properties. However, DPT also caused seizures at high doses, showing that DPT has complex dose-dependent in vivo polypharmacology.

Evaluation of novel epibatidine analogs in the rat nicotine drug discrimination assay and in the rat chronic constriction injury neuropathic pain model.

Nicotine is the primary psychoactive component responsible for maintaining tobacco dependence in humans. Chronic pain is often a consequence of tobacco-related pathologies, and the development of a dual therapeutic that could treat chronic pain and tobacco dependence would be advantageous. Epibatidine reliably substitutes for nicotine in the drug discrimination assay, and is a potent analgesic, but has a side-effect profile that limits its therapeutic potential. Thus, considerable efforts to produce epibatidine derivatives are underway. Here we tested three epibatidine derivatives, 2'-fluoro-3'-(4-nitrophenyl)deschloroepibatidine (RTI-7527-102; i.e., RTI-102), 2'-fluorodeschloroepibatidine (RTI-7527-36; i.e., RTI-36), and 3'-(3″-dimethylaminophenyl)-epibatidine (RTI-7527-76; i.e., RTI-76) in both the rat nicotine drug discrimination assay as well as in the rat chronic constriction injury (CCI) of the sciatic nerve neuropathic pain model. Male and female Sprague-Dawley rats were trained on a fixed-ratio 10 schedule to discriminate nicotine (0.32mg/kg base) from vehicle. All compounds dose-dependently substituted for nicotine, without significant decreases in response rates. In the discrimination assay the rank order potency was RTI-36 > nicotine > RTI-102 > RTI-76. Evidence suggests the α4β2* subtype is particularly important to nicotine-related abuse potential. Thus, here we utilized the antagonist dihydro-β-erythroidine (DHβE) to examine relative β2 subunit contribution. DHβE (3.2mg/kg, s.c.) antagonized the discriminative stimulus effects of nicotine. However, relative to antagonism of nicotine, DHβE produced less antagonism of RTI-102 and RTI-76 and greater antagonism of RTI-36. It is likely that at nicotinic receptor subunits RTI-102, RTI-76 and RTI-36 possess differing activity. To confirm that the full discriminative stimulus of these compounds was due to nAChR activity beyond the β2 subunit, we examined these compounds in the presence of the non-selective nicotinic receptor antagonist mecamylamine. Mecamylamine (0.56mg/kg, s.c.) pretreatment abolished nicotine-paired lever responding for all compounds. In a separate cohort, male and female Sprague-Dawley rats underwent CCI surgery and tested for CCI-induced mechanical allodynia via the von Frey assay. Each compound produced CCI-induced mechanical allodynia reversal. RTI-36 displayed higher potency than either RTI-102 or RTI-76. These novel epibatidine analogs may prove to be useful tools in the fight against nicotine dependence as well as novel neuropathic pain analgesics.

Effects on cell cycle progression and cytoskeleton organization of five Bothrops spp. venoms in cell culture-based assays

Snake envenomation is a neglected tropical disease. In Brazil, the Bothrops genus is responsible for about 86% of snakebite accidents. Despite extensive evidence of the cytotoxicity of snake venoms, the cellular and molecular mechanisms involved are not fully understood, especially regarding the effects on cell cycle progression and cytoskeleton organization. Traditionally, the effectiveness and quality control tests of venoms and antivenoms are assessed by in vivo assays. Despite this, there is a rising effort to develop surrogate in vitro models according to the 3R principle (Replacement, Reduction, and Refinement). In this study, we treated rat liver cells (BRL-3A) with venoms from five Bothrops species (B. jararaca, B. jararacussu, B. moojeni, B. alternatus, and B. neuwiedi) and analyzed cell viability and IC50 by MTT assay, cell cycle phases distribution by flow cytometry, and morphology and cytoskeleton alterations by immunofluorescence. In addition, we evaluated the correlation between IC50 and the enzymatic and biological activities of each venom. Our results indicated that Bothrops spp. venoms decreased the cell viability of rat liver BRL-3A cells. The rank order of potency was B. jararacussu > B. moojeni > B. alternatus > B. jararaca > B. neuwiedi. The mechanisms of cytotoxicity were related to microtubules and actin network disruption, but not to cell cycle arrest. No clear correlation was found between the IC50 and retrieved literature data of in vitro enzymatic and in vivo biological activities. This work contributed to understanding cellular and molecular mechanisms underlying the Bothrops spp. venom cytotoxicity, which can help to improve envenomation treatment, as well as disclose potential therapeutic properties of snake venoms.

Fentanyl analog structure-activity relationships demonstrate determinants of diverging potencies for antinociception and respiratory depression

Opioid overdoses, particularly those involving fentanyl-related substances (FRS), present a significant public health challenge in the United States. This structure-activity relationship (SAR) study evaluated the relationship between the chemical structure of seventeen FRS and their in vivo mu-opioid-receptor (MOR) mediated effects. SAR evaluations included fluorine substitutions on the aniline or phenethyl ring and variations in N-acyl chain length. Adult male Swiss Webster mice were administered fluorinated regioisomers of fentanyl, butyrylfentantyl and valerylfentanyl, and compared to MOR standards including morphine, buprenorphine, and fentanyl to determine if they would elicit prototypical opioid-like effects including hyperlocomotion (open-field test), antinociception (warm-water tail-withdrawal test), and hypoventilation (whole-body plethysmography test). To determine if the MOR was the pharmacological mechanism responsible for these effects, naltrexone or naloxone pretreatments were administered to evaluate their actions on FRS-induced antinociception and hypoventilation. There were three main findings. First, FRS elicited hyperlocomotion, antinociception, and hypoventilation in mice to varying degrees, similar to prototypical MOR standards. Second, the rank order of potencies for hypoventilatory effects of FRS were different for each series including FRS with increasing N-acyl chain length (i.e., acetylfentanyl, fentanyl, butyrylfentanyl, valerylfentanyl, hexanoylfentanyl), phenethyl-fluorinated regioisomers (e.g., 2′-fluorofentanyl, 3′-fluorofentanyl, 4′-fluorofentanyl), and aniline-fluorinated regioisomers (e.g., ortho-fluorofentanyl, meta-fluorofentanyl, para-fluorofentanyl). Third, the degree of separation in potencies observed for the antinociceptive and hypoventilatory effects of these drugs did not always follow that which was observed for their antinociceptive and hyperlocomotor effects. This study clarifies the in vivo activities for these FRS and elucidates a SAR for MOR-mediated effects among structural isomers.

Differential and substrate-specific inhibition of γ-secretase by the C-terminal region of ApoE2, ApoE3, and ApoE4

Aberrant low γ-secretase activity is associated with most of the presenilin mutations that underlie familial Alzheimer's disease (fAD). However, the role of γ-secretase in the more prevalent sporadic AD (sAD) remains unaddressed. Here, we report that human apolipoprotein E (ApoE), the most important genetic risk factor of sAD, interacts with γ-secretase and inhibits it with substrate specificity in cell-autonomous manners through its conserved C-terminal region (CT). This ApoE CT-mediated inhibitory activity is differentially compromised in different ApoE isoforms, resulting in an ApoE2>ApoE3>ApoE4 potency rank order inversely correlating to their associated AD risk. Interestingly, in an AD mouse model, neuronal ApoE CT migrates to amyloid plaques in the subiculum from other regions and alleviates the plaque burden. Together, our data reveal a hidden role of ApoE as a γ-secretase inhibitor with substrate specificity and suggest that this precision γ-inhibition by ApoE may protect against the risk of sAD.

Repertoire of P-glycoprotein drug transporters in the zoonotic nematode Toxocara canis

Toxocara canis has a complex lifecycle including larval stages in the somatic tissue of dogs that tolerate macrocyclic lactones. In this study, we investigated T. canis permeability glycoproteins (P-gps, ABCB1) with a putative role in drug tolerance. Motility experiments demonstrated that while ivermectin failed to abrogate larval movement, the combination of ivermectin and the P-gp inhibitor verapamil induced larval paralysis. Whole organism assays revealed functional P-gp activity in larvae which were capable of effluxing the P-gp substrate Hoechst 33342 (H33342). Further investigation of H33342 efflux demonstrated a unique rank order of potency for known mammalian P-gp inhibitors, suggesting that one or more of the T. canis transporters has nematode-specific pharmacological properties. Analysis of the T. canis draft genome resulted in the identification of 13 annotated P-gp genes, enabling revision of predicted gene names and identification of putative paralogs. Quantitative PCR was used to measure P-gp mRNA expression in adult worms, hatched larvae, and somatic larvae. At least 10 of the predicted genes were expressed in adults and hatched larvae, and at least 8 were expressed in somatic larvae. However, treatment of larvae with macrocyclic lactones failed to significantly increase P-gp expression as measured by qPCR. Further studies are needed to understand the role of individual P-gps with possible contributions to macrocyclic lactone tolerance in T. canis.

Mechanistic in vitro studies indicate that the clinical drug-drug interactions between protease inhibitors and rosuvastatin are driven by inhibition of intestinal BCRP and hepatic OATP1B1 with minimal contribution from OATP1B3, NTCP and OAT3.

Previous use of a mechanistic static model to accurately quantify the increased rosuvastatin exposure due to drug-drug interaction (DDI) with coadministered atazanavir underpredicted the magnitude of area under the plasma concentration-time curve ratio (AUCR) based on inhibition of breast cancer resistance protein (BCRP) and organic anion transporting polypeptide (OATP) 1B1. To reconcile the disconnect between predicted and clinical AUCR, atazanavir and other protease inhibitors (darunavir, lopinavir and ritonavir) were evaluated as inhibitors of BCRP, OATP1B1, OATP1B3, sodium taurocholate cotransporting polypeptide (NTCP) and organic anion transporter (OAT) 3. None of the drugs inhibited OAT3, nor did darunavir and ritonavir inhibit OATP1B3 or NTCP. All drugs inhibited BCRP-mediated estrone 3-sulfate transport or OATP1B1-mediated estradiol 17β-D-glucuronide transport with the same rank order of inhibitory potency (lopinavir>ritonavir>atazanavir>>darunavir) and mean IC50 values ranging from 15.5 ± 2.80 μM to 143 ± 14.7 μM or 0.220 ± 0.0655 μM to 9.53 ± 2.50 μM, respectively. Atazanavir and lopinavir also inhibited OATP1B3- or NTCP-mediated transport with a mean IC50 of 1.86 ± 0.500 μM or 65.6 ± 10.7 μM and 5.04 ± 0.0950 μM or 20.3 ± 2.13 μM, respectively. Following integration of a combined hepatic transport component into the previous mechanistic static model using the in vitro inhibitory kinetic parameters determined above for atazanavir, the newly predicted rosuvastatin AUCR reconciled with the clinically observed AUCR confirming additional minor involvement of OATP1B3 and NTCP inhibition in its DDI. The predictions for the other protease inhibitors confirmed inhibition of intestinal BCRP and hepatic OATP1B1 as the principal pathways involved in their clinical DDI with rosuvastatin.

Verification of In Vivo Estrogenic Activity for Four Per- and Polyfluoroalkyl Substances (PFAS) Identified as Estrogen Receptor Agonists via New Approach Methodologies.

Given concerns about potential toxicological hazards of the thousands of data-poor per- and polyfluorinated alkyl substances (PFAS) currently in commerce and detected in the environment, tiered testing strategies that employ high-throughput in vitro screening as an initial testing tier have been implemented. The present study evaluated the effectiveness of previous in vitro screening for identifying PFAS capable, or incapable, of inducing estrogenic responses in fish exposed in vivo. Fathead minnows (Pimephales promelas) were exposed for 96 h to five PFAS (perfluorooctanoic acid [PFOA]; 1H,1H,8H,8H-perfluorooctane-1,8-diol [FC8-diol]; 1H,1H,10H,10H-perfluorodecane-1,10-diol [FC10-diol]; 1H,1H,8H,8H-perfluoro-3,6-dioxaoctane-1,8-diol [FC8-DOD]; and perfluoro-2-methyl-3-oxahexanoic acid [HFPO-DA]) that showed varying levels of in vitro estrogenic potency. In agreement with in vitro screening results, exposure to FC8-diol, FC10-diol, and FC8-DOD caused concentration-dependent increases in the expression of transcript coding for vitellogenin and estrogen receptor alpha and reduced expression of insulin-like growth factor and apolipoprotein eb. Once differences in bioconcentration were accounted for, the rank order of potency in vivo matched that determined in vitro. These results provide a screening level benchmark for worst-case estimates of potential estrogenic hazards of PFAS and a basis for identifying structurally similar PFAS to scrutinize for putative estrogenic activity.

Pharmacological characterization of novel small molecule agonists and antagonists for the orphan receptor GPR139

The orphan G protein-coupled receptor GPR139 is predominantly expressed in the central nervous system and has attracted considerable interest as a therapeutic target. However, the biological role of this receptor remains somewhat elusive, in part due to the lack of quality pharmacological tools to investigate GPR139 function. In an effort to understand GPR139 signaling and to identify improved compounds, in this study we performed virtual screening and analog searches, in combination with multiple pharmacological assays. We characterized GPR139-dependent signaling using previously published reference agonists in Ca2+ mobilization and inositol monophosphate accumulation assays, as well as a novel real-time GPR139 internalization assay. For the four reference agonists tested, the rank order of potency was conserved across signaling and internalization assays: JNJ-63533054 > Compound 1a » Takeda > AC4 > DL43, consistent with previously reported values. We noted an increased efficacy of JNJ-63533054-mediated inositol monophosphate signaling and internalization, relative to Compound 1a. We then performed virtual screening for GPR139 agonist and antagonist compounds that were screened and validated in GPR139 functional assays. We identified four GPR139 agonists that were active in all assays, with similar or reduced potency relative to known compounds. Likewise, compound analogs selected based on GPR139 agonist and antagonist substructure searches behaved similarly to their parent compounds. Thus, we have characterized GPR139 signaling for multiple new ligands using G protein-dependent assays and a new real-time internalization assay. These data add to the GPR139 tool compound repertoire, which could be optimized in future medical chemistry campaigns.

PS-B03-6: COMPARISON OF SUPPRESSIVE EFFECTS ON CATECHOLAMINE SECRETION BETWEEN OLMESARTAN AND ENALAPRIL

Objective: The present study was designed to compare olmesartan, an angiotensin II receptor blocker (ARB) with enalapril, an angiotensin converting enzyme inhibitor (ACEI) in the suppressive effects on the secretion of catecholamines (CA) from the perfused model of the rat adrenal gland. Design and Methods: The adrenal gland was isolated and perfused with Krebs-bicarbonate. CA was measured directly by using the fluorospectrophotometer. Results: Both olmesartan (50 μM) and enalapril (50 μM) during perfusion into an adrenal vein for 90 min inhibited the CA secretory responses evoked by ACh (5.32 μM), DMPP (a selective neuronal nicotinic Nn receptor agonist, 100 μM), high K+ (a direct membrane-depolarizer, 56 μM), and McN-A-343 (a selective muscarinic M1 receptor agonist, 100 μM) in a time-dependent manner. Also, in the presence of enalapril or olmesartan, the secretory responses of CA evoked by veratridine (an activator of voltage-dependent Na+ channels, 100 μM), Bay-K-8644 (a L-type dihydropyridine Ca2+ channel activator, 10 μM), and cyclopiazonic acid (a cytoplasmic Ca2+-ATPase inhibitor, 10 μM) were significantly reduced. Based on the same concentration (50 μM) of enalapril and olmesartan, for the CA release evoked by Ach, high K+, DMPP, McN-A-343, angiotensin II, veratridine, Bay-K-8644 and cyclopiazonic acid, the following rank order of inhibitory potency was obtained: olmesartan > enalapril. In the simultaneous presence of enalapril and olmesartan, Ach-evoked CA secretory responses were more strongly inhibited in comparison with that of enalapril- or olmesartan-treated alone. Conclusion: Collectively, these results demonstrate that both enalapril and olmesartan inhibit the CA secretory responses evoked by activation of both cholinergic and angiotensin II receptors stimulation as well as by direct membrane depolarization in the perfused model of the isolated rat adrenal medulla. When these two drugs were used in combination, their effects were enhanced, which may also be of clinical benefit. Based on concentration used in this study, it seems that the inhibitory effect of olmesartan on the CA secretion is more potent than that of enalapril.

Imaging of Gαq Proteins in Mouse and Human Organs and Tissues.

G protein-coupled receptors (GPCRs) transfer extracellular signals across cell membranes by activating intracellular heterotrimeric G proteins. Several studies suggested G proteins as novel drug targets for the treatment of complex diseases, e.g., asthma and cancer. Recently, we developed specific radiotracers, [³H]PSB-15900-FR and [³H]PSB-16254-YM, for the Gαq family of G proteins by tritiation of the macrocyclic natural products FR900359 (FR) and YM-254890 (YM). In the present study, we utilized these potent radioligands to perform autoradiography studies in tissues of healthy mice, mouse models of disease, and human tissues. Specific binding was high, while non-specific binding was extraordinarily low, giving nearly identical results for both radioligands. High expression levels of Gαq proteins were detected in healthy mouse organs showing the following rank order of potency: kidney > liver > brain > pancreas > lung > spleen, while expression in the heart was low. Organ sub-structures, e.g., of mouse brain and lung, were clearly distinguishable. Whereas an acute asthma model in mice did not result in altered Gαq protein expressions as compared to control animals, a cutaneous melanoma model displayed significantly increased expression in comparison to healthy skin. These results suggest the future development of Gαq-protein-binding radio-tracers as novel diagnostics.

The atypic antipsychotic clozapine inhibits multiple cardiac ion channels.

Clozapine is an atypical neuroleptic used to manage treatment-resistant schizophrenia which is known to inhibit cardiac hERG/KV11.1 potassium channels, a pharmacological property associated with increased risk of potentially fatal Torsades de Pointes (TdP) and sudden cardiac death (SCD). Yet, the long-standing clinical practice of clozapine does not show a consistent association with increased incidence of TdP, although SCD is considerably higher among schizophrenic patients than in the general population. Here, we have established the inhibitory profile of clozapine at the seven cardiac ion currents proposed by the ongoing comprehensive in vitro pro-arrhythmia (CiPA) initiative to better predict new drug cardio-safety risk. We found that clozapine inhibited all CiPA currents tested with the following rank order of potency: KV11.1 > NaV1.5 (late current) ≈ CaV1.2 ≈ NaV1.5 (peak current) ≈ KV7.1 > KV4.3 > Kir2.1 (outward current). Half-maximal inhibitory concentrations (IC50) at the repolarizing KV11.1 and KV7.1 channels, and at the depolarizing CaV1.2 and NaV1.5 channels fell within a narrow half-log 3-10µM concentration range, suggesting that mutual compensation could explain the satisfactory arrhythmogenic cardio-safety profile of clozapine. Although the IC50 values determined herein using an automated patch-clamp (APC) technique are at the higher end of clozapine plasmatic concentrations at target therapeutic doses, this effective antipsychotic appears prone to distribute preferentially into the cardiac tissue, which supports the clinical relevance of our in vitro pharmacological findings.

In vitro and in vivo pharmacology of nine novel synthetic cannabinoid receptor agonists

Synthetic cannabinoid receptor agonists (SCRAs) are novel psychoactive substances that bind to and activate CB1 receptors in the brain. The structural manipulations observed in newer SCRAs suggest that manufacturers have incorporated modern drug development techniques into their repertoire, often producing higher CB1 receptor affinity than Δ9-tetrahydrocannabinol (Δ9-THC). This study examined nine SCRAs recently detected by forensic surveillance, some of which caused fatalities: 5F-MDMB-PICA, FUB-144, 5F-MMB-PICA, MMB-4en-PICA, MMB-FUBICA, 5F-EDMB-PINACA, APP-BINACA, MDMB-4en-PINACA, and FUB-AKB48. Compounds were evaluated for CB1 and CB2 receptor binding affinity and functional activation and for their effects on body temperature, time course, and pharmacological equivalence with Δ9-THC in Δ9-THC drug discrimination in mice. All SCRAs bound to and activated CB1 and CB2 receptors with high affinity, with similar or greater affinity for CB2 than CB1 receptors and stimulated [35S]GTPγS binding in CB1 and CB2 expressing cell membranes. All compounds produced hypothermia, with shorter latency to peak effects for SCRAs than Δ9-THC. All SCRAs fully substituted for Δ9-THC in drug discrimination at one or more doses. Rank order potency in producing in vivo effects mostly aligned with rank order CB1 receptor affinities. Potencies for Δ9-THC-like discriminative stimulus effects were similar across sex except Δ9-THC was more potent in females and 5F-MMB-PICA was more potent in males. In summary, 5F-EMDB-PINACA, 5F-MDMB-PICA, MDMB-4en-PINACA, FUB-144, FUB-AKB48, 5F-MMB-PICA, MMB-4en-PICA, and MMB-FUBICA are potent and efficacious SCRAs with pharmacology like that of past SCRAs that have been abused in humans. In contrast, APP-BINACA was efficacious, but had lower potency than most past SCRAs.