Knowledge, Attitudes, and Practices (KAP) of community pharmacists regarding antibiotic use and resistance: a cross-sectional study from the United Arab Emirates.

One of the main contributing factors of antibiotic resistance is the dispensing of antibiotics without prescription. This study investigated community pharmacists' knowledge, attitudes, and practices in relation to antibiotic dispensing and resistance in United Arab Emirates (UAE). A cross-sectional survey was conducted using validated questionnaire. (40.1%) had an overall positive KAP score. A total of (88%) respondents were aware of the illegality of dispensing antibiotics without a prescription. Only (31%) had good knowledge regarding amoxicillin dosage for upper respiratory tract infection. The primary misconduct found numerous pharmacists prescribing antibiotics without a prescription, even though they were aware that this should never be done. Pharmacists who attended events focused on antibiotic use and resistance were more likely to have good knowledge about antibiotics (Adjusted Odd Ratio (AOR): 1.673; 95%CI: 1.029-2.719; p = 0.038), more likely to have positive attitude (AOR: 1.889; 95%CI: 1.133-3.149; p = 0.015), and more likely to have good practice (AOR: 3.182; 95%CI: 1.541-6.572; p = 0.002).

Novel methyllycaconitine analogues selective for the α4ß2 over α7 nicotinic acetylcholine receptors.

Analogues of methyllycaconitine (MLA) based on a (3-ethyl-9-methylidene-3-azabicyclo[3.3.1]nonan-1-yl)methanol template have been designed and synthesised that incorporate the modified ester sidechains distinct from that present in the natural product. Electrophysiology experiments using Xenopus oocytes expressing nicotinic acetylcholine receptors (nAChRs) revealed selected analogues served as non-competitive inhibitors that showed selectivity for the α4ß2 over α7 nAChR subtypes, and selectivity for the (α4)3(ß2)2 over (α4)2(ß2)3 stoichiometry. This study more clearly defines the biological effects of MLA analogues and identifies strategies for the development of MLA analogues as selective ligands for the α4ß2 nAChR subtype.

Revisiting autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) mutations in the nicotinic acetylcholine receptor reveal an increase in efficacy regardless of stochiometry.

Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is a genetic form of epilepsy that is caused by mutations in several genes, including genes encoding for the α4 and ß2 subunits of the nicotinic acetylcholine (nACh) receptor. Pentameric α4ß2 nACh receptors are the most abundant nicotinic receptor in the mammalian brain and form two stoichiometries, the (α4)3(ß2)2 and (α4)2(ß2)3 receptors that differ in their physiological and pharmacological properties. The purpose of this study was to investigate how ADNFLE mutations ß2V287M, ß2V287L or α4T293I manifest themselves in different receptor stoichiometries. We expressed wild-type and mutant receptors in Xenopus oocytes and measured the response to ACh and other agonists at both receptor stoichiometries. For all three mutations, the efficacy of ACh at (α4)2(ß2)3 receptors was increased. At (α4)3(ß2)2 receptors, the efficacy of activation was increased both when two molecules of agonist, either ACh or the site-selective agonist sazetidine-A, were bound at the α4-ß2 interfaces, and when a third ACh molecule was bound at the α4-α4 site. Regardless of stoichiometry, the mutations increased the current elicited by low concentrations of ACh. Further, the smoking cessation agents, nicotine, varenicline and cytisine increased activation of mutant (α4)3(ß2)2 receptors, while only nicotine increased activation of mutant (α4)2(ß2)3 receptors. Chronic exposure of all agonists reduced ACh-activation levels at low and high ACh concentrations. From this, we concluded that mutations that cause ADNFLE manifest themselves in a change in efficacy regardless of the stoichiometry of the receptor.

Covalent trapping of methyllycaconitine at the α4-α4 interface of the α4ß2 nicotinic acetylcholine receptor: antagonist binding site and mode of receptor inhibition revealed.

The α4ß2 nicotinic acetylcholine receptors (nAChRs) are widely expressed in the brain and are implicated in a variety of physiological processes. There are two stoichiometries of the α4ß2 nAChR, (α4)2(ß2)3 and (α4)3(ß2)2, with different sensitivities to acetylcholine (ACh), but their pharmacological profiles are not fully understood. Methyllycaconitine (MLA) is known to be an antagonist of nAChRs. Using the two-electrode voltage clamp technique and α4ß2 nAChRs in the Xenopus oocyte expression system, we demonstrate that inhibition by MLA occurs via two different mechanisms; that is, a direct competitive antagonism and an apparently insurmountable mechanism that only occurs after preincubation with MLA. We hypothesized an additional MLA binding site in the α4-α4 interface that is unique to this stoichiometry. To prove this, we covalently trapped a cysteine-reactive MLA analog at an α4ß2 receptor containing an α4(D204C) mutation predicted by homology modeling to be within reach of the reactive probe. We demonstrate that covalent trapping results in irreversible reduction of ACh-elicited currents in the (α4)3(ß2)2 stoichiometry, indicating that MLA binds to the α4-α4 interface of the (α4)3(ß2)2 and providing direct evidence of ligand binding to the α4-α4 interface. Consistent with other studies, we propose that the α4-α4 interface is a structural target for potential therapeutics that modulate (α4)3(ß2)2 nAChRs.

AE Succinimide, an Analogue of Methyllycaconitine, When Bound Generates a Nonconducting Conformation of the α4ß2 Nicotinic Acetylcholine Receptor.

Nicotinic acetylcholine (nACh) receptors are pentameric ligand-gated ion channels that mediate fast synaptic transmission. The α4ß2 nACh receptor is highly expressed in the brain and exists in two functional stoichiometries: the (α4)2(ß2)3 and (α4)3(ß2)2 that differ by an ACh-binding site at the α4-α4 interface of (α4)3(ß2)2 receptors. Methyllycaconitine (MLA) is an nACh receptor antagonist, and while potent at both α7 and α4ß2 nACh receptors, it has a higher selectivity for the α7 nACh receptor. The anthranilate-succinimide ester side-chain is important for its activity and selectivity. Here we identify a simplified MLA analogue that contains only the A and E ring skeleton of MLA, AE succinimide, that binds close to the channel lumen to display insurmountable inhibition at α4ß2 nACh receptors. Although inhibition by AE succinimide was found to be voltage-dependent indicating a possible pore channel blocker, substituted-cysteine accessibility experiments indicated it did not bind between 2'-16' region of the channel pore. Instead, we found that upon binding and in the presence of ACh, there is a conformational change to the channel membrane that was identified when the compound was assessed against (α4 V13'C)ß2 nACh receptors. It was found that in the 3:2 stoichiometry the two adjacent α4 subunits containing 13' cysteine mutations formed a disulfide bond and occluded ion conductance. This was reversed by treatment with the reducing agent, dithiothreitol. Thus, AE succinimide has a different mechanism of inhibition to both MLA and other AE analogues, such as AE bicyclic alcohol, in that upon binding to an as yet unidentified site, AE succinimide in the presence of ACh induces a conformational change to the channel that generates a ligand-bound closed state.