Adenosine A1 Receptor Subtype Research Articles

Covalently Binding Adenosine A3 Receptor Agonist ICBM Irreversibly Reduces Voltage-Gated Ca2+ Currents in Dorsal Root Ganglion Neurons

Interest has been focused in recent years on the analgesic effects exerted by adenosine and its receptors, A1, A2A, A2B, and A3 adenosine receptor (AR) subtypes, in different in vivo models of chronic pain. In particular, it was demonstrated that selective A3AR agonists reduced pro-nociceptive N-type Ca2+ channels in dorsal root ganglion (DRG) neurons isolated from rats and, by this mechanism, inhibit post inflammatory visceral hypersensitivity. In the present study, we investigate the effect of a previously reported irreversibly binding A3AR agonist, ICBM, on Ca2+ currents (ICa) in rat DRG neurons. Present data demonstrate that ICBM, an isothiocyanate derivative designed for covalent binding to the receptor, concentration-dependently inhibits ICa. This effect is irreversible, since it persists after drug removal, differently from the prototypical A3AR agonist, Cl-IB-MECA. ICBM pre-exposure inhibits the effect of a subsequent Cl-IB-MECA application. Thus, covalent A3AR agonists such as ICBM may represent an innovative, beneficial, and longer-lasting strategy to achieve efficacious chronic pain control versus commonly used, reversible, A3AR agonists. However, the possible limitations of this drug and other covalent drugs may be, for example, a characteristic adverse effect profile, suggesting that more pre-clinical studies are needed.

Adenosine Receptors Expression in Human Retina and Choroid with Age-related Macular Degeneration.

Adenosine signaling modulates ocular inflammatory processes, and its antagonism mitigates neovascularization in both newborns and preclinical models of ocular neovascularization including age-related macular degeneration (AMD). The adenosine receptor expression patterns have not been well characterized in the human retina and choroid. Here we examined the expression of adenosine receptor subtypes within the retina and choroid of human donor eyes with and without AMD. Antibodies specifically targeting adenosine receptor subtypes A1, A2A, A2B, and A3 were used to assess their expression patterns. Quantitative real-time PCR analysis was used to confirm gene expression of these receptors within the normal human retina and choroid. We found that all four receptor subtypes were expressed in several layers of the retina, and within the retinal pigment epithelium and choroid. The expression of A1 receptors was more prominent in the inner and outer plexiform layers, where microglia normally reside, and supported by RNA expression in the retina. A2A and A2B showed similar expression patterns with prominent expression in the vasculature and retinal pigment epithelium. No dramatic differences in expression of these receptors were observed in eyes from patients with dry or wet AMD compared to control, with the exception A3 receptors. Eyes with dry AMD lost expression of A3 in the photoreceptor outer segments compared with eyes from control or wet AMD. The ocular presence of adenosine receptors is consistent with their proposed role in modulation of inflammation in both the retina and choroid, and their potential targeting for AMD treatment.

Species dependence of A3 adenosine receptor pharmacology and function.

Efforts to fully understand pharmacological differences between G protein-coupled receptor (GPCR) species homologues are generally not pursued in detail during the drug development process. To date, many GPCRs that have been successfully targeted are relatively well-conserved across species in amino acid sequence and display minimal variability of biological effects. However, the A3 adenosine receptor (AR), an exciting drug target for a multitude of diseases associated with tissue injury, ischemia, and inflammation, displays as little as 70% sequence identity among mammalian species (e.g., rodent vs. primate) commonly used in drug development. Consequently, the pharmacological properties of synthetic A3AR ligands vary widely, not only in binding affinity, selectivity, and signaling efficacy, but to the extent that some function as agonists in some species and antagonists in others. Numerous heterocyclic antagonists that have nM affinity at the human A3AR are inactive or weakly active at the rat and mouse A3ARs. Positive allosteric modulators, including the imidazo [4,5-c]quinolin-4-amine derivative LUF6000, are only active at human and some larger animal species that have been evaluated (rabbit and dog), but not rodents. A3AR agonists evoke systemic degranulation of rodent, but not human mast cells. The rat A3AR undergoes desensitization faster than the human A3AR, but the human homologue can be completely re-sensitized and recycled back to the cell surface. Thus, comprehensive pharmacological evaluation and awareness of potential A3AR species differences are critical in studies to further understand the basic biological functions of this unique AR subtype. Recombinant A3ARs from eight different species have been pharmacologically characterized thus far. In this review, we describe in detail current knowledge of species differences in genetic identity, G protein-coupling, receptor regulation, and both orthosteric and allosteric A3AR pharmacology.

Targeting the A3 adenosine receptor to prevent and reverse chemotherapy-induced neurotoxicities in mice

Cisplatin is used to combat solid tumors. However, patients treated with cisplatin often develop cognitive impairments, sensorimotor deficits, and peripheral neuropathy. There is no FDA-approved treatment for these neurotoxicities. We investigated the capacity of a highly selective A3 adenosine receptor (AR) subtype (A3AR) agonist, MRS5980, to prevent and reverse cisplatin-induced neurotoxicities. MRS5980 prevented cisplatin-induced cognitive impairment (decreased executive function and impaired spatial and working memory), sensorimotor deficits, and neuropathic pain (mechanical allodynia and spontaneous pain) in both sexes. At the structural level, MRS5980 prevented the cisplatin-induced reduction in markers of synaptic integrity. In-situ hybridization detected Adora3 mRNA in neurons, microglia, astrocytes and oligodendrocytes. RNAseq analysis identified 164 genes, including genes related to mitochondrial function, of which expression was changed by cisplatin and normalized by MRS5980. Consistently, MRS5980 prevented cisplatin-induced mitochondrial dysfunction and decreased signs of oxidative stress. Transcriptomic analysis showed that the A3AR agonist upregulates genes related to repair pathways including NOTCH1 signaling and chromatin modification in the cortex of cisplatin-treated mice. Importantly, A3AR agonist administration after completion of cisplatin treatment resolved cognitive impairment, neuropathy and sensorimotor deficits. Our results highlight the efficacy of a selective A3AR agonist to prevent and reverse cisplatin-induced neurotoxicities via preventing brain mitochondrial damage and activating repair pathways. An A3AR agonist is already in cancer, clinical trials and our results demonstrate management of neurotoxic side effects of chemotherapy as an additional therapeutic benefit.

Effects of Shenfu injection on myocardial adenosine receptors in rats with myocardial ischemia-reperfusion postconditioning.

Shenfu injection (SFI) has been reported to have a protection against myocardial ischemia-reperfusion (MI/R) injury. However, the changes of adenosine receptors in MI/R postconditioning when pretreated with SFI are unclear. Forty-five rats were randomly divided into sham group (sham), MI/R postconditioning group (MI/R-post), low-dose SFI group (1mL/kg), middle-dose SFI group (2.5mL/kg), and high-dose SFI group (5mL/kg). In SFI groups, SFI was intravenously injected before reperfusion, and rats were treated with ischemic postconditioning after ischemia for 30min. After 24h of reperfusion, the levels of Ca2+ and cAMP in blood platelets were analyzed. Myocardial infarct volume and myocardial pathology were observed. The levels of adenosine receptor subtypes A1, A2b, and A3 in myocardium were analyzed using immunohistochemistry and Western blot. The oxidative stress-related indicators were also observed. Compared with the MI/R-post group, SFI ameliorated the MI/R injury by decreasing the myocardial infarct area, oxidative stress, and concentration of Ca2+ and cAMP (p < 0.01). Pretreatment with SFI enhanced the expression of adenosine receptors A1 and A2b in a dose manner compared with the MI/R-post group. In contrast, the levels of adenosine receptor A3 were increased after MI/R postconditioning compared with the sham group, and its expression continued to increase with the increase of SFI. Furthermore, the oxidative stress reduced with the concentrations of SFI. These results demonstrated that pretreatment with SFI might regulate the expression of adenosine receptors to improve the MI/R postconditioning.

Adenosine and Ketogenic Treatments.

It is well known that the neuromodulator adenosine, acting through the adenosine A1 receptor subtype, can limit or stop seizures. In 2008, adenosine was proposed as a key component of the anticonvulsant mechanism of the ketogenic diet (KD), a very low carbohydrate diet that can be highly effective in drug-refractory epilepsy. In this study, we review the accumulated data on the intersection among adenosine, ketosis, and anticonvulsant/antiepileptogenic effects. In several rodent models of epilepsy and seizures, antiseizure effects of ketogenic treatments (the KD itself, exogenous ketone bodies, medium-chain triglycerides or fatty acids) are reversed by administration of an adenosine A1 receptor antagonist. In addition, KD treatment elevates extracellular adenosine and tissue adenosine content in brain. Efforts to maintain or mimic a ketogenic milieu in brain slices reveal a state of reduced excitability produced by pre- and postsynaptic adenosine A1 receptor-based effects. Long-lasting seizure reduction may be due to adenosine-based epigenetic effects. In conclusion, there is accumulating evidence for an adenosinergic anticonvulsant action in the ketogenic state. In some cases, the main trigger is mildly but consistently lowered glucose in the brain. More research is needed to investigate the importance of adenosine in the antiepileptogenic and neuroprotective effects of these treatments. Future research may begin to investigate alternative adenosine-promoting strategies to enhance the KD or to find use as treatments themselves.

The Adenosine System at the Crossroads of Intestinal Inflammation and Neoplasia.

Adenosine is a purine nucleoside, resulting from the degradation of adenosine triphosphate (ATP). Under adverse conditions, including hypoxia, ischemia, inflammation, or cancer, the extracellular levels of adenosine increase significantly. Once released, adenosine activates cellular signaling pathways through the engagement of the four known G-protein-coupled receptors, adenosine A1 receptor subtype (A1), A2A, A2B, and A3. These receptors, expressed virtually on all immune cells, mitigate all aspects of immune/inflammatory responses. These immunosuppressive effects contribute to blunt the exuberant inflammatory responses, shielding cells, and tissues from an excessive immune response and immune-mediated damage. However, a prolonged persistence of increased adenosine concentrations can be deleterious, participating in the creation of an immunosuppressed niche, ideal for neoplasia onset and development. Based on this evidence, the present review has been conceived to provide a comprehensive and critical overview of the involvement of adenosine system in shaping the molecular mechanisms underlying the enteric chronic inflammation and in promoting the generation of an immunosuppressive niche useful for the colorectal tumorigenesis.

Selective Antagonism of A1 Adenosinergic Receptors Strengthens the Neuromodulation of the Sensorimotor Network During Epidural Spinal Stimulation.

Although epidural spinal stimulation (ESS) results in promising therapeutic effects in individuals with spinal cord injury (SCI), its potential to generate functional motor recovery varies between individuals and remains largely unclear. However, both preclinical and clinical studies indicate the capacity of electrical and pharmacological interventions to synergistically increase the engagement of spinal sensorimotor networks and regain motor function after SCI. This study explored whether selective pharmacological antagonism of the adenosine A1 receptor subtype synergizes with ESS, thereby increasing motor response. We hypothesized that selective pharmacological antagonism of A1 receptors during ESS would produce facilitatory effects in spinal sensorimotor networks detected as an increased amplitude of spinally-evoked motor potentials and sustained duration of ESS induced activity. Terminal experiments were performed in adult rats using trains of stereotyped pulses at 40 Hz delivered at L5 with the local administration to the cord of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). We demonstrated that ESS combined with the blockage of A1 receptors increased the magnitude of the endogenous modulation and postponed the decay of responses that occur during ESS alone. Although DPCPX significantly increased the yield of repetitive stimulation in intact spinal cords, the effects of A1 antagonism on motor evoked responses after an acute spinal transection was not detected. These studies support the future investigation of the optimal dosage, methods of delivery, and systemic effects of the synergistic application of A1 antagonists and spinal stimulation in the intact and injured spinal cord.

Sexually dimorphic therapeutic response in bortezomib-induced neuropathic pain reveals altered pain physiology in female rodents.

Chemotherapy-induced neuropathic pain (CINP) in both sexes compromises many current chemotherapeutics and lacks an FDA-approved therapy. We recently identified the sphingosine-1-phosphate receptor subtype 1 (S1PR1) and A3 adenosine receptor subtype (A3AR) as novel targets for therapeutic intervention. Our work in male rodents using paclitaxel, oxaliplatin, and bortezomib showed robust inhibition of CINP with either S1PR1 antagonists or A3AR agonists. The S1PR1 functional antagonist FTY720 (Gilenya) is FDA-approved for treating multiple sclerosis, and selective A3AR agonists are in advanced clinical trials for cancer and inflammatory disorders, underscoring the need for their expedited trials in patients with CINP as chemotherapy adjuncts. Our findings reveal that S1PR1 antagonists and A3AR agonists mitigate paclitaxel and oxaliplatin CINP in female and male rodents, but failed to block or reverse bortezomib-induced neuropathic pain (BINP) in females. Although numerous mechanisms likely underlie these differences, we focused on receptor levels. We found that BINP in male rats, but not in female rats, was associated with increased expression of A3AR in the spinal cord dorsal horn, whereas S1PR1 levels were similar in both sexes. Thus, alternative mechanisms beyond receptor expression may account for sex differences in response to S1PR1 antagonists. Morphine and duloxetine, both clinical analgesics, reversed BINP in female mice, demonstrating that the lack of response is specific to S1PR1 and A3AR agents. Our findings suggest that A3AR- and S1PR1-based therapies are not viable approaches in preventing and treating BINP in females and should inform future clinical trials of these drugs as adjuncts to chemotherapy.

Free-Energy Calculations for Bioisosteric Modifications of A3 Adenosine Receptor Antagonists.

Adenosine receptors are a family of G protein-coupled receptors with increased attention as drug targets on different indications. We investigate the thermodynamics of ligand binding to the A3 adenosine receptor subtype, focusing on a recently reported series of diarylacetamidopyridine inhibitors via molecular dynamics simulations. With a combined approach of thermodynamic integration and one-step perturbation, we characterize the impact of the charge distribution in a central heteroaromatic ring on the binding affinity prediction. Standard charge distributions according to the GROMOS force field yield values in good agreement with the experimental data and previous free energy calculations. Subsequently, we examine the thermodynamics of inhibitor binding in terms of the energetic and entropic contributions. The highest entropy penalties are found for inhibitors with methoxy substituents in meta position of the aryl groups. This bulky group restricts rotation of aromatic rings attached to the pyrimidine core which leads to two distinct poses of the ligand. Our predictions support the previously proposed binding pose for the o-methoxy ligand, yielding in this case a very good correlation with the experimentally measured affinities with deviations below 4 kJ/mol.

Multiple adenosine receptor subtypes stimulate wound healing in human EA.hy926 endothelial cells.

Wound healing is an important outcome of tissue damage and can be stimulated by adenosine released from cells during events such as tissue injury, ischaemia or tumour growth. The aim of this research was to determine the potency and efficacy of adenosine A1, A2A and A2B receptor agonists on the rate of wound healing and cell proliferation in human EA.hy926 endothelial cells. Real-time PCR data showed that only adenosine A1, A2A and A2B receptor mRNA were expressed in this cell line. All three adenosine receptor agonists, CPA, CGS21680 and NECA, significantly increased the rate of wound healing in human EAhy926 endothelial cells with the following order of potency CGS21680>CPA>NECA and efficacy CPA>NECA>CGS21680. The selective adenosine A1, A2A and A2B receptor antagonists, DPCPX, ZM241385 and MRS1754 (all at 10nM), reversed the effects of their respective agonists. EAhy926 endothelial cell proliferation was also significantly increased with the adenosine A1 and A2B receptor agonists, CPA and NECA. Western blot analysis demonstrated that adenosine A2A and A1 receptor protein levels were highly expressed compared with the adenosine A2B receptors in the EAhy926 endothelial cell lines. While all three adenosine A1, A2A and A2B receptor subtypes contribute to cell proliferation and wound healing in human EAhy926 endothelial cells, treatments selectively targeting receptor subtypes may further enhance wound healing.

2-Aryladenine derivatives as a potent scaffold for A1, A3 and dual A1/A3 adenosine receptor antagonists: Synthesis and structure-activity relationships

From a collection containing more than 1500 academic compounds, in silico screening identified a hit for the human A1 adenosine receptor containing a new purine scaffold. To study the structure activity relationships of this new chemical series for adenosine receptors, a library of 24 purines was synthesized and tested in radioligand binding assays at human A1, A2A, A2B and A3 adenosine receptor subtypes. Fourteen molecules showed potent antagonism at A1, A3 or dual A1/A3 adenosine receptors. This purine scaffold is an important source for novel biochemical tools and/or therapeutic drugs.

1,2,4]Triazolo[1,5-c]pyrimidines as adenosine receptor antagonists: Modifications at the 8 position to reach selectivity towards A3 adenosine receptor subtype

[1,2,4]Triazolo[1,5-c]pyrimidine is a promising platform to develop adenosine receptor antagonists. Here, we tried to investigate the effect of the substituent at the 8 position of [1,2,4]triazolo[1,5-c]pyrimidine derivatives on affinity and selectivity at the human A3 adenosine receptor subtype. In particular, we have introduced both esters and amides, principally with a benzylic nature. In addition, a small series of 5-substituted [1,2,4]triazolo[1,5-c]pyrimidines was designed in order to complete the structure-activity relationship analysis. Several of these new compounds showed affinity towards human A3 adenosine receptor in the low nanomolar range, with the most potent derivative of the series bringing a 4-ethylbenzylester at the 8 position (compound 18, hA3AR Ki = 1.21 nM). Docking studies performed on the synthesized compounds inside models of human A1, A2A and A3 adenosine receptors showed similar binding modes, comparable with the typical crystallographic binding mode of the inverse agonist ZM-241,385.

A3 Adenosine Receptors as Modulators of Inflammation: From Medicinal Chemistry to Therapy.

The A3 adenosine receptor (A3 AR) subtype is a novel, promising therapeutic target for inflammatory diseases, such as rheumatoid arthritis (RA) and psoriasis, as well as liver cancer. A3 AR is coupled to inhibition of adenylyl cyclase and regulation of mitogen-activated protein kinase (MAPK) pathways, leading to modulation of transcription. Furthermore, A3 AR affects functions of almost all immune cells and the proliferation of cancer cells. Numerous A3 AR agonists, partial agonists, antagonists, and allosteric modulators have been reported, and their structure-activity relationships (SARs) have been studied culminating in the development of potent and selective molecules with drug-like characteristics. The efficacy of nucleoside agonists may be suppressed to produce antagonists, by structural modification of the ribose moiety. Diverse classes of heterocycles have been discovered as selective A3 AR blockers, although with large species differences. Thus, as a result of intense basic research efforts, the outlook for development of A3 AR modulators for human therapeutics is encouraging. Two prototypical selective agonists, N6-(3-Iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA; CF101) and 2-chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (Cl-IB-MECA; CF102), have progressed to advanced clinical trials. They were found safe and well tolerated in all preclinical and human clinical studies and showed promising results, particularly in psoriasis and RA, where the A3 AR is both a promising therapeutic target and a biologically predictive marker, suggesting a personalized medicine approach. Targeting the A3 AR may pave the way for safe and efficacious treatments for patient populations affected by inflammatory diseases, cancer, and other conditions.

The role of 5-arylalkylamino- and 5-piperazino- moieties on the 7-aminopyrazolo[4,3-d]pyrimidine core in affecting adenosine A1 and A2A receptor affinity and selectivity profiles

New 7-amino-2-phenylpyrazolo[4,3-d]pyrimidine derivatives, substituted at the 5-position with aryl(alkyl)amino- and 4-substituted-piperazin-1-yl- moieties, were synthesized with the aim of targeting human (h) adenosine A1 and/or A2A receptor subtypes. On the whole, the novel derivatives 1–24 shared scarce or no affinities for the off-target hA2B and hA3 ARs. The 5-(4-hydroxyphenethylamino)- derivative 12 showed both good affinity (Ki = 150 nM) and the best selectivity for the hA2A AR while the 5-benzylamino-substituted 5 displayed the best combined hA2A (Ki = 123 nM) and A1 AR affinity (Ki = 25 nM). The 5-phenethylamino moiety (compound 6) achieved nanomolar affinity (Ki = 11 nM) and good selectivity for the hA1 AR. The 5-(N4-substituted-piperazin-1-yl) derivatives 15–24 bind the hA1 AR subtype with affinities falling in the high nanomolar range. A structure-based molecular modeling study was conducted to rationalize the experimental binding data from a molecular point of view using both molecular docking studies and Interaction Energy Fingerprints (IEFs) analysis.

β-Nicotinamide adenine dinucleotide acts at prejunctional adenosine A1 receptors to suppress inhibitory musculomotor neurotransmission in guinea pig colon and human jejunum.

Intracellular microelectrodes were used to record neurogenic inhibitory junction potentials in the intestinal circular muscle coat. Electrical field stimulation was used to stimulate intramural neurons and evoke contraction of the smooth musculature. Exposure to β-nicotinamide adenine dinucleotide (β-NAD) did not alter smooth muscle membrane potential in guinea pig colon or human jejunum. ATP, ADP, β-NAD, and adenosine, as well as the purinergic P2Y1 receptor antagonists MRS 2179 and MRS 2500 and the adenosine A1 receptor agonist 2-chloro-N6-cyclopentyladenosine, each suppressed inhibitory junction potentials in guinea pig and human preparations. β-NAD suppressed contractile force of twitch-like contractions evoked by electrical field stimulation in guinea pig and human preparations. P2Y1 receptor antagonists did not reverse this action. Stimulation of adenosine A1 receptors with 2-chloro-N6-cyclopentyladenosine suppressed the force of twitch contractions evoked by electrical field stimulation in like manner to the action of β-NAD. Blockade of adenosine A1 receptors with 8-cyclopentyl-1,3-dipropylxanthine suppressed the inhibitory action of β-NAD on the force of electrically evoked contractions. The results do not support an inhibitory neurotransmitter role for β-NAD at intestinal neuromuscular junctions. The data suggest that β-NAD is a ligand for the adenosine A1 receptor subtype expressed by neurons in the enteric nervous system. The influence of β-NAD on intestinal motility emerges from adenosine A1 receptor-mediated suppression of neurotransmitter release at inhibitory neuromuscular junctions.

Novel thiazole-thiophene conjugates as adenosine receptor antagonists: synthesis, biological evaluation and docking studies.

Here we report novel thiazole-thiophene conjugates as adenosine receptor antagonists. All the molecules were evaluated for their binding affinity for adenosine receptors. Most of the molecules were found to interact with the A1, A2A and A3 adenosine receptor subtypes with good affinity values. The most potent and selective compound 8n showed an A3Ki value of 0.33μM with selectivity ratios of >90 versus the A1 and >30 versus the A2 subtypes. For compound 8n docking studies into the binding site of the A3 adenosine receptor are provided to visualize its binding mode.

Role of adenosine receptor subtypes in methamphetamine reward and reinforcement

The neurobiology of methamphetamine (MA) remains largely unknown despite its high abuse liability. The present series of studies explored the role of adenosine receptors on MA reward and reinforcement and identified alterations in the expression of adenosine receptors in dopamine terminal areas following MA administration in rats. We tested whether stimulating adenosine A1 or A2A receptor subtypes would influence MA-induced place preference or MA self-administration on fixed and progressive ratio schedules in male Sprague–Dawley rats. Stimulation of either adenosine A1 or A2A receptors significantly reduced the development of MA-induced place preference. Stimulating adenosine A1, but not A2A, receptors reduced MA self-administration responding. We next tested whether repeated experimenter-delivered MA administration would alter the expression of adenosine receptors in the striatal areas using immunoblotting. We observed no change in the expression of adenosine receptors. Lastly, rats were trained to self-administer MA or saline for 14 days and we detected changes in adenosine A1 and A2A receptor expression using immunoblotting. MA self-administration significantly increased adenosine A1 in the nucleus accumbens shell, caudate-putamen and prefrontal cortex. MA self-administration significantly decreased adenosine A2A receptor expression in the nucleus accumbens shell, but increased A2A receptor expression in the amygdala. These findings demonstrate that MA self-administration produces selective alterations in adenosine receptor expression in the nucleus accumbens shell and that stimulation of adenosine receptors reduces several behavioral indices of MA addiction. Together, these studies shed light onto the neurobiological alterations incurred through chronic MA use that may aid in the development of treatments for MA addiction.

Characterization of the Adenosine Pharmacology of Ticagrelor Reveals Therapeutically Relevant Inhibition of Equilibrative Nucleoside Transporter 1

Studies have shown that ticagrelor has a further adenosine-mediated mechanism of action in addition to its potent inhibition of the P2Y12 receptor, which may explain some of ticagrelor's clinical characteristics. This study aimed to further characterize the adenosine pharmacology of ticagrelor, its major metabolites, and other P2Y12 receptor antagonists. Inhibition of nucleoside transporter-mediated [(3)H]adenosine uptake by ticagrelor, its major metabolites, and alternative P2Y12 antagonists was examined in recombinant Madin-Darby canine kidney (MDCK) cells. The pharmacology of ticagrelor and its major metabolites at adenosine A1, A2A, A2B, and A3 receptor subtypes was examined using in vitro radioligand binding and functional assays and ex vivo C-fiber experiments in rat and guinea pig vagus nerves. Ticagrelor (and less effectively its metabolites) and the main cangrelor metabolite inhibited [(3)H]adenosine uptake in equilibrative nucleoside transporter (ENT) 1-expressing MDCK cells, whereas cangrelor and the active metabolites of prasugrel or clopidogrel had no effect. No significant inhibitory activity was observed in MDCK cells expressing ENT2 or concentrative nucleoside transporters 2/3. Ticagrelor demonstrated high affinity (inhibition constant [Ki] = 41 nmol/L) for ENT1. In adenosine receptor-binding experiments, ticagrelor and its major circulating metabolite, AR-C124910XX, had low affinity (Ki > 6 µmol/L) for each of the adenosine A1, A2A, and A2B receptors, whereas ticagrelor had a submicromolar (Ki = 190 nmol/L) affinity for the adenosine A3 receptor. However, in functional assays, at high concentrations (10 µmol/L) ticagrelor only partially inhibited 3 mmol/L adenosine-induced depolarizations in the guinea pig and rat vagus nerve preparations (by 35% and 49%, respectively). Ticagrelor inhibits cellular adenosine uptake selectively via ENT1 inhibition at concentrations of clinical relevance. However, the low-binding affinity and functional inhibition of adenosine receptors observed with ticagrelor or its metabolites indicate that they possess a negligible adenosine-like activity at clinically relevant concentrations.

Chapter Eleven - Adenosine Receptors and Epilepsy: Current Evidence and Future Potential

Adenosine receptors are a powerful therapeutic target for regulating epileptic seizures. As a homeostatic bioenergetic network regulator, adenosine is perfectly suited to establish or restore an ongoing balance between excitation and inhibition, and its anticonvulsant efficacy is well established. There is evidence for the involvement of multiple adenosine receptor subtypes in epilepsy, but in particular the adenosine A1 receptor subtype can powerfully and bidirectionally regulate seizure activity. Mechanisms that regulate adenosine itself are increasingly appreciated as targets to thus influence receptor activity and seizure propensity. Taken together, established evidence for the powerful potential of adenosine-based epilepsy therapies and new strategies to influence receptor activity can combine to capitalize on this endogenous homeostatic neuromodulator.