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.

Adenosine A1R/A3R (Adenosine A1 and A3 Receptor) Agonist AST-004 Reduces Brain Infarction in a Nonhuman Primate Model of Stroke.

BACKGROUND AND PURPOSE: Treatment with A1R/A3R (adenosine A1 and A3 receptor) agonists in rodent models of acute ischemic stroke results in significantly reduced lesion volume, indicating activation of adenosine A1R or A3R is cerebroprotective. However, dosing and timing required for cerebroprotection has yet to be established, and whether adenosine A1R/A3R activation will lead to cerebroprotection in a gyrencephalic species has yet to be determined. METHODS: The current study used clinical study intervention timelines in a nonhuman primate model of transient, 4-hour middle cerebral artery occlusion to investigate a potential cerebroprotective effect of the dual adenosine A1R/A3R agonist AST-004. Bolus and then 22 hours intravenous infusion of AST-004 was initiated 2 hours after transient middle cerebral artery occlusion. Primary outcome measures included lesion volume, lesion growth kinetics, penumbra volume as well as initial pharmacokinetic-pharmacodynamic relationships measured up to 5 days after transient middle cerebral artery occlusion. Secondary outcome measures included physiological parameters and neurological function. RESULTS: Administration of AST-004 resulted in rapid and statistically significant decreases in lesion growth rate and total lesion volume. In addition, penumbra volume decline over time was significantly less under AST-004 treatment compared with vehicle treatment. These changes correlated with unbound AST-004 concentrations in the plasma and cerebrospinal fluid as well as estimated brain A1R and A3R occupancy. No relevant changes in physiological parameters were observed during AST-004 treatment. CONCLUSIONS: These findings suggest that administration of AST-004 and combined A1R/A3R agonism in the brain are efficacious pharmacological interventions in acute ischemic stroke and warrant further clinical evaluation.

Lack of efficacy of a partial adenosine A1 receptor agonist in neuropathic pain models in mice.

Previous studies suggest that adenosine A1 receptors (A1R) modulate the processing of pain. The aim of this study was to characterize the distribution of A1R in nociceptive tissues and to evaluate whether targeting A1R with the partial agonist capadenoson may reduce neuropathic pain in mice. The cellular distribution of A1R in dorsal root ganglia (DRG) and the spinal cord was analyzed using fluorescent in situ hybridization. In behavioral experiments, neuropathic pain was induced by spared nerve injury or intraperitoneal injection of paclitaxel, and tactile hypersensitivities were determined using a dynamic plantar aesthesiometer. Whole-cell patch-clamp recordings were performed to assess electrophysiological properties of dissociated DRG neurons. We found A1R to be expressed in populations of DRG neurons and dorsal horn neurons involved in the processing of pain. However, administration of capadenoson at established in vivo doses (0.03-1.0 mg/kg) did not alter mechanical hypersensitivity in the spared nerve injury and paclitaxel models of neuropathic pain, whereas the standard analgesic pregabalin significantly inhibited the pain behavior. Moreover, capadenoson failed to affect potassium currents in DRG neurons, in contrast to a full A1R agonist. Despite expression of A1R in nociceptive neurons, our data do not support the hypothesis that pharmacological intervention with partial A1R agonists might be a valuable approach for the treatment of neuropathic pain.

The adenosine A1 receptor agonist WAG 994 suppresses acute kainic acid-induced status epilepticus in vivo.

Status epilepticus (SE) is a neurological emergency characterized by continuous seizure activity lasting longer than 5 min, often with no recovery between seizures (Trinka et al., 2015). SE is refractory to benzodiazepine and second-line treatments in about 30% cases. Novel treatment approaches are urgently needed as refractory SE is associated with mortality rates of up to 70%. Robust adenosinergic anticonvulsant effects have been known for decades, but translation into seizure treatments was hampered by cardiovascular side effects. However, the selective adenosine A1 receptor agonist SDZ WAG 994 (WAG) displays diminished cardiovascular side effects compared to classic A1R agonists and was safely administered systemically in human clinical trials. Here, we investigate the anticonvulsant efficacy of WAG in vitro and in vivo. WAG robustly inhibited high-K+-induced continuous epileptiform activity in rat hippocampal slices (IC50 = 52.5 nM). Importantly, WAG acutely suppressed SE in vivo induced by kainic acid (20 mg/kg i.p.) in mice. After SE was established, mice received three i.p. injections of WAG or diazepam (DIA, 5 mg/kg). Interestingly, DIA did not attenuate SE while the majority of WAG-treated mice (1 mg/kg) were seizure-free after three injections. Anticonvulsant effects were retained when a lower dose of WAG (0.3 mg/kg) was used. Importantly, all WAG-treated mice survived kainic acid induced SE. In summary, we report for the first time that an A1R agonist with an acceptable human side-effect profile can acutely suppress established SE in vivo. Our results suggest that WAG stops or vastly attenuates SE while DIA fails to mitigate SE in this model.

Safety and efficacy of the partial adenosine A1 receptor agonist neladenoson bialanate in patients with chronic heart failure with reduced ejection fraction: a phase IIb, randomized, double-blind, placebo-controlled trial.

AIMS: Neladenoson bialanate is a partial adenosine A1 receptor agonist with demonstrated beneficial effects on cardiac function in animal models. We aimed to assess the dose-response effect of neladenoson bialanate on cardiac structure and function, clinical outcome, and safety in patients with heart failure (HF) with reduced ejection fraction (HFrEF). METHODS AND RESULTS: PANTHEON was a dose-finding, phase IIb, randomized, double-blind, placebo-controlled trial conducted in 92 centres in 11 countries including 462 patients with chronic HFrEF, randomized to once daily oral dose of neladenoson bialanate (5, 10, 20, 30, and 40 mg) or placebo. The primary endpoints were change from baseline to 20 weeks in left ventricular ejection fraction (LVEF) (echocardiography) and in N-terminal pro-B-type natriuretic peptide (NT-proBNP). Mean age of the patients was 67 years, 17% were female, mean LVEF was 28%, mean NT-proBNP was 2085 ng/L. After 20 weeks of treatment, there was no dose-effect of neladenoson bialanate on changes in NT-proBNP or LVEF (primary endpoints). No effect of neladenoson bialanate was found on left ventricular volumes, high-sensitivity troponin T, or cardiovascular mortality, HF hospitalization, and urgent visits for HF (secondary endpoints). There was a dose-dependent increase in creatinine and cystatin C, and a dose-dependent decrease in estimated glomerular filtration rate and heart rate. CONCLUSIONS: In patients with chronic HFrEF, treatment with neladenoson bialanate was not associated with dose-dependent favourable effects on cardiac structure and function, cardiac risk markers, or clinical outcome but was associated with a dose-dependent decrease in renal function. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov Identifier NCT02992288.

In Vivo Evaluation of A1 Adenosine Agonists as Novel Anticonvulsant Medical Countermeasures to Nerve Agent Intoxication in a Rat Soman Seizure Model.

Organophosphorus nerve agents (NAs) irreversibly inhibit acetylcholinesterase, which results in the accumulation of acetylcholine and widespread excitotoxic seizure activity. Because current medical countermeasures (anticholinergics, AChE reactivators, and benzodiazepines) lack sufficient anti-seizure efficacy when treatment is delayed, those intoxicated are at risk for severe brain damage or death if treatment is not immediately available. Toward developing a more effective anti-seizure treatment for NA intoxication, this study evaluated the efficacy of A1 adenosine (ADO) receptor (A1AR) agonists in a rat soman seizure model. One minute after exposure to soman (1.6 × LD50, subcutaneous), rats were treated intraperitoneally with one of the following agonists at increasing dose levels until anti-seizure efficacy was achieved: N6-cyclopentaladenosine (CPA), 2-chloro-N6-cyclopentyladenosine (CCPA), and (±)-5'-chloro-5'-deoxy-ENBA (ENBA). All A1AR agonists were efficacious in preventing seizure and promoting survival. The effective doses for the A1AR agonists were 60 mg/kg CPA, 36 mg/kg CCPA, and 62 mg/kg ENBA. Whereas vehicle-treated rats experienced 100% seizure and 21% survival (N = 28), ADO treatments reduced seizure occurrence and improved survival rates: 8% seizure and 83% survival with CPA (60 mg/kg, N = 12), 17% seizure and 75% survival with CCPA (36 mg/kg, N = 12), and 8% seizure, 83% survival with ENBA (62 mg/kg, N = 12). The brains of ADO-treated rats were also protected from damage as indicated by neurohistopathological analysis. While all ADO agonists provided neuroprotection, rats receiving CCPA and ENBA experienced less severe ADO-induced side effects (e.g., sedation, hypothermia, bradycardia) than with CPA. The data from this study suggest that the ADO signaling pathway is a promising mechanism for countering seizure activity induced by NAs.

Reduction of repetitive behavior by co-administration of adenosine receptor agonists in C58 mice.

Repetitive behaviors are diagnostic for autism spectrum disorder (ASD) and commonly observed in other neurodevelopmental disorders. Currently, there are no effective pharmacological treatments for repetitive behavior in these clinical conditions. This is due to the lack of information about the specific neural circuitry that mediates the development and expression of repetitive behavior. Our previous work in mouse models has linked repetitive behavior to decreased activation of the subthalamic nucleus, a brain region in the indirect and hyperdirect pathways in the basal ganglia circuitry. The present experiments were designed to further test our hypothesis that pharmacological activation of the indirect pathway would reduce repetitive behavior. We used a combination of adenosine A1 and A2A receptor agonists that have been shown to alter the firing frequency of dorsal striatal neurons within the indirect pathway of the basal ganglia. This drug combination markedly and selectively reduced repetitive behavior in both male and female C58 mice over a six-hour period, an effect that required both A1 and A2A agonists as neither alone reduced repetitive behavior. The adenosine A1 and A2A receptor agonist combination also significantly increased the number of Fos transcripts and Fos positive cells in dorsal striatum. Fos induction was found in both direct and indirect pathway neurons suggesting that the drug combination restored the balance of activation across these complementary basal ganglia pathways. The adenosine A1 and A2A receptor agonist combination also maintained its effectiveness in reducing repetitive behavior over a 7-day period. These findings point to novel potential therapeutic targets for development of drug therapies for repetitive behavior in clinical disorders.

Adenosine A1 receptor: A neuroprotective target in light induced retinal degeneration.

Light induced retinal degeneration (LIRD) is a useful model that resembles human retinal degenerative diseases. The modulation of adenosine A1 receptor is neuroprotective in different models of retinal injury. The aim of this work was to evaluate the potential neuroprotective effect of the modulation of A1 receptor in LIRD. The eyes of rats intravitreally injected with N6-cyclopentyladenosine (CPA), an A1 agonist, which were later subjected to continuous illumination (CI) for 24 h, showed retinas with a lower number of apoptotic nuclei and a decrease of Glial Fibrillary Acidic Protein (GFAP) immunoreactive area than controls. Lower levels of activated Caspase 3 and GFAP were demonstrated by Western Blot (WB) in treated animals. Also a decrease of iNOS, TNFα and GFAP mRNA was demonstrated by RT-PCR. A decrease of Iba 1+/MHC-II+ reactive microglial cells was shown by immunohistochemistry. Electroretinograms (ERG) showed higher amplitudes of a-wave, b-wave and oscillatory potentials after CI compared to controls. Conversely, the eyes of rats intravitreally injected with dipropylcyclopentylxanthine (DPCPX), an A1 antagonist, and subjected to CI for 24 h, showed retinas with a higher number of apoptotic nuclei and an increase of GFAP immunoreactive area compared to controls. Also, higher levels of activated Caspase 3 and GFAP were demonstrated by Western Blot. The mRNA levels of iNOS, nNOS and inflammatory cytokines (IL-1ß and TNFα) were not modified by DPCPX treatment. An increase of Iba 1+/MHC-II+ reactive microglial cells was shown by immunohistochemistry. ERG showed that the amplitudes of a-wave, b-wave, and oscillatory potentials after CI were similar to control values. A single pharmacological intervention prior illumination stress was able to swing retinal fate in opposite directions: CPA was neuroprotective, while DPCPX worsened retinal damage. In summary, A1 receptor agonism is a plausible neuroprotective strategy in LIRD.

Biochemical and Pharmacological Role of A1 Adenosine Receptors and Their Modulation as Novel Therapeutic Strategy.

Adenosine, the purine nucleoside, mediates its effects through activation of four G-protein coupled adenosine receptors (ARs) named as A1, A2A, A2B and A3. In particular, A1ARs are distributed through the body, primarily inhibitory in the regulation of adenylyl cyclase activity and able to reduce the cyclic AMP levels. Considerable advances have been made in the pharmacological and molecular characterization of A1ARs, which had been proposed as targets for the discovery and drug design of antagonists, agonists and allosteric enhancers. Several lines of evidence indicate that adenosine interacting with A1ARs may be an endogenous protective agent in the human body since it prevents the damage caused by various pathological conditions, such as in ischemia/hypoxia, epileptic seizures, excitotoxic neuronal injury and cardiac arrhythmias in cardiovascular system. It has also been reported that one of the most promising targets for the development of new anxiolytic drugs could be A1ARs, and that their activation may reduce pain signaling in the spinal cord. A1AR antagonists induce diuresis and natriuresis in various experimental models, mediating the inhibition of A1ARs in the proximal tubule which is primarily responsible for reabsorption and fluid uptake. In addition, the results of various studies indicate that adenosine is present within pancreatic islets and is implicated through A1ARs in the regulation of insulin secretion and in glucose concentrations. In the present paper it will become apparent that A1ARs could be implicated in the pharmacological treatment of several pathologies with an important influence on human health.

Adenosine A1 receptor activation increases myocardial protein S-nitrosothiols and elicits protection from ischemia-reperfusion injury in male and female hearts.

Nitric oxide (NO) plays an important role in cardioprotection, and recent work from our group and others has implicated protein S-nitrosylation (SNO) as a critical component of NO-mediated protection in different models, including ischemic pre- and post-conditioning and sex-dependent cardioprotection. However, studies have yet to examine whether protein SNO levels are similarly increased with pharmacologic preconditioning in male and female hearts, and whether an increase in protein SNO levels, which is protective in male hearts, is sufficient to increase baseline protection in female hearts. Therefore, we pharmacologically preconditioned male and female hearts with the adenosine A1 receptor agonist N6-cyclohexyl adenosine (CHA). CHA administration prior to ischemia significantly improved functional recovery in both male and female hearts compared to baseline in a Langendorff-perfused heart model of ischemia-reperfusion injury (% of preischemic function ± SE: male baseline: 37.5±3.4% vs. male CHA: 55.3±3.2%; female baseline: 61.4±5.7% vs. female CHA: 76.0±6.2%). In a separate set of hearts, we found that CHA increased p-Akt and p-eNOS levels. We also used SNO-resin-assisted capture with LC-MS/MS to identify SNO proteins in male and female hearts, and determined that CHA perfusion induced a modest increase in protein SNO levels in both male (11.4%) and female (12.3%) hearts compared to baseline. These findings support a potential role for protein SNO in a model of pharmacologic preconditioning, and provide evidence to suggest that a modest increase in protein SNO levels is sufficient to protect both male and female hearts from ischemic injury. In addition, a number of the SNO proteins identified with CHA treatment were also observed with other forms of cardioprotective stimuli in prior studies, further supporting a role for protein SNO in cardioprotection.

Exploring the Role of N6-Substituents in Potent Dual Acting 5'-C-Ethyltetrazolyladenosine Derivatives: Synthesis, Binding, Functional Assays, and Antinociceptive Effects in Mice ∇.

Structural determinants of affinity of N6-substituted-5'-C-(ethyltetrazol-2-yl)adenosine and 2-chloroadenosine derivatives at adenosine receptor (AR) subtypes were studied with binding and molecular modeling. Small N6-cycloalkyl and 3-halobenzyl groups furnished potent dual acting A1AR agonists and A3AR antagonists. 4 was the most potent dual acting human (h) A1AR agonist (Ki = 0.45 nM) and A3AR antagonist (Ki = 0.31 nM) and highly selective versus A2A; 11 and 26 were most potent at both h and rat (r) A3AR. All N6-substituted-5'-C-(ethyltetrazol-2-yl)adenosine derivatives proved to be antagonists at hA3AR but agonists at the rA3AR. Analgesia of 11, 22, and 26 was evaluated in the mouse formalin test (A3AR antagonist blocked and A3AR agonist strongly potentiated). N6-Methyl-5'-C-(ethyltetrazol-2-yl)adenosine (22) was most potent, inhibiting both phases, as observed combining A1AR and A3AR agonists. This study demonstrated for the first time the advantages of a single molecule activating two AR pathways both leading to benefit in this acute pain model.

Role of A1 and A2A adenosine receptor agonists in adipose tissue inflammation induced by obesity in mice.

Adenosine receptors are expressed in adipose tissue and control physiological and pathological events such as lipolysis and inflammation. The aim of this study was to evaluate the activity of N6-cyclopentyladenosine (CPA), a potent and selective A1 adenosine receptor agonist; 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxyamidoadenosine hydrochloride (CGS-21680), an A2A adenosine receptor agonist; and 5'-N-ethylcarboxamidoadenosine (NECA), a potent non-selective adenosine receptor agonist on adipose tissue inflammatory alterations induced by obesity in mice. Swiss mice were fed with a high-fat diet for 12 weeks and agonists were administered in the last two weeks. Body weight, adiposity and glucose homeostasis were evaluated. Inflammation in adipose tissue was assessed by evaluation of adipokine production and macrophage infiltration. Adenosine receptor signaling in adipose tissue was also evaluated. Mice that received CGS21680 presented an improvement in glucose homeostasis in association with systemically reduced inflammatory markers (TNF-α, PAI-1) and in the visceral adipose tissue (TNF-α, MCP-1, macrophage infiltration). Activation of p38 signaling was found in adipose tissue of this group of mice. NECA-treated mice presented some improvements in glucose homeostasis associated with an observed weight loss. Mice that received CPA presented only a reduction in the ex vivo basal lipolysis rate measured within visceral adipose tissue. In conclusion, administration of the A2A receptor agonist to obese mice resulted in improvements in glucose homeostasis and adipose tissue inflammation, corroborating the idea that new therapeutics to treat obesity could emerge from these compounds.

Partial Adenosine A1 Agonist in Heart Failure.

Adenosine exerts a variety of physiological effects by binding to cell surface G-protein-coupled receptor subtypes, namely, A1, A2a, A2b, and A3. The central physiological role of adenosine is to preclude tissue injury and promote repair in response to stress. In the heart, adenosine acts as a cytoprotective modulator, linking cardiac function to metabolic demand predominantly via activation of adenosine A1 receptors (A1Rs), which leads to inhibition of adenylate cyclase activity, modulation of protein kinase C, and opening of ATP-sensitive potassium channels. Activation of myocardial adenosine A1Rs has been shown to modulate a variety of pathologies associated with ischemic cardiac injury, including arrhythmogenesis, coronary and ventricular dysfunction, apoptosis, mitochondrial dysfunction, and ventricular remodeling. Partial A1R agonists are agents that are likely to elicit favorable pharmacological responses in heart failure (HF) without giving rise to the undesirable cardiac and extra-cardiac effects observed with full A1R agonism. Preclinical data have shown that partial adenosine A1R agonists protect and improve cardiac function at doses that do not result in undesirable effects on heart rate, atrioventricular conduction, and blood pressure, suggesting that these compounds may constitute a valuable new therapy for chronic HF. Neladenoson bialanate (BAY1067197) is the first oral partial and highly selective A1R agonist that has entered clinical development for the treatment of HF. This review provides an overview of adenosine A1R-mediated signaling in the heart, summarizes the results from preclinical and clinical studies of partial A1R agonists in HF, and discusses the potential benefits of these drugs in the clinical setting.

A1 Adenosine Receptor Activation Modulates Central Nervous System Development and Repair.

Adenosine is an endogenous, autacoid purine nucleoside which performs many important biological roles, particularly during stressful events. Adenosine can signal through four adenosine receptor (AR) subtypes: A1, A2A, A2B, and A3. Of these, adenosine A1 receptor (A1AR) has a broad, wide distribution throughout different vertebrate cell types and the highest affinity to adenosine. The A1AR-dependent action of adenosine is well documented in reports from numerous studies that have used different selective A1AR agonists and antagonists as well as in animals that have a genetically manipulated A1AR gene. Despite its wide distribution and function, A1AR homo/hetero-oligomerization with other adenosine and non-adenosine receptors extends its biological role during developmental, physiological, and pathological situations. In this review, we initially discuss the A1AR structure and most important signaling pathway triggered by its activation. Next, we summarize some of the most well-known biological effects of A1AR in the central nervous system (CNS) during development and adulthood, in addition to its role in nervous system regeneration and repair.

Development of C-Methyl Branched Purine Ribonucleoside Analogs: Chemistry, Biological Activity and Therapeutic Potential.

In this review, we first highlighted on C-methyl-branched nucleosides and nucleotides approved as anti-hepatitis C infection (HCV) drugs, their mechanism of action and recent progress in the development of new clinical candidates. Then, we report on our attempt to develop several C-methyl nucleosides/tides potentially useful for treatment of various diseases such cancer, pain, epilepsy and glaucoma. Design, synthesis and pharmacological screening of 1'-C-, 2'-C-, 3'-C-methyladenosine or other purine/pyrimidine nucleosides allowed us to discover some promising new molecules. 3'-C-Methyladenosine showed antitumor activity against several human tumor cell lines. We have investigated the mechanism of action of 3;-C-methyladenosine that proved to be an effective inhibitor of ribonucleotide reductase. Moreover, we will also summarize the chemical and biological properties of some of the recent N6-substituted and 5', N6-disubstituted 2'-C-methyladenosine derivatives that were synthetized in our laboratory and evaluated as A1 adenosine receptor agonists. 2-Chloro-2'- C-methyl-N6-cyclopentyladenosine (2'-Me-CCPA), 5'-chloro-5'-deoxy-N6-(±)-(endo-norborn- 2-yl)adenosine (5'Cl5'd-(±)-ENBA) and 2'-C-methyl-5'-chloro-5'-deoxy-N6-(±)-(endonorborn- 2-yl)adenosine (2'-Me-5'Cl5'd-(±)-ENBA) displayed high hA1AR affinity and selectivity. 2'-Me-CCPA and 5'Cl5'd-(±)-ENBA showed significant analgesic properties.

A Positive Allosteric Modulator of the Adenosine A1 Receptor Selectively Inhibits Primary Afferent Synaptic Transmission in a Neuropathic Pain Model.

In the spinal cord and periphery, adenosine inhibits neuronal activity through activation of the adenosine A1 receptor (A1R), resulting in antinociception and highlighting the potential of therapeutically targeting the receptor in the treatment of neuropathic pain. This study investigated the changes in adenosine tone and A1R signaling, together with the actions of a novel A1R positive allosteric modulator (PAM), VCP171 [(2-amino-4-(3-(trifluoromethyl)phenyl)thiophen-3-yl)(phenyl)methanone], on excitatory and inhibitory neurotransmission at spinal cord superficial dorsal horn synapses in a rat partial nerve-injury model of neuropathic pain. In the absence of A1R agonists, superfusion of the A1R antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 1 µM), produced a significantly greater increase in electrically evoked α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor-mediated synaptic current (eEPSC) amplitude in both lamina I and II neurons from nerve-injured animals than in controls, suggesting that endogenous adenosine tone is increased in the dorsal horn. Inhibitory GABAergic and glycinergic synaptic currents were also significantly increased by DPCPX in controls but there was no difference after nerve injury. The A1R agonist, N6-cyclopentyladenosine, produced greater inhibition of eEPSC amplitude in lamina II but not lamina I of the spinal cord dorsal horn in nerve-injured versus control animals, suggesting a functional increase in A1R sensitivity in lamina II neurons after nerve injury. The A1R PAM, VCP171, produced a greater inhibition of eEPSC amplitude of nerve-injury versus control animals in both lamina I and lamina II neurons. Enhanced adenosine tone and A1R sensitivity at excitatory synapses in the dorsal horn after nerve injury suggest that new generation PAMs of the A1R can be effective treatments for neuropathic pain.

Effect of adenosine A1 receptor agonist on the enhanced excitability of spinal dorsal horn neurons after peripheral nerve injury.

Neuronal hyperactivity has been implicated in abnormal pain sensation following peripheral nerve injuries. Previous studies have indicated that the activation of adenosine A1 receptors (A1R) in the central and peripheral nervous systems produces an antinociceptive effect. However, the mechanisms involved in the peripheral effect are still not fully understood. The effects of the local application of the selective A1R agonist, 2-chloro-N(6)-cyclopentyladenosine (CCPA) on neuronal hyperactivity were examined in this study using a neuropathic pain model induced by a tibial nerve injury. We utilized Fos protein-like immunoreactivity induced by noxious heat stimulation to examine changes in the number of Fos protein like immunoreactive (Fos-LI) neuron profiles in the spinal dorsal horn, and behavioral analysis for mechanical and thermal sensitivities. The nerve injury induced an exaggerated Fos response to noxious heat stimulation. The number of Fos-LI neuron profiles was significantly decreased and their distribution was restricted to the central terminal field of the spared peroneal nerve 3 days after the injury. The number of Fos-LI neuron profiles returned to control levels and a large number of these profiles were observed in the central terminal field of the injured tibial nerve 14 days after the injury. These enhanced Fos responses were attenuated by the local application of CCPA. The nerve injury also resulted in mechanical allodynia and thermal hyperalgesia. The local application of CCPA inhibited thermal hyperalgesia, but was less effective against mechanical allodynia. These results indicated that activation of peripheral A1R plays a role in the regulation of nerve injury-induced hyperalgesia.

IL-11 is required for A1 adenosine receptor-mediated protection against ischemic AKI.

A1 adenosine receptor activation ameliorates ischemic AKI through the induction of renal proximal tubular sphingosine kinase-1. However, systemic adverse effects may limit A1 adenosine receptor-based therapy for ischemic AKI, indicating a need to identify alternative therapeutic targets within this pathway. Here, we evaluated the function of renal proximal tubular IL-11, a clinically approved hematopoietic cytokine, in A1 adenosine receptor-mediated induction of sphingosine kinase-1 and renal protection. Treatment of human proximal tubule epithelial (HK-2) cells with a selective A1 adenosine receptor agonist, chloro-N(6)-cyclopentyladenosine (CCPA), induced the expression of IL-11 mRNA and protein in an extracellular signal-regulated kinase-dependent manner, and administration of CCPA in mice induced renal synthesis of IL-11. Pretreatment with CCPA protected against renal ischemia-reperfusion injury in wild-type mice, but not in IL-11 receptor-deficient mice. Administration of an IL-11-neutralizing antibody abolished the renal protection provided by CCPA. Similarly, CCPA did not induce renal IL-11 expression or protect against renal ischemia-reperfusion injury in mice lacking the renal proximal tubular A1 adenosine receptor. Finally, treatment with CCPA induced sphingosine kinase-1 in HK-2 cells and wild-type mice, but not in IL-11 receptor-deficient or renal proximal tubule A1 adenosine receptor-deficient mice. Taken together, these results suggest that induction of renal proximal tubule IL-11 is a critical intermediary in A1 adenosine receptor-mediated renal protection that warrants investigation as a novel therapeutic target for the treatment of ischemic AKI.