The Bifunctional Dimer Caffeine-Indan Attenuates α-Synuclein Misfolding, Neurodegeneration and Behavioral Deficits after Chronic Stimulation of Adenosine A1 Receptors.

We previously found that chronic adenosine A1 receptor stimulation with N6-Cyclopentyladenosine increased α-synuclein misfolding and neurodegeneration in a novel α-synucleinopathy model, a hallmark of Parkinson's disease. Here, we aimed to synthesize a dimer caffeine-indan linked by a 6-carbon chain to cross the blood-brain barrier and tested its ability to bind α-synuclein, reducing misfolding, behavioral abnormalities, and neurodegeneration in our rodent model. Behavioral tests and histological stains assessed neuroprotective effects of the dimer compound. A rapid synthesis of the 18F-labeled analogue enabled Positron Emission Tomography and Computed Tomography imaging for biodistribution measurement. Molecular docking analysis showed that the dimer binds to α-synuclein N- and C-termini and the non-amyloid-ß-component (NAC) domain, similar to 1-aminoindan, and this binding promotes a neuroprotective α-synuclein "loop" conformation. The dimer also binds to the orthosteric binding site for adenosine within the adenosine A1 receptor. Immunohistochemistry and confocal imaging showed the dimer abolished α-synuclein upregulation and aggregation in the substantia nigra and hippocampus, and the dimer mitigated cognitive deficits, anxiety, despair, and motor abnormalities. The 18F-labeled dimer remained stable post-injection and distributed in various organs, notably in the brain, suggesting its potential as a Positron Emission Tomography tracer for α-synuclein and adenosine A1 receptor in Parkinson's disease therapy.

Impact of Coffee Intake on Measures of Wellbeing in Mice.

Coffee intake is increasingly recognized as a life-style factor associated with the preservation of health, but there is still a debate on the relative effects of caffeinated and decaffeinated coffee. We now tested how the regular drinking of caffeinated and decaffeinated coffee for 3 weeks impacted on the behavior of male and female adult mice. Males drinking caffeinated coffee displayed statistically significant lower weight gain, increased sensorimotor coordination, greater motivation in the splash test, more struggling in the forced swimming test, faster onset of nest building, more marble burying and greater sociability. Females drinking caffeinated coffee displayed statistically significant increased hierarchy fighting, greater self-care and motivation in the splash test and faster onset of nest building. A post-hoc two-way ANOVA revealed sex-differences in the effects of caffeinated coffee (p values for interaction between the effect of caffeinated coffee and sex) on the hierarchy in the tube test (p = 0.044; dominance), in the time socializing (p = 0.044) and in the latency to grooming (p = 0.048; selfcare), but not in the marble burying test (p = 0.089). Intake of decaffeinated coffee was devoid of effects in males and females. Since caffeine targets adenosine receptors, we verified that caffeinated but not decaffeinated coffee intake increased the density of adenosine A1 receptors (A1R) and increased A1R-mediated tonic inhibition of synaptic transmission in the dorsolateral striatum and ventral but not dorsal hippocampus, the effects being more evident in the ventral hippocampus of females and striatum of males. In contrast, caffeinated and decaffeinated coffee both ameliorated the antioxidant status in the frontal cortex. It is concluded that caffeinated coffee increases A1R-mediated inhibition in mood-related areas bolstering wellbeing of both males and females, with increased sociability in males and hierarchy struggling and self-care in females.

Putative Role of Adenosine A1 Receptors in Exogenous Ketone Supplements-Evoked Anti-Epileptic Effect.

Approximately 30% of patients with epilepsy are drug-refractory. There is an urgent need to elucidate the exact pathophysiology of different types of epilepsies and the mechanisms of action of both antiseizure medication and metabolic therapies to treat patients more effectively and safely. For example, it has been demonstrated that exogenous ketone supplement (EKS)-generated therapeutic ketosis, as a metabolic therapy, may decrease epileptic activity in both animal models and humans, but its exact mechanism of action is unknown. However, it was demonstrated that therapeutic ketosis, among others, can increase adenosine level, which may enhance activity of A1 adenosine receptors (A1Rs) in the brain. It has also been demonstrated previously that adenosine has anti-epileptic effect through A1Rs in different models of epilepsies. Thus, it is possible that (i) therapeutic ketosis generated by the administration of EKSs may exert its anti-epileptic effect through, among other mechanisms, increased adenosine level and A1R activity and that (ii) the enhanced activity of A1Rs may be a necessary anti-epileptic mechanism evoked by EKS administration-generated ketosis. Moreover, EKSs can evoke and maintain ketosis without severe side effects. These results also suggest that the therapeutic application of EKS-generated ketosis may be a promising opportunity to treat different types of epilepsies. In this literature review, we specifically focus on the putative role of A1Rs in the anti-epileptic effect of EKS-induced ketosis.

Extracellular cAMP elicits contraction of rat vas deferens: Involvement of ecto-5'-nucleotidase and adenosine A1 receptors.

AIMS: It is well established that intracellular cAMP contributes to the relaxation of vas deferens smooth muscle. In many tissues, intracellular cAMP is actively transported to the extracellular space, where it exerts regulatory functions, via its metabolite adenosine. These actions take place through the cAMP conversion to adenosine by ectoenzymes, a process called "extracellular cAMP-adenosine pathway". Herein, we investigated whether, in addition to ATP, extracellular cAMP might be an alternative source of adenosine, influencing the contraction of vas deferens smooth muscle. MAIN METHODS: The effects of cAMP, 8-Br-cAMP and adenosine were analyzed in the isometric contractions of rat vas deferens. cAMP efflux was analyzed by measuring extracellular cAMP levels after exposure of vas deferens segments to isoproterenol and forskolin in the presence or absence of MK-571, an inhibitor of MRP/ABCC transporters. KEY FINDINGS: While 8-Br-cAMP, a cell-permeable cAMP analog, induced relaxation of KCl-precontracted vas deferens, the non-permeant cAMP increased the KCl-induced contractile response, which was mimicked by adenosine, but prevented by inhibitors of ecto-5'-nucleotidase or A1 receptors. Our results also showed that isoproterenol and forskolin increases cAMP efflux via an MRP/ABCC transporter-dependent mechanism, since it is inhibited by MK-571. SIGNIFICANCE: Our data show that activation of ß-adrenoceptors and adenylyl cyclase increases cAMP efflux from vas deferens tissue, which modulates the vas deferens contractile response via activation of adenosine A1 receptors. Assuming that inhibition of vas deferens contractility has been proposed as a strategy for male contraception, the extracellular cAMP-adenosine pathway emerges as a potential pharmacological target that should be considered in studies of male fertility.

Cordycepin suppresses steroidogenic acute regulatory protein expression by reducing SP1 in human granulosa-lutein cells.

In brief: Cordycepin (COR), a compound derived from Cordyceps, is recognized as an adenosine analog with numerous beneficial effects on human health. However, its impact on steroidogenic acute regulatory protein (STAR) expression in ovarian granulosa cells is not well understood. This study demonstrates that COR downregulates STAR expression by reducing the expression of the SP1 transcription factor. Abstract: Cordycepin (COR), a pure compound of Cordyceps, is known as an adenosine analog that exerts many beneficial effects on human health. The steroidogenesis mediated by ovarian granulosa cells is pivotal in maintaining normal female reproductive function. The steroidogenic acute regulatory protein (STAR) regulates the rate-limiting step in steroidogenesis. COR has been shown to stimulate STAR expression in mouse Leydig cells, the steroidogenic cells in the testes. However, the effect of COR on STAR expression in ovarian granulosa cells remains undetermined. In the present study, we show that treatment with COR downregulates STAR expression in a steroidogenic human granulosa-like tumor cell line, KGN, and primary culture of human granulosa-lutein (hGL) cells obtained from patients undergoing in vitro fertilization. We used specific adenosine receptor (AR) antagonists, and our results reveal that the inhibitory effect of COR on STAR expression is mediated by AR-A1, AR-A2A, and AR-A3. In both KGN and primary hGL cells, COR activates ERK1/2 and AKT signaling pathways, but only activation of ERK1/2 is required for the COR-induced downregulation of STAR expression. In addition, our results demonstrate that COR downregulates STAR expression by reducing the expression of the SP1 transcription factor. These results provide a better understanding of the biological function of COR on STAR expression in the ovary, which may lead to the development of alternative therapeutic approaches for female reproductive disorders.

Interaction of Purine and its Derivatives with A1, A2-Adenosine Receptors and Vascular Endothelial Growth Factor Receptor-1 (Vegf-R1) as a Therapeutic Alternative to Treat Cancer.

BACKGROUND: There are several studies that indicate that cancer development may be conditioned by the activation of some biological systems that involve the interaction of different biomolecules, such as adenosine and vascular endothelial growth factor. These biomolecules have been targeted of some drugs for treat of cancer; however, there is little information on the interaction of purine derivatives with adenosine and vascular endothelial growth factor receptor (VEGF-R1). OBJECTIVE: The aim of this research was to determine the possible interaction of purine (1: ) and their derivatives (2-31: ) with A1, A2-adenosine receptors, and VEGF-R1. METHODS: Theoretical interaction of purine and their derivatives with A1, A2-adenosine receptors and VEGF-R1 was carried out using the 5uen, 5mzj and 3hng proteins as theoretical tools. Besides, adenosine, cgs-15943, rolofylline, cvt-124, wrc-0571, luf-5834, cvt-6883, AZD-4635, cabozantinib, pazopanib, regorafenib, and sorafenib drugs were used as controls. RESULTS: The results showed differences in the number of aminoacid residues involved in the interaction of purine and their derivatives with 5uen, 5mzj and 3hng proteins compared with the controls. Besides, the inhibition constants (Ki) values for purine and their derivatives 5: , 9: , 10: , 14: , 15: , 16: , and 20: were lower compared with the controls CONCLUSIONS: Theoretical data suggest that purine and their derivatives 5: , 9: , 10: , 14: , 15: , 16: , and 20: could produce changes in cancer cell growth through inhibition of A1, A2-adenosine receptors and VEGFR-1 inhibition. These data indicate that these purine derivatives could be a therapeutic alternative to treat some types of cancer.

Hypnotic effect of AR-001 through adenosine A1 receptor.

Insomnia is one of the most common sleep disorders, affecting 10-15% of the global population. Because classical remedies used to treat insomnia have various side effects, new therapeutics for insomnia are attracting attention. In the present study, we found that N2-Ethyl-N4-(furan-2-ylmethyl) quinazoline-2,4-diamine (AR-001) has adenosine A1 receptor agonistic activity and exhibits hypnotic efficacy by decreasing sleep onset latency and increasing total sleep time in a pentobarbital-induced sleep model. This hypnotic effect of AR-001 was significantly inhibited by the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). As a result of immunohistochemistry, AR-001 was shown to increase neural activity in the sleep-promoting region, ventrolateral preoptic nucleus (VLPO), and decrease neural activity in the wake-promoting region, basal forebrain (BF), and lateral hypothalamus (LH), and that these effects of AR-001 were significantly inhibited by DPCPX treatment. In addition, AR-001 increased adenosine A1 receptor mRNA levels in the hypothalamus. In conclusion, this study suggests that AR-001 has a hypnotic effect, at least partially, through adenosine A1 receptor and may have therapeutic potential for insomnia.

Adenosine triggers early astrocyte reactivity that provokes microglial responses and drives the pathogenesis of sepsis-associated encephalopathy in mice.

Molecular pathways mediating systemic inflammation entering the brain parenchyma to induce sepsis-associated encephalopathy (SAE) remain elusive. Here, we report that in mice during the first 6 hours of peripheral lipopolysaccharide (LPS)-evoked systemic inflammation (6 hpi), the plasma level of adenosine quickly increased and enhanced the tone of central extracellular adenosine which then provoked neuroinflammation by triggering early astrocyte reactivity. Specific ablation of astrocytic Gi protein-coupled A1 adenosine receptors (A1ARs) prevented this early reactivity and reduced the levels of inflammatory factors (e.g., CCL2, CCL5, and CXCL1) in astrocytes, thereby alleviating microglial reaction, ameliorating blood-brain barrier disruption, peripheral immune cell infiltration, neuronal dysfunction, and depression-like behaviour in the mice. Chemogenetic stimulation of Gi signaling in A1AR-deficent astrocytes at 2 and 4 hpi of LPS injection could restore neuroinflammation and depression-like behaviour, highlighting astrocytes rather than microglia as early drivers of neuroinflammation. Our results identify early astrocyte reactivity towards peripheral and central levels of adenosine as an important pathway driving SAE and highlight the potential of targeting A1ARs for therapeutic intervention.

Antidepressant effects of selective adenosine receptor antagonists targeting the A1 and A2A receptors administered jointly with NMDA receptor ligands: behavioral, biochemical and molecular investigations in mice.

BACKGROUND: The objective of the study was to ascertain the antidepressant potential of the co-administration of NMDA receptor ligands and selective adenosine A1 and A2A receptor antagonists. METHODS: The forced swim test (FST) and spontaneous locomotor activity test were carried out in adult male naïve mice. Before the behavioral testing, animals received DPCPX (a selective adenosine A1 receptor antagonist, 1 mg/kg) or istradefylline (a selective adenosine A2A receptor antagonist, 0.5 mg/kg) in combination with L-701,324 (a potent NMDA receptor antagonist, 1 mg/kg), D-cycloserine (a partial agonist at the glycine recognition site of NMDA receptor, 2.5 mg/kg), CGP 37849 (a competitive NMDA receptor antagonist, 0.3 mg/kg) or MK-801 (a non-competitive NMDA receptor antagonist, 0.05 mg/kg). Additionally, serum BDNF level and the mRNA level of the Adora1, Comt, and Slc6a15 genes in the murine prefrontal cortex were determined. RESULTS: The obtained results showed that DPCPX and istradefylline administered jointly with NMDA receptor ligands (except for DPCPX + D-cycloserine combination) produced an antidepressant effect in the FST in mice without enhancement in spontaneous motility of animals. An elevation in BDNF concentration was noted in the D-cycloserine-treated group. Adora1 expression increased with L-701,324, DPCPX + D-cycloserine, and DPCPX + CGP 37849, while D-cycloserine, CGP 37849, and MK-801 led to a decrease. Comt mRNA levels dropped with DPCPX + L-701,324, istradefylline + L-701,324/CGP 37849 but increased with D-cycloserine, MK-801, CGP 37849 and DPCPX + MK-801/ CGP 37849. Slc6a15 levels were reduced by D-cycloserine, DPCPX + L-701,324 but rose with DPCPX + CGP 37849/MK-801 and istradefylline + D-cycloserine/MK-801/CGP 37849. CONCLUSION: Our study suggests that selective antagonists of adenosine receptors may enhance the antidepressant efficacy of NMDA receptor ligands highlighting a potential synergistic interaction between the adenosinergic and glutamatergic systems. Wherein, A2A receptor antagonists are seen as more promising candidates in this context. Given the intricate nature of changes in BDNF levels and the expression of Adora1, Comt, and Slc6a15 seen after drug combinations exerting antidepressant properties, further research and integrative approaches are crucial understand better the mechanisms underlying their antidepressant action.

Hyperbaric Oxygen Attenuates Chronic Postsurgical Pain by Regulating the CD73/Adenosine/A1R Axis of the Spinal Cord in Rats.

Chronic postsurgical pain (CPSP) affects postoperative rehabilitation and quality of life in patients, but its mechanisms are still poorly understood. Hyperbaric oxygen (HBO) attenuates neuropathic pain in animal and human studies, but its efficacy for CPSP treatment and its underlying mechanism have not been elucidated. This study aimed to investigate the analgesic effect of HBO in a CPSP rat model and the role of spinal cord adenosine circulation in HBO-induced analgesia. A skin/muscle incision and retraction (SMIR) rat model was used to mimic CPSP, and HBO treatment (2.5 atmospheric absolute, 60 minutes) was administered once daily for 5 consecutive days beginning 3 days after surgery. The role of spinal cord adenosine circulation in HBO-induced analgesia was investigated using ß-methylene ADP (a CD73 inhibitor), 8-cyclopentyl-1,3-dipropylxanthine (an A1R antagonist), or an intrathecal injection of adenosine. The mechanical paw withdrawal threshold was determined at different timepoints before and after surgery. The spinal cord adenosine and adenosine triphosphate (ATP) contents were analyzed using high-performance liquid chromatography, and the spinal cord expression of adenosine-1 receptor (A1R), extracellular 5'-nucleotidase (CD73), and adenosine kinase (ADK) was examined by Western blotting and immunofluorescence staining. The results showed that the mechanical paw withdrawal threshold of the ipsilateral hind paw and the adenosine content decreased, and the spinal cord expression of A1R, CD73, and ADK and ATP content increased within 14 days after surgery. HBO treatment alleviated mechanical allodynia, reduced ATP content, and increased adenosine content by activating CD73 but downregulated the spinal cord expression of A1R, CD73, and ADK. Intrathecal adenosine alleviated mechanical allodynia after SMIR and downregulated the spinal cord expression of A1R and CD73, and intrathecal ß-methylene ADP or 8-cyclopentyl-1,3-dipropylxanthine attenuated the analgesic effect of HBO treatment on SMIR-induced CPSP. PERSPECTIVE: Spinal cord adenosine is involved in the occurrence and development of CPSP, and HBO treatment alleviates CPSP by regulating adenosine production/metabolism in the spinal cord. Thus, HBO may be employed for the treatment of CPSP with favorable efficacy.

Ligand-Directed Labeling of the Adenosine A1 Receptor in Living Cells.

The study of protein function and dynamics in their native cellular environment is essential for progressing fundamental science. To overcome the requirement of genetic modification of the protein or the limitations of dissociable fluorescent ligands, ligand-directed (LD) chemistry has most recently emerged as a complementary, bioorthogonal approach for labeling native proteins. Here, we describe the rational design, development, and application of the first ligand-directed chemistry approach for labeling the A1AR in living cells. We pharmacologically demonstrate covalent labeling of A1AR expressed in living cells while the orthosteric binding site remains available. The probes were imaged using confocal microscopy and fluorescence correlation spectroscopy to study A1AR localization and dynamics in living cells. Additionally, the probes allowed visualization of the specific localization of A1ARs endogenously expressed in dorsal root ganglion (DRG) neurons. LD probes developed here hold promise for illuminating ligand-binding, receptor signaling, and trafficking of the A1AR in more physiologically relevant environments.

Analgesia by fascia manipulation is mediated by peripheral and spinal adenosine A1 receptor in a mouse model of peripheral inflammation.

The role of adenosine receptors in fascial manipulation-induced analgesia has not yet been investigated. The purpose of this study was to evaluate the involvement of the adenosine A1 receptor (A1R) in the antihyperalgesic effect of plantar fascia manipulation (PFM), specifically in mice with peripheral inflammation. Mice injected with Complete Freund's Adjuvant (CFA) underwent behavioral, i.e. mechanical hyperalgesia and edema. The mice underwent PFM for either 3, 9 or 15 min. Response frequency to mechanical stimuli was then assessed at 24 and 96 h after plantar CFA injection. The adenosinergic receptors were assessed by systemic (intraperitoneal, i.p.), central (intrathecal, i.t.), and peripheral (intraplantar, i.pl.) administration of caffeine. The participation of the A1R was investigated using the 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), a selective A1R subtype antagonist. PFM inhibited mechanical hyperalgesia induced by CFA injection and did not reduce paw edema. Furthermore, the antihyperalgesic effect of PFM was prevented by pretreatment of the animals with caffeine given by i.p., i.pl., and i.t. routes. In addition, i.pl. and i.t. administrations of DPCPX blocked the antihyperalgesia caused by PFM. These observations indicate that adenosine receptors mediate the antihyperalgesic effect of PFM. Caffeine's inhibition of PFM-induced antihyperalgesia suggests that a more precise understanding of how fascia-manipulation and caffeine interact is warranted.

Effect of Xanthohumol, a Bioactive Natural Compound from Hops, on Adenosine Pathway in Rat C6 Glioma and Human SH-SY5Y Neuroblastoma Cell Lines.

Xanthohumol (Xn) is an antioxidant flavonoid mainly extracted from hops (Humulus lupulus), one of the main ingredients of beer. As with other bioactive compounds, their therapeutic potential against different diseases has been tested, one of which is Alzheimer's disease (AD). Adenosine is a neuromodulatory nucleoside that acts through four different G protein-coupled receptors: A1 and A3, which inhibit the adenylyl cyclases (AC) pathway, and A2A and A2B, which stimulate this activity, causing either a decrease or an increase, respectively, in the release of excitatory neurotransmitters such as glutamate. This adenosinergic pathway, which is altered in AD, could be involved in the excitotoxicity process. Therefore, the aim of this work is to describe the effect of Xn on the adenosinergic pathway using cell lines. For this purpose, two different cellular models, rat glioma C6 and human neuroblastoma SH-SY5Y, were exposed to a non-cytotoxic 10 µM Xn concentration. Adenosine A1 and A2A, receptor levels, and activities related to the adenosine pathway, such as adenylate cyclase, protein kinase A, and 5'-nucleotidase, were analyzed. The adenosine A1 receptor was significantly increased after Xn exposure, while no changes in A2A receptor membrane levels or AC activity were reported. Regarding 5'-nucleotidases, modulation of their activity by Xn was noted since CD73, the extracellular membrane attached to 5'-nucleotidase, was significantly decreased in the C6 cell line. In conclusion, here we describe a novel pathway in which the bioactive flavonoid Xn could have potentially beneficial effects on AD as it increases membrane A1 receptors while modulating enzymes related to the adenosine pathway in cell cultures.

Repeated caffeine intake suppresses cerebral grey matter responses to chronic sleep restriction in an A1 adenosine receptor-dependent manner: a double-blind randomized controlled study with PET-MRI.

Evidence has shown that both sleep loss and daily caffeine intake can induce changes in grey matter (GM). Caffeine is frequently used to combat sleepiness and impaired performance caused by insufficient sleep. It is unclear (1) whether daily use of caffeine could prevent or exacerbate the GM alterations induced by 5-day sleep restriction (i.e. chronic sleep restriction, CSR), and (2) whether the potential impact on GM plasticity depends on individual differences in the availability of adenosine receptors, which are involved in mediating effects of caffeine on sleep and waking function. Thirty-six healthy adults participated in this double-blind, randomized, controlled study (age = 28.9 ± 5.2 y/; F:M = 15:21; habitual level of caffeine intake < 450 mg; 29 homozygous C/C allele carriers of rs5751876 of ADORA2A, an A2A adenosine receptor gene variant). Each participant underwent a 9-day laboratory visit consisting of one adaptation day, 2 baseline days (BL), 5-day sleep restriction (5 h time-in-bed), and a recovery day (REC) after an 8-h sleep opportunity. Nineteen participants received 300 mg caffeine in coffee through the 5 days of CSR (CAFF group), while 17 matched participants received decaffeinated coffee (DECAF group). We examined GM changes on the 2nd BL Day, 5th CSR Day, and REC Day using magnetic resonance imaging and voxel-based morphometry. Moreover, we used positron emission tomography with [18F]-CPFPX to quantify the baseline availability of A1 adenosine receptors (A1R) and its relation to the GM plasticity. The results from the voxel-wise multimodal whole-brain analysis on the Jacobian-modulated T1-weighted images controlled for variances of cerebral blood flow indicated a significant interaction effect between caffeine and CSR in four brain regions: (a) right temporal-occipital region, (b) right dorsomedial prefrontal cortex (DmPFC), (c) left dorsolateral prefrontal cortex (DLPFC), and (d) right thalamus. The post-hoc analyses on the signal intensity of these GM clusters indicated that, compared to BL, GM on the CSR day was increased in the DECAF group in all clusters  but decreased in the thalamus, DmPFC, and DLPFC in the CAFF group. Furthermore, lower baseline subcortical A1R availability predicted a larger GM reduction in the CAFF group after CSR of all brain regions except for the thalamus. In conclusion, our data suggest an adaptive GM upregulation after 5-day CSR, while concomitant use of caffeine instead leads to a GM reduction. The lack of consistent association with individual A1R availability may suggest that CSR and caffeine affect thalamic GM plasticity predominantly by a different mechanism. Future studies on the role of adenosine A2A receptors in CSR-induced GM plasticity are warranted.

Development of Putative Bivalent Dicovalent Ligands for the Adenosine A1 Receptor.

Accumulating evidence suggests that G protein-coupled receptors (GPCRs) can exist and function in homodimer and heterodimer forms. The adenosine A1 receptor (A1R) has been shown to form both homodimers and heterodimers, but there is a lack of chemical tools to study these dimeric receptor populations. This work describes the synthesis and pharmacological evaluation of a novel class of bivalent GPCR chemical tools, where each ligand moiety of the bivalent compound contains a sulfonyl fluoride covalent warhead designed to be capable of simultaneously reacting with each A1R of an A1R homodimer. The novel compounds were characterised using radioligand binding assays, including washout assays, and functionally in cAMP assays. The bivalent dicovalent compounds were competitive A1R antagonists and showed evidence of covalent binding and simultaneous binding across an A1R homodimer. Greater selectivity for A1R over the adenosine A3 receptor was observed for bivalent dicovalent over the equivalent monovalent compounds, indicating subtype selectivity can be achieved with dual occupation by a bivalent dicovalent ligand.

Lack of Interactions Between Alteplase/Tenecteplase and the Adenosine A1R/A3R Agonist AST-004.

BACKGROUND: AST-004, a small molecule agonist of the adenosine A1 and A3 receptors, is a potential cerebroprotectant for patients with acute stroke and is currently in clinical trials. Drug-drug interactions are critically important to assess in the context of acute stroke care. Lytic therapy with tPA (tissue-type plasminogen activator)-induced plasmin formation (alteplase) is the only available pharmacotherapy for acute stroke. Consequently, it is imperative to evaluate potential interactions between AST-004 and tPAs such as alteplase and tenecteplase. METHODS: The interactions between AST-004 and tPAs were evaluated in 3 ways in preparation for AST-004 phase II trials. First, the metabolic stability of AST-004 was determined in the presence of alteplase and plasmin. Second, the potential for AST-004 to influence the thrombolytic efficacy of alteplase and tenecteplase was evaluated with an in vitro assay system utilizing a fluorogenic substrate of plasmin. Finally, the potential for AST-004 to influence the thrombolytic efficacy of alteplase was also determined with an in vitro thrombolysis assay of human blood thrombi. RESULTS: Neither alteplase nor plasmin affected the stability of AST-004 in vitro. In 2 different in vitro systems, AST-004 had no effect on the ability of alteplase or tenecteplase to generate plasmin, and AST-004 had no effect on the thrombolytic efficacy of alteplase to lyse blood clots in human blood. CONCLUSIONS: These studies indicate that there will be no interactions between AST-004 and tPAs such as alteplase or tenecteplase in patients with stroke undergoing thrombolytic therapy.

Lactuca sativa L. Extract Enhances Sleep Duration Through Upregulation of Adenosine A1 Receptor and GABAA Receptors Subunits in Pentobarbital-Injected Mice.

We investigated the effects of Lactuca sativa L. extracts (Lactuc) on pentobarbital-induced sleep in mice to elucidate the mechanisms underlying its impact on sleep quality. Mice were randomly assigned to five groups: control, positive control (diazepam 2 mg/kg b.w.), and three groups orally administered with Lactuc (50, 100, and 200 mg/kg b.w.). After 2 weeks of oral administration and intraperitoneal injections, the mice were killed. We found that the Lactuc-administered groups had significantly reduced sleep latency and increased sleep duration compared with the control group. Furthermore, the oral administration of Lactuc induced a significant increase in mRNA expression and protein expression of adenosine A1 receptor in the brains compared with the expressions in the control group. In addition, the Lactuc-administered groups exhibited significantly higher levels of mRNA expressions of GABAA receptors subunits α2, ß2, γ1, and, γ2 in the brain tissue. Therefore, we suggest that Lactuc could be used to develop natural products that effectively improve sleep quality and duration.

8-Bicycloalkyl-CPFPX derivatives as potent and selective tools for in vivo imaging of the A1 adenosine receptor.

Imaging of the A1 adenosine receptor (A1R) by positron emission tomography (PET) with 8-cyclopentyl-3-(3-[18F]fluoropropyl)-1-propyl-xanthine ([18F]CPFPX) has been widely used in preclinical and clinical studies. However, this radioligand suffers from rapid peripheral metabolism and subsequent accumulation of radiometabolites in the vascular compartment. In the present work, we prepared four derivatives of CPFPX by replacement of the cyclopentyl group with norbornane moieties. These derivatives were evaluated by competition binding studies, microsomal stability assays and LC-MS analysis of microsomal metabolites. In addition, the 18F-labeled isotopologue of 8-(1-norbornyl)-3-(3-fluoropropyl)-1-propylxanthine (1-NBX) as the most promising candidate was prepared by radiofluorination of the corresponding tosylate precursor and the resulting radioligand ([18F]1-NBX) was evaluated by permeability assays with Caco-2 cells and in vitro autoradiography in rat brain slices. Our results demonstrate that 1-NBX exhibits significantly improved A1R affinity and selectivity when compared to CPFPX and that it does not give rise to lipophilic metabolites expected to cross the blood-brain-barrier in microsomal assays. Furthermore, [18F]1-NBX showed a high passive permeability (Pc = 6.9 ± 2.9 × 10-5 cm/s) and in vitro autoradiography with this radioligand resulted in a distribution pattern matching A1R expression in the brain. Moreover, a low degree of non-specific binding (5%) was observed. Taken together, these findings identify [18F]1-NBX as a promising candidate for further preclinical evaluation as potential PET tracer for A1R imaging.

Activation of hepatic adenosine A1 receptor ameliorates MASH via inhibiting SREBPs maturation.

Metabolic (dysfunction)-associated steatohepatitis (MASH) is the advanced stage of metabolic (dysfunction)-associated fatty liver disease (MAFLD) lacking approved clinical drugs. Adenosine A1 receptor (A1R), belonging to the G-protein-coupled receptors (GPCRs) superfamily, is mainly distributed in the central nervous system and major peripheral organs with wide-ranging physiological functions; however, the exact role of hepatic A1R in MAFLD remains unclear. Here, we report that liver-specific depletion of A1R aggravates while overexpression attenuates diet-induced metabolic-associated fatty liver (MAFL)/MASH in mice. Mechanistically, activation of hepatic A1R promotes the competitive binding of sterol-regulatory element binding protein (SREBP) cleavage-activating protein (SCAP) to sequestosome 1 (SQSTM1), rather than protein kinase A (PKA) leading to SCAP degradation in lysosomes. Reduced SCAP hinders SREBP1c/2 maturation and thus suppresses de novo lipogenesis and inflammation. Higher hepatic A1R expression is observed in patients with MAFL/MASH and high-fat diet (HFD)-fed mice, which is supposed to be a physiologically adaptive response because A1R agonists attenuate MAFL/MASH in an A1R-dependent manner. These results highlight that hepatic A1R is a potential target for MAFL/MASH therapy.

Caffeine Impaired Acupuncture Analgesia in Inflammatory Pain by Blocking Adenosine A1 Receptor.

Caffeine consumption inhibits acupuncture analgesic effects by blocking adenosine signaling. However, existing evidence remains controversial. Hence, this study aimed to examine the adenosine A1 receptor (A1R) role in moderate-dose caffeine-induced abolishing effect on acupuncture analgesia using A1R knockout mice (A1R-/-). We assessed the role of A1R in physiological sensory perception and its interaction with caffeine by measuring mechanical and thermal pain thresholds and administering A1R and adenosine 2A receptor antagonists in wild-type (WT) and A1R-/- mice. Formalin- and complete Freund's adjuvant (CFA)-induced inflammatory pain models were recruited to explore moderate-dose caffeine effect on pain perception and acupuncture analgesia in WT and A1R-/- mice. Moreover, a C-fiber reflex electromyogram in the biceps femoris was conducted to validate the role of A1R in the caffeine-induced blockade of acupuncture analgesia. We found that A1R was dispensable for physiological sensory perception and formalin- and CFA-induced hypersensitivity. However, genetic deletion of A1R impaired the antinociceptive effect of acupuncture in A1R-/- mice under physiological or inflammatory pain conditions. Acute moderate-dose caffeine administration induced mechanical and thermal hyperalgesia under physiological conditions but not in formalin- and CFA-induced inflammatory pain. Moreover, caffeine significantly inhibited electroacupuncture (EA) analgesia in physiological and inflammatory pain in WT mice, comparable to that of A1R antagonists. Conversely, A1R deletion impaired the EA analgesic effect and decreased the caffeine-induced inhibitory effect on EA analgesia in physiological conditions and inflammatory pain. Moderate-dose caffeine administration diminished the EA-induced antinociceptive effect by blocking A1R. Overall, our study suggested that caffeine consumption should be avoided during acupuncture treatment. PERSPECTIVE: Moderate-dose caffeine injection attenuated EA-induced antinociceptive effect in formalin- and CFA-induced inflammatory pain mice models by blocking A1R. This highlights the importance of monitoring caffeine intake during acupuncture treatment.