Phencyclidine (PCP)-like discriminative stimulus effects of metaphit and of 2-amino-5-phosphonovalerate in pigeons: generality across different training doses of PCP

Pigeons were trained to discriminate either a fixed dose of PCP (1 mg/kg; n =3) or a progressively decreasing dose (1-0.56–0.32 mg/kg; n =4) from saline. Lowering of the training dose shifted the dose-effect curve for PCP's discriminative stimulus effects about 5-fold to the left, in a parallel manner, but did not decrease the accuracy of the discrimination performance and did not significantly increase the extent to which pentobarbital and chlordiazepoxide produced PCP-appropriate responding. Dose-effect curves based on binary generalization data were evaluated statistically with new methods that may be more appropriate than those used previously. Metaphit, a proposed PCP-receptor acylator, and 2-amino-5-phosphonovalerate (AP5), an N-methyl- d -aspartate (NMDA) antagonist, produced complete PCP-appropriate responding in the high training dose group only at doses that suppressed the rate of responding and that produced ataxia. However, 4-fold lower doses of metaphit and AP5, which did not produce directly observable behavioral effects, were found to substitute completely for PCP in the low training dose group. These data support the notion that PCP, metaphit, and AP5 have a common discriminative effect in pigeons.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46447/1/213_2004_Article_BF00207232.pd

Compounds of novel structure having kappa-agonist behavioral effects in Rhesus monkeys

The kappa-agonist behavioral effects of several compounds were studied in rhesus monkeys and mice. Rhesus monkeys trained to discriminate ethylketazocine from saline responded as if ethylketazocine had been administered when given bridged oripavines with either N-allyl or N-cyclopropylmethyl, but not N-methyl, substituents. These compounds had C7 substitutions of either 2-hydroxy-2-pentyl or 2-hydroxy-5-methyl-2-hexyl. Monkeys also showed ethylketazocine-like responding when given U-50, 488 (-3,4-dichloro-N-methyl-N-[2- (1-pyrrolidinyl) cyclohexyl]- benzeneacetamide), a compound with an atypical structure not resembling any known narcotic. Additionally, ethylketazocine-like responding was produced by two 5,9-alpha dimethyl 6-7-benzomorphans with either an N-2-methoxyisobutyl or an N-2-methoxypropyl substituent. The latter compound was the only compound active in producing ethylketazocine-like discriminative effects that also reversed morphine-withdrawal signs. The N-methyl bridged oripavines that were inactive in producing ethylketazocine-like discriminative effects reversed morphine withdrawal signs.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/23805/1/0000043.pd

Excitatory amino acid antagonists induce a phencyclidine-like catalepsy in pigeons: Structure-activity studies

The excitatory amino acid antagonists -2-amino-5-phosphonovalerate (-AP5), its isomers -(-)-AP5 and -(+)-AP5, -2-amino-4-phosphonobutyrate (AP4), -2-amino-7-phosphonoheptanoate (AP7), [beta]--aspartylaminomethylphosphonic acid (ASP-AMP), cis-2,3-piperidinedi-carboxylic acid (cis-PDA), and [gamma]--glutamylaminomethylsulphonic acid (GAMS) were tested for their ability to produce a phencyclidine (PCP)-like catalepsy in pigeons when administered intracerebro-ventricularly. Each of the antagonists produced catalepsy, although -AP5, and the non-selective antagonists GAMS and cis-PDA, produced the effect only at toxic doses. The rank order of potency to produce catalepsy was AP7 > -AP5 > -AP5 > cis-pda > ASP-AMP > AP4 > -AP5 > GAMS; there was a strong positive correlation between this rank order of potency vivo and the potency order of these compounds in vitro as NMDA antagonists. The antagonists did not displace significant amounts of [3H]N[1-(2-thienyl)cyclohexyl]piperidine (a congener of phencyclidine) from its recognition site in the brain of pigeon. Thus, the PCP-like catalepsy that is produced by the excitatory neurotransmission at NMDA-preferring receptors that are distinct from, but related to, PCP receptors. The results strongly support the hypothesis that a reduction of neurotransmission at excitatory synapses, utilizing NMDA-preferring receptors, may underlie catalepsy in pigeons induced by PCP.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26604/1/0000145.pd

Characterization and optimization of heroin hapten-BSA conjugates: method development for the synthesis of reproducible hapten-based vaccines

A potential new treatment for drug addiction is immunization with vaccines that induce antibodies that can abrogate the addictive effects of the drug of abuse. One of the challenges in the development of a vaccine against drugs of abuse is the availability of an optimum procedure that gives reproducible and high yielding hapten-protein conjugates. In this study, a heroin/morphine surrogate hapten (MorHap) was coupled to bovine serum albumin (BSA) using maleimide-thiol chemistry. MorHap-BSA conjugates with 3, 5, 10, 15, 22, 28, and 34 haptens were obtained using different linker and hapten ratios. Using this optimized procedure, MorHap-BSA conjugates were synthesized with highly reproducible results and in high yields. The number of haptens attached to BSA was compared by 2,4,6-trinitrobenzenesulfonic acid (TNBS) assay, modified Ellman’s test and matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Among the three methods, MALDI-TOF MS discriminated subtle differences in hapten density. The effect of hapten density on enzyme-linked immunosorbent assay (ELISA) performance was evaluated with seven MorHap-BSA conjugates of varying hapten densities, which were used as coating antigens. The highest antibody binding was obtained with MorHap-BSA conjugates containing 3–5 haptens. This is the first report that rigorously analyzes, optimizes and characterizes the conjugation of haptens to proteins that can be used for vaccines against drugs of abuse. The effect of hapten density on the ELISA detection of antibodies against haptens demonstrates the importance of careful characterization of the hapten density by the analytical techniques described. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00216-014-8035-x) contains supplementary material, which is available to authorized users

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

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

Phencyclidine-induced catalepsy in pigeons: Specificity and stereoselectivity

A procedure is described for the rapid assessment of cataleptic activity (loss of righting, without head-drop and without eye closure) of phencyclidine-type drugs. Single- and cumulative-dosing procedures with phencyclidine and ketamine produced similar results. Pentobarbital produced loss of righting at doses which also induced head-drop and eye closure. Catalepsy was induced exclusively by the d-isomers of ketamine, 1-(1-phenylcyclohexyl)-3-methylpiperidine and [alpha]-dioxadrol. The procedure is suitable for studying compounds which may interact with phencyclidine receptors.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/24637/1/0000048.pd

Financing Direct Democracy: Revisiting the Research on Campaign Spending and Citizen Initiatives

The conventional view in the direct democracy literature is that spending against a measure is more effective than spending in favor of a measure, but the empirical results underlying this conclusion have been questioned by recent research. We argue that the conventional finding is driven by the endogenous nature of campaign spending: initiative proponents spend more when their ballot measure is likely to fail. We address this endogeneity by using an instrumental variables approach to analyze a comprehensive dataset of ballot propositions in California from 1976 to 2004. We find that both support and opposition spending on citizen initiatives have strong, statistically significant, and countervailing effects. We confirm this finding by looking at time series data from early polling on a subset of these measures. Both analyses show that spending in favor of citizen initiatives substantially increases their chances of passage, just as opposition spending decreases this likelihood

The Intriguing Effects of Substituents in the N-Phenethyl Moiety of Norhydromorphone: A Bifunctional Opioid from a Set of “Tail Wags Dog” Experiments

This work is licensed under a Creative Commons Attribution 4.0 International License.(−)-N-Phenethyl analogs of optically pure N-norhydromorphone were synthesized and pharmacologically evaluated in several in vitro assays (opioid receptor binding, stimulation of [35S]GTPγS binding, forskolin-induced cAMP accumulation assay, and MOR-mediated β-arrestin recruitment assays). “Body” and “tail” interactions with opioid receptors (a subset of Portoghese’s message-address theory) were used for molecular modeling and simulations, where the “address” can be considered the “body” of the hydromorphone molecule and the “message” delivered by the substituent (tail) on the aromatic ring of the N-phenethyl moiety. One compound, N-p-chloro-phenethynorhydromorphone ((7aR,12bS)-3-(4-chlorophenethyl)-9-hydroxy-2,3,4,4a,5,6-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-7(7aH)-one, 2i), was found to have nanomolar binding affinity at MOR and DOR. It was a potent partial agonist at MOR and a full potent agonist at DOR with a δ/μ potency ratio of 1.2 in the ([35S]GTPγS) assay. Bifunctional opioids that interact with MOR and DOR, the latter as agonists or antagonists, have been reported to have fewer side-effects than MOR agonists. The p-chlorophenethyl compound 2i was evaluated for its effect on respiration in both mice and squirrel monkeys. Compound 2i did not depress respiration (using normal air) in mice or squirrel monkeys. However, under conditions of hypercapnia (using air mixed with 5% CO2), respiration was depressed in squirrel monkeys.NIDA grant P30 DA13429NIDA grant DA039997NIDA grant DA018151NIDA grant DA035857NIDA grant DA047574NIH Intramural Research Programs of the National Institute on Drug AbuseNational Institute of Alcohol Abuse and AlcoholismNIH Intramural Research Programs of the National Institute on Drug AbuseNIH Intramural Research Program through the Center for Information TechnologyNIH Intramural Research Programs of the National Institute on Drug Abus

Informing the design of a national screening and treatment programme for chronic viral hepatitis in primary care: qualitative study of at-risk immigrant communities and healthcare professionals

n Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise statedThis paper presents independent research funded by the National Institute for Health Research (NIHR) under the Programme Grants for Applied Research programme (RP-PG-1209-10038).