In vitro permeation of nicotine and tobacco specific nitrosamines from smokeless tobacco product extracts in a 3D buccal tissue model.

Tobacco product use is a risk factor in the development of oral cancer, although epidemiology studies show this risk is far less with smokeless tobacco product use than cigarette smoking. While smokeless tobacco contains harmful and potentially harmful constituents (HPHCs), the oral permeation of HPHCs in oral tobacco products is not completely understood. To improve the understanding, three different extract concentrations of the CORESTA reference products (CRP) for snus (CRP1.1) and moist snuff (CRP2.1) were applied to cellular tissue derived from two donors of EpiOral™ model, a 3D human buccal model, and permeation of nicotine and tobacco-specific nitrosamines (TSNAs) were measured over two hours. Permeation of 0.15% caffeine in complete artificial saliva and cell viability were also measured. Results showed that a consistent and concentration dependent cumulative permeation of nicotine and TSNAs was observed with high percent recovery in all conditions. A high degree of sensitivity was seen for all analytes, with minimal cytotoxicity for both CRPs. The data presented here show the EpiOral™ model is fit-for-purpose to evaluate the permeation of nicotine and TSNAs in nicotine-containing snus and moist snuff oral tobacco.

Key Challenges and Recommendations for In Vitro Testing of Tobacco Products for Regulatory Applications: Consideration of Test Materials and Exposure Parameters.

The Institute for In Vitro Sciences (IIVS) is sponsoring a series of workshops to identify, discuss and develop recommendations for optimal scientific and technical approaches for conducting in vitro assays, to assess potential toxicity within and across tobacco and various next generation nicotine and tobacco products (NGPs), including heated tobacco products (HTPs) and electronic nicotine delivery systems (ENDS). The third workshop (24-26 February 2020) summarised the key challenges and made recommendations concerning appropriate methods of test article generation and cell exposure from combustible cigarettes, HTPs and ENDS. Expert speakers provided their research, perspectives and recommendations for the three basic types of tobacco-related test articles: i) pad-collected material (PCM); ii) gas vapour phase (GVP); and iii) whole smoke/aerosol. These three types of samples can be tested individually, or the PCM and GVP can be combined. Whole smoke/aerosol can be bubbled through media or applied directly to cells at the air-liquid interface. Summaries of the speaker presentations and the recommendations developed by the workgroup are presented. Following discussion, the workshop concluded the following: that there needs to be greater standardisation in aerosol generation and collection processes; that methods for testing the NGPs need to be developed and/or optimised, since simply mirroring cigarette smoke testing approaches may be insufficient; that understanding and quantitating the applied dose is fundamental to the interpretation of data and conclusions from each study; and that whole smoke/aerosol approaches must be contextualised with regard to key information, including appropriate experimental controls, environmental conditioning, analytical monitoring, verification and performance criteria.

TC-8831, a nicotinic acetylcholine receptor agonist, reduces L-DOPA-induced dyskinesia in the MPTP macaque.

Long-term L-DOPA treatment for Parkinson's disease (PD) is limited by motor complications, particularly L-DOPA-induced dyskinesia (LID). A therapy with the ability to ameliorate LID without reducing anti-parkinsonian benefit would be of great value. We assessed the ability of TC-8831, an agonist at nicotinic acetylcholine receptors (nAChR) containing α6ß2/α4ß2 subunit combinations, to provide such benefits in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine- (MPTP) lesioned macaques with established LID. Animals were treated orally for consecutive 14-day periods with twice-daily vehicle (weeks 1-2) or TC-8831 (0.03, 0.1 or 0.3 mg/kg, weeks 3-8). L-DOPA was also administered, once-daily, (weeks 1-12, median-dose 30 mg/kg, p.o.). For the following two-weeks (weeks 9-10), TC-8831 was washed out, while once-daily L-DOPA treatment was maintained. The effects of once-daily amantadine (3 mg/kg, p.o.) were then assessed over weeks 11-12. LID, parkinsonism, duration and quality of ON-time were assessed weekly by a neurologist blinded to treatment. TC-8831 reduced the duration of 'bad' ON-time (ON-time with disabling dyskinesia) by up to 62% and decreased LID severity (median score 18 cf. 34 (vehicle), 0.1 mg/kg, 1-3 h period). TC-8831 also significantly reduced choreiform and dystonic dyskinesia (median scores 6 and 31 cf. 19 and 31 respectively (vehicle), both 0.03 mg/kg, 1-3 h). At no time did TC-8831 treatment result in a reduction in anti-parkinsonian benefit of L-DOPA. By comparison, amantadine also significantly reduced dyskinesia and decreased 'bad' ON-time (up to 61%) but at the expense of total ON-time (reduced by up to 23%). TC-8831 displayed robust anti-dyskinetic actions and improved the quality of ON-time evoked by L-DOPA without any reduction in anti-parkinsonian benefit.

Novel nicotinic acetylcholine receptor agonists containing carbonyl moiety as a hydrogen bond acceptor.

A novel series of α4ß2 nAChR agonists lacking common pyridine or its bioisosteric heterocycle have been disclosed. Essential pharmacophoric elements of the series are exocyclic carbonyl moiety as a hydrogen bond acceptor and secondary amino group within diaza- or azabicyclic scaffold. Computer modeling studies suggested that molecular shape of the ligand also contributes to promotion of agonism. Proof of concept for improving working memory performance in a novel object recognition task has been demonstrated on a representative of the series, 3-propionyl-3,7-diazabicyclo[3.3.0]octane (34).

Systemic administration of an alpha-7 nicotinic acetylcholine agonist reverses neuropathic pain in male Sprague Dawley rats.

UNLABELLED: Alpha-7 nicotinic acetylcholine receptor (α7 nAChR) agonists attenuate pain and inflammation in preclinical models. This study tested whether systemic delivery of an α7 nAChR agonist attenuates neuropathic pain and associated immune-mediated pro-inflammation. Hind paw response thresholds to mechanical stimuli in male Sprague Dawley rats were assessed before and after sciatic chronic constriction injury (CCI) or sham surgery. Osmotic mini-pumps containing TC-7020, an α7 nAChR selective agonist, were implanted 10 to 14 days after surgery. TC-7020 (1, 3, and 10 mg/kg/d; s.c.) significantly attenuated CCI-induced allodynia, which lasted through 2 weeks of test compound administration. Spinal cords were collected after 2 weeks and processed for microglial and astrocyte activation markers within the ipsilateral L4-L6 dorsal horn. In addition, ipsilateral L4-5 dorsal root ganglia (DRGs) were processed for neuronal injury and satellite cell activation markers. CCI-induced central glial cell activation markers were not suppressed by TC-7020, even though TC-7020 is mildly blood-brain barrier permeable. However, TC-7020 downregulated the integrated density of activation transcription factor 3 (ATF3) but not the number of ATF positive cells. TC-7020 also downregulated phosphorylated extracellular signal kinase (p-ERK) and satellite cell activation in the CCI-affected DRGs. Therefore, systemic α7 nAChR agonist may be effective in treating neuropathic pain via reducing neuronal injury and immune cells activation occurring in the periphery. PERSPECTIVE: These studies demonstrated that TC-7020, an alpha7 nicotinic acetylcholine receptor agonist with partial blood-brain barrier permeability, reversed neuropathic pain in rats, likely via attenuation of inflammation in the DRG and/or the site of sciatic injury.

Analgesic effects mediated by neuronal nicotinic acetylcholine receptor agonists: correlation with desensitization of α4ß2* receptors.

Nicotinic α4ß2* agonists are known to be effective in a variety of preclinical pain models, but the underlying mechanisms of analgesic action are not well-understood. In the present study, we characterized activation and desensitization properties for a set of seventeen novel α4ß2*-selective agonists that display druggable physical and pharmacokinetic attributes, and correlated the in vitro pharmacology results to efficacies observed in a mouse formalin model of analgesia. ABT-894 and Sazetidine-A, two compounds known to be effective in the formalin assay, were included for comparison. The set of compounds displayed a range of activities at human (α4ß2)(2)ß2 (HS-α4ß2), (α4ß2)(2)α5 (α4ß2α5) and (α4ß2)(2)α4 (LS-α4ß2) receptors. We report the novel finding that desensitization of α4ß2* receptors may drive part of the antinociceptive outcome. Our molecular modeling approaches revealed that when receptor desensitization rather than activation activitiesat α4ß2* receptors are considered, there is a better correlation between analgesia scores and combined in vitro properties. Our results suggest that although all three α4ß2 subtypes assessed are involved, it is desensitization of α4ß2α5 receptors that plays a more prominent role in the antinociceptive action of nicotinic compounds. For modulation of Phase I responses, correlations are significantly improved from an r(2) value of 0.53 to 0.67 and 0.66 when HS- and LS-α4ß2 DC(50) values are considered, respectively. More profoundly, considering the DC(50) at α4ß2α5 takes the r(2) from 0.53 to 0.70. For Phase II analgesia scores, adding HS- or LS-α4ß2 desensitization potencies did not improve the correlations significantly. Considering the α4ß2α5 DC(50) value significantly increased the r(2) from 0.70 to 0.79 for Phase II, and strongly suggested a more prominent role for α4ß2α5 nAChRs in the modulation of pain in the formalin assay. The present studies demonstrate that compounds which are more potent at desensitization of α4ß2* receptors display better analgesia scores in the formalin test. Consideration of desensitization propertiesat α4ß2* receptors, especially at α4ß2α5, in multiple linear regression analyses significantly improves correlations with efficacies of analgesia. Thus, α4ß2* nicotinic acetylcholine receptor desensitization may contribute to efficacy in the mediation of pain, and represent a mechanism for analgesic effects mediated by nicotinic agonists.

Discovery of 3-(5-chloro-2-furoyl)-3,7-diazabicyclo[3.3.0]octane (TC-6683, AZD1446), a novel highly selective α4ß2 nicotinic acetylcholine receptor agonist for the treatment of cognitive disorders.

Diversification of essential nicotinic cholinergic pharmacophoric elements, i.e., cationic center and hydrogen bond acceptor, resulted in the discovery of novel potent α4ß2 nAChR selective agonists comprising a series of N-acyldiazabicycles. Core characteristics of the series are an exocyclic carbonyl moiety as a hydrogen bond acceptor and endocyclic secondary amino group. These features are positioned at optimal distance and with optimal relative spatial orientation to provide near optimal interactions with the receptor. A novel potent and highly selective α4ß2 nAChR agonist 3-(5-chloro-2-furoyl)-3,7-diazabicyclo[3.3.0]octane (56, TC-6683, AZD1446) with favorable pharmaceutical properties and in vivo efficacy in animal models has been identified as a potential treatment for cognitive deficits associated with psychiatric or neurological conditions and is currently being progressed to phase 2 clinical trials as a treatment for Alzheimer's disease.

TC-5214 (S-(+)-mecamylamine): a neuronal nicotinic receptor modulator with antidepressant activity.

Both clinical and preclinical data support a potential therapeutic benefit of modulating the activity of CNS neuronal nicotinic receptors (NNRs) to treat depression and anxiety disorders. Based on the notion that the depressive states involve hypercholinergic tone, we have examined the potential palliative role of NNR antagonism in these disorders, using TC-5214 (S-(+) enantiomer of mecamylamine), a noncompetitive NNR antagonist. TC-5214 demonstrated positive effects in a number of animal models of depression and anxiety. TC-5214 was active in the forced swim test in rats (minimum effective dose (MED)=3 mg/kg i.p.), a classical depression model. It was also active in the behavioral despair test in mice (0.1-3.0 mg/kg i.p.), another model of depression. In the social interaction paradigm in rats, a model of generalized anxiety disorder (GAD), TC-5214 was active at a dose of 0.05 mg/kg s.c. In the light/dark chamber paradigm in rats, a model of GAD and phobia, TC-5214 was also active at a dose of 0.05 mg/kg s.c. Although TC-5214 shows modest selectivity among NNR subtypes, the antidepressant and anxiolytic effects seen in these studies are likely attributable to antagonist effects at the alpha4beta2 NNRs. This is supported by the observation of similar effects with alpha4beta2-selective partial agonists such as cytisine and with alpha4beta2-selective antagonists such as TC-2216. TC-5214 was well tolerated in acute and chronic toxicity studies in mice, rats, and dogs, showed no mutagenicity and displayed safety pharmacology, pharmacokinetic and metabolic profiles appropriate for therapeutic development. Overall, the results support a novel nicotinic cholinergic antagonist mechanism for antidepressant and anxiolytic effects and highlight the potential of NNR antagonists such as TC-5214 as therapeutics for the treatment of anxiety and depression.

Therapeutic potential of novel selective drugs targeting nicotinic acetylcholine receptors.

The potential therapeutic benefit of nicotinic ligands in a variety of neurodegenerative pathologies involving the CNS has energized research efforts to develop nicotinic acetylcholine receptor (nAChR) subtype-selective ligands (Bencherif and Schmitt, 2005). In particular, there has been a concerted effort to develop nicotinic compounds with selectivity for CNS nAChRs as potential pharmaceutical tools in the management of these disorders. Clinical and experimental data demonstrate a central role for alpha7 and alpha4beta2 nAChRs in cognitive function, sensory processing, mood, and neuroprotection (Bencherif and Schmitt, 2005; Buccafusco et al., 2005). The development of safe alpha7-selective ligands has been hampered by their lack of discrimination with hERG channels and 5-HT3 receptors. We have developed a number of compounds that display nanomolar affinity to the alpha7 and/or the alpha4beta2 receptor. Investigation of alpha7 functional activity showed a full range of activities from antagonists to full agonists without any significant activity at the human 5-HT3 receptor, P450 isozymes, hERG channels, or in the AMES test. Our findings demonstrate that potent and highly selective nAChR ligands can be designed.