Abstract
IntroductionThe cholinergic anti-inflammatory pathway can downregulate inflammation via the release of acetylcholine (ACh) by the vagus nerve. This neurotransmitter binds to the α7 subunit of nicotinic acetylcholine receptors (α7nAChR), expressed on macrophages and other immune cells. We tested the pharmacological and functional profile of two novel compounds, PMP-311 and PMP-072 and investigated their role in modulating collagen-induced arthritis (CIA) in mice.MethodsBoth compounds were characterized with binding, electrophysiological, and pharmacokinetic studies. For in vivo efficacy studies in the CIA model the compounds were administered daily by oral gavage from day 20 till sacrifice at day 34. Disease progression was monitored by visual clinical scoring and measurement of paw swelling. Inflammation and joint destruction were examined by histology and radiology.ResultsTreatment with PMP-311 was effective in preventing disease onset, reducing clinical signs of arthritis, and reducing synovial inflammation and bone destruction. PMP-072 also showed a trend in arthritis reduction at all concentrations tested. The data showed that while both compounds bind to α7nAChR with high affinity, PMP-311 acts like a classical agonist of ion channel activity, and PMP-072 can actually act as an ion channel antagonist. Moreover, PMP-072 was clearly distinct from typical competitive antagonists, since it was able to act as a silent agonist. It synergizes with the allosteric modulator PNU-120596, and subsequently activates desensitized α7nAChR. However, PMP-072 was less efficacious than PMP-311 at both channel activation and desensitization, suggesting that both conducting and non-conducting states maybe of importance in driving an anti-inflammatory response. Finally, we found that the anti-arthritic effect can be observed despite limited penetration of the central nervous system.ConclusionsThese data provide direct evidence that the α7nAChR in immune cells does not require typical ion channel activation to exert its antiinflammatory effects.
Highlights
The cholinergic anti-inflammatory pathway can downregulate inflammation via the release of acetylcholine (ACh) by the vagus nerve
The introduction of anti-tumor necrosis factor (TNF) therapy and other new biologicals has played a major role in improving patient outcomes, Rheumatoid arthritis (RA) is still associated with long-term morbidity and early mortality
[3] there is still a need for the identification of new pathways involved in the modulation of inflammation, which could help to increase the efficacy of the RA treatment
Summary
Both compounds were characterized with binding, electrophysiological, and pharmacokinetic studies. Binding studies with rat α7 nAChR were done using the rat pheochromocytoma cell line PC12 that endogenously expresses the α7 nAChR (American Type Culture Collection, Manassas, VA). Test compounds were diluted in binding buffer, to 2.2 times the desired final concentration, and 55 μl was added to the cells; 55 μl binding buffer was added to the cells in the control wells (total binding, non-specific binding, and cell controls; n = 1–3). Biotinylated α-bungarotoxin (BTx) (Invitrogen) was added to the cells (excluding the cell control) for a final concentration of 10 nM. An excess of unlabeled BTx was added to the non-specific binding (NSB) control at a final concentration of 1.5 μM. The samples were incubated at room temperature for 1.0 to 1.5 hour (s) and thereafter the cells were washed one time with binding buffer to remove unbound BTx
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