Antagonism of triazolam self-administration in rhesus monkeys responding under a progressive-ratio schedule: In vivo apparent pA2 analysis.

BACKGROUND: Conventional benzodiazepines bind non-selectively to GABAA receptors containing α1, α2, α3, and α5 subunits (α1GABAA, α2GABAA, α3GABAA, and α5GABAA receptors, respectively), and the role of these different GABAA receptor subtypes in the reinforcing effects of benzodiazepines has not been characterized fully. We used a pharmacological antagonist approach with available subtype-selective ligands to evaluate the role of GABAA receptor subtypes in the reinforcing effects of the non-selective conventional benzodiazepine, triazolam. METHODS: Rhesus monkeys (n=4) were trained under a progressive-ratio schedule of intravenous midazolam delivery and dose-response functions were determined for triazolam, in the absence and presence of flumazenil (non-selective antagonist), βCCT and 3-PBC (α1GABAA-preferring antagonists), and XLi-093 (α5GABAA-selective antagonist). RESULTS: Flumazenil, βCCT and 3-PBC shifted the dose-response functions for triazolam to the right in a surmountable fashion, whereas XLi-093 was ineffective. Schild analyses revealed rank orders of potencies of flumazenil=βCCT\u3e3-PBC. Comparison of potencies between self-administration and previous binding studies with human cloned GABAA receptor subtypes suggested that the potencies for βCCT and 3-PBC were most consistent with binding at α2GABAA and α3GABAA receptors, but not α1GABAA or α5GABAA receptor subtypes. CONCLUSIONS: Our findings were not entirely consistent with blockade of α1GABAA receptors and are consistent with the possibility of α2GABAA and/or α3GABAA subtype involvement in antagonism of the reinforcing effects of triazolam. The α5GABAA receptor subtype likely does not play a substantial role in self-administration under these conditions

Role of gamma-aminobutyric acid type A (GABAA) receptor subtypes in acute benzodiazepine physical dependence-like effects: evidence from squirrel monkeys responding under a schedule of food presentation.

RATIONALE: Assays of schedule-controlled responding can be used to characterize the pharmacology of benzodiazepines and other GABAA receptor modulators, and are sensitive to changes in drug effects that are related to physical dependence. OBJECTIVE: The present study used this approach to investigate the role of GABAA receptor subtypes in mediating dependence-like effects following benzodiazepine administration. METHODS: Squirrel monkeys (n = 6) were trained on a fixed-ratio schedule of food reinforcement. Initially, the response rate-decreasing effects of chlordiazepoxide (0.1-10 mg/kg; nonselective GABAA receptor agonist), zolpidem (0.032-1.0 mg/kg; α1 subunit-containing GABAA subtype-preferring agonist), and HZ-166 (0.1-10 mg/kg; functionally selective α2 and α3 subunit-containing GABAA receptor agonist) were assessed. Next, acute dependence-like effects following single injections of chlordiazepoxide, zolpidem, and HZ-166 were assessed with flumazenil (0.1-3.2 mg/kg; nonselective GABAA receptor antagonist). Finally, acute dependence-like effects following zolpidem administration were assessed with βCCt and 3-PBC (0.1-3.2 mg/kg and 0.32-10 mg/kg, respectively; α1 subunit-containing GABAA receptor antagonists). RESULTS: Chlordiazepoxide, zolpidem, and HZ-166 produced dose- and time-dependent decreases in response rates, whereas flumazenil, βCCT, and 3-PBC were ineffective. After the drug effects waned, flumazenil produced dose-dependent decreases in response rates following administration of 10 mg/kg chlordiazepoxide and 1.0 mg/kg zolpidem, but not following any dose of HZ-166. Further, both βCCT and 3-PBC produced dose-dependent decreases in response rates when administered after 1.0 mg/kg zolpidem. CONCLUSIONS: These data raise the possibility that α1 subunit-containing GABAA receptors play a major role in physical dependence-related behaviors following a single injection of a benzodiazepine

The relationship between extreme inter-individual variation in macrophage gene expression and genetic susceptibility to inflammatory bowel disease

The differentiation of resident intestinal macrophages from blood monocytes depends upon signals from the macrophage colony-stimulating factor receptor (CSF1R). Analysis of genome-wide association studies (GWAS) indicates that dysregulation of macrophage differentiation and response to microorganisms contributes to susceptibility to chronic inflammatory bowel disease (IBD). Here, we analyzed transcriptomic variation in monocyte-derived macrophages (MDM) from affected and unaffected sib pairs/trios from 22 IBD families and 6 healthy controls. Transcriptional network analysis of the data revealed no overall or inter-sib distinction between affected and unaffected individuals in basal gene expression or the temporal response to lipopolysaccharide (LPS). However, the basal or LPS-inducible expression of individual genes varied independently by as much as 100-fold between subjects. Extreme independent variation in the expression of pairs of HLA-associated transcripts (HLA-B/C, HLA-A/F and HLA-DRB1/DRB5) in macrophages was associated with HLA genotype. Correlation analysis indicated the downstream impacts of variation in the immediate early response to LPS. For example, variation in early expression of IL1B was significantly associated with local SNV genotype and with subsequent peak expression of target genes including IL23A, CXCL1, CXCL3, CXCL8 and NLRP3. Similarly, variation in early IFNB1 expression was correlated with subsequent expression of IFN target genes. Our results support the view that gene-specific dysregulation in macrophage adaptation to the intestinal milieu is associated with genetic susceptibility to IBD.</p

Finding effective support-tree preconditioners

In 1995, Gremban, Miller, and Zagha introduced support-tree preconditioners and a parallel algorithm called support-tree conjugate gradient (STCG) for solving linear systems of the form Ax = b, where A is an n × n Laplacian matrix. A Laplacian is a symmetric matrix in which the off-diagonal entries are non-positive, and the row and column sums are zero. A Laplacian A with 2m non-zeros can be interpreted as an undirected positively-weighted graph G with n vertices and m edges, where there is an edge between two nodes i and j with weight c((i, j)) = −Ai,j = −Aj,i if Ai,j = Aj,i &lt; 0. Gremban et al. showed experimentally that STCG performs well on several classes of graphs commonly used in scientific computations. In his thesis, Gremban also proved upper bounds on the number of iterations required for STCG to converge for certain classes of graphs. In this paper, we present an algorithm for finding a preconditioner for an arbitrary graph G = (V, E) with n nodes, m edges, and a weight function c&gt; 0 on the edges, where w.l.o.g., mine∈E c(e) = 1. Equipped with this preconditioner, STCG requires O(log 4 n · � ∆/α) iterations, where α = min U⊂V,|U|≤|V |/2 c(U, V \U)/|U | is the minimum edge expansion of the graph, and ∆ = maxv∈V c(v) is the maximum incident weight on any vertex. Each iteration requires O(m) work and can be implemented in O(log n) steps in parallel, using only O(m) space. Our results generalize to matrices that are symmetric and diagonally-dominant (SDD).

beta CCT, an antagonist selective for alpha(1)GABA(A) receptors, reverses diazepam withdrawal-induced anxiety in rats

The abrupt discontinuation of prolonged benzodiazepine treatment elicits a withdrawal syndrome with increased anxiety as a major symptom. The neural mechanisms underlying benzodiazepine physical dependence are still insufficiently understood. Flumazenil, the non-selective antagonist of the benzodiazepine binding site of GABA(A) receptors was capable of preventing and reversing the increased anxiety during benzodiazepine withdrawal in animals and humans in some, but not all studies. On the other hand, a number of data suggest that GABA(A) receptors containing alpha(1) subunits are critically involved in processes developing during prolonged use of benzodiazepines, such are tolerance to sedative effects, liability to physical dependence and addiction. Hence, we investigated in the elevated plus maze the level of anxiety 24 h following 21 days of diazepam treatment and the influence of flumazenil or a preferential alpha(1)-subunit selective antagonist beta CCt on diazepam withdrawal syndrome in rats. Abrupt cessation of protracted once-daily intraperitoneal administration of 2 mg/kg diazepam induced a withdrawal syndrome, measured by increased anxiety-like behavior in the elevated plus maze 24 h after treatment cessation. Acute challenge with either flumazenil (10 mg/kg) or beta CCt (1.25, 5 and 20 mg/kg) alleviated the diazepam withdrawal-induced anxiety. Moreover, both antagonists induced an anxiolytic-like response close, though not identical, to that seen with acute administration of diazepam. These findings imply that the mechanism by which antagonism at GABA(A) receptors may reverse the withdrawal-induced anxiety involves the alpha(1) subunit and prompt further studies aimed at linking the changes in behavior with possible adaptive changes in subunit expression and function of GABA(A) receptors

Duration of treatment and activation of alpha(1)-containing GABA(A) receptors variably affect the level of anxiety and seizure susceptibility after diazepam withdrawal in rats

Long-term use of benzodiazepine-type drugs may lead to physical dependence, manifested by withdrawal syndrome after abrupt cessation of treatment. The aim of the present study was to investigate the influence of duration of treatment, as well as the role of alpha(1)-containing GABA(A) receptors, in development of physical dependence to diazepam, assessed through the level of anxiety and susceptibility to pentylenetetrazole (PTZ)-induced seizures, 24 h after withdrawal from protracted treatment in rats. Withdrawal of 2 mg/kg diazepam after 28, but not after 14 or 21 days of administration led to an anxiety-like behavior in the elevated plus maze. Antagonism of the diazepam effects at alpha(1)-containing GABA(A) receptors, achieved by daily administration of the neutral modulator beta CCt (5 mg/kg), did not affect the anxiety level during withdrawal. An increased susceptibility to PTZ-induced seizures was observed during diazepam withdrawal after 21 and 28 days of treatment. Daily co-administration of beta CCt further decreased the PTZ-seizure threshold after 21 days of treatment, whilst it prevented the diazepam withdrawal-elicited decrease of the PTZ threshold after 28 days of treatment. In conclusion, the current study suggests that the role of alpha(1)-containing GABA(A) receptors in mediating the development of physical dependence may vary based on the effect being studied and duration of protracted treatment. Moreover, the present data supports previous findings that the lack of activity at alpha(1)-containing GABA(A) receptors is not sufficient to eliminate physical dependence liability of ligands of the benzodiazepine type

Design, synthesis, and subtype selectivity of 3,6-disubstituted β-carbolines at Bz/GABA(A)ergic receptors. SAR and studies directed toward agents for treatment of alcohol abuse

A series of 3,6-disubstituted β-carbolines was synthesized and evaluated for their in vitro affinities at αxβ3γ2 GABAA/benzodiazepine receptor subtypes by radioligand binding assays in search of α1 subtype selective ligands to treat alcohol abuse. Analogues of β-carboline-3-carboxylate-t-butyl ester (βCCt, 1) were synthesized via a CDI-mediated process and the related 6-substituted β-carboline-3-carboxylates 6 including WYS8 (7) were synthesized via a Sonogashira or Stille coupling processes from 6-iodo βCCt (5). The bivalent ligands of βCCt (32 and 33) were also designed and prepared via a palladium-catalyzed homocoupling process to expand the structure-activity relationships (SAR) to larger ligands. Based on the pharmacophore/receptor model, a preliminary SAR study on 34 analogues illustrated that large substituents at position -6 of the β-carbolines were well tolerated. As expected, these groups are proposed to project into the extracellular domain (LDi region) of GABAA/Bz receptors (see 32 and 33). Moreover, substituents located at position -3 of the β-carboline nucleus exhibited a conserved stereo interaction in lipophilic pocket L1, while N(2) presumably underwent a hydrogen bonding interaction with H1. Three novel β-carboline ligands (βCCt, 3PBC and WYS8), which preferentially bound to α1 BzR subtypes permitted a comparison of the pharmacological efficacies with a range of classical BzR antagonists (flumazenil, ZK93426) from several different structural groups and indicated these β-carbolines were “near GABA neutral antagonists”. Based on the SAR, the most potent (in vitro) α1 selective ligand was the 6-substituted acetylenyl βCCt (WYS8, 7). Earlier both βCCt and 3PBC had been shown to reduce alcohol self-administration in alcohol preferring (P) and high alcohol drinking (HAD) rats but had little or no effect on sucrose self-administration.1–3 These data prompted the synthesis of the β-carbolines presented here

Sh-I-048A, an in vitro non-selective super-agonist at the benzodiazepine site of GABAA receptors: The approximated activation of receptor subtypes may explain behavioral effects

Enormous progress in understanding the role of four populations of benzodiazepine-sensitive GABA(A) receptors was paralleled by the puzzling findings suggesting that substantial separation of behavioral effects may be accomplished by apparently nonselective modulators. We report on SH-I-048A, a newly synthesized chiral positive modulator of GABAA receptors characterized by exceptional subnanomolar affinity, high efficacy and non-selectivity. Its influence on behavior was assessed in Wistar rats and contrasted to that obtained with 2 mg/kg diazepam. SH-I-048A reached micromolar concentrations in brain tissue, while the unbound fraction in brain homogenate was around 1.5%. The approximated electrophysiological responses, which estimated free concentrations of SH-I-048A or diazepam are able to elicit, suggested a similarity between the 10 mg/kg dose of the novel ligand and 2 mg/kg diazepam; however, SH-I-048A was relatively more active at and as-containing GABAA receptors. Behaviorally, SH-I-048A induced sedative, muscle relaxant and ataxic effects, reversed mechanical hyperalgesia 24 h after injury, while it was devoid of clear anxiolytic actions and did not affect water-maze performance. While lack of clear anxiolytic actions may be connected with an enhanced potentiation at al-containing GABAA receptors, the observed behavior in the rotarod, water maze and peripheral nerve injury tests was possibly affected by its prominent action at receptors containing the as subunit. The current results encourage further innovative approaches aimed at linking in vitro an in vivo data in order to help define fine-tuning mechanisms at four sensitive receptor populations that underlie subtle differences in behavioral profiles of benzodiazepine site ligands

Search for α3β2/3γ2 subtype selective ligands that are stable on human liver microsomes

Selective modulation of specific benzodiazepine receptor (BzR) gamma amino butyric acid-A (GABAA) receptor ion channels has been identified as an important method for separating out the variety of pharmacological effects elicited by BzR-related drugs. Importantly, it has been demonstrated that both α2β(2/3)γ2 (α2BzR) and α3BzR (and/or α2/α3) BzR subtype selective ligands exhibit anxiolytic effects with little or no sedation. Previously we have identified several such ligands; however, three of our parent ligands exhibited significant metabolic liability in rodents in the form of a labile ester group. Here eight analogs are reported which were designed to circumvent this liability by utilizing a rational replacement of the ester moiety based on medicinal chemistry precedents. In a metabolic stability study using human liver microsomes, four compounds were found to undergo slower metabolic transformation, as compared to their corresponding ester analogs. These compounds were also evaluated in in vitro binding as well as efficacy assays. Additionally, bioisostere 11 was evaluated in a rodent model of anxiety. It exhibited anxiolytic activity at doses of 10 and 100 mg/kg and was devoid of sedative properties

Optimization and Stability Testing of Four Commercially Available Dried Blood Spot Devices for Estimating Measles and Rubella IgG Antibodies.

Blood collection using dried blood spots (DBS) provides an easier alternative to venipuncture for sample collection, transport, and storage but requires additional processing that can cause variability in results. Whole-blood samples spotted on four DBS devices and respective paired serum samples were tested for antimeasles and antirubella IgG antibody concentrations by enzyme immunoassay. Elution protocols for DBS devices were optimized for comparability relative to serum samples using 12 adult volunteers. Stability of DBS collected on HemaSpot HF was assessed under various temperature conditions (+4, 22 to 25, and 45°C) at six time points (0, 7, 15, 30, 60, and 90 days) in a controlled laboratory setting using six adult volunteers. Devices were shipped and stored for 30 days at four settings with variable temperature and humidity conditions to assess the impact on antibody concentrations. Three DBS devices demonstrated comparable antibody concentrations with paired sera following optimization. Antibodies recovered from DBS were stable for at least 90 days at 4°C and for 30 days at ambient temperature (22 to 25°C) using the HemaSpot HF device. A drastic decline in antibody concentrations was observed at 45°C, resulting in quantitative and qualitative discrepancies by day 7. HemaSpot HF devices shipped to field sites and stored at ambient temperature and humidity resulted in quantitative, but not qualitative, variability. Measurement of antimeasles and antirubella IgG antibodies with DBS devices is an accurate alternative to testing serum, provided elution protocols are optimized. Stability of HemaSpot HF devices at ambient temperature enables broader use in surveys when serum processing and cold storage are not feasible. IMPORTANCE Dried blood spot (DBS) collection offers various advantages over conventional methods of blood collection, especially when collecting and transporting samples for a serosurvey. Yet use of DBS requires additional processing steps in the laboratory that can add to variability in results. We optimized a protocol to elute IgG antibodies against measles and rubella viruses in four DBS devices, demonstrating high concordance with paired venous sera for most devices. Extensive stability studies with various temperature and storage conditions in the laboratory and in the field were conducted using HemaSpot HF DBS devices prior to its use in one of the largest community-based measles and rubella serological surveys in the world