2-Aryladenine Derivatives as a Potent Scaffold for Adenosine Receptor Antagonists: The 6-Morpholino Derivatives.

A set of 2-aryl-9-H or methyl-6-morpholinopurine derivatives were synthesized and assayed through radioligand binding tests at human A1, A2A, A2B, and A3 adenosine receptor subtypes. Eleven purines showed potent antagonism at A1, A3, dual A1/A2A, A1/A2B, or A1/A3 adenosine receptors. Additionally, three compounds showed high affinity without selectivity for any specific adenosine receptor. The structure-activity relationships were made for this group of new compounds. The 9-methylpurine derivatives were generally less potent but more selective, and the 9H-purine derivatives were more potent but less selective. These compounds can be an important source of new biochemical tools and/or pharmacological drugs.

A2B adenosine receptor signaling and regulation.

The A2B adenosine receptor (A2BR) is one of the four adenosine-activated G protein-coupled receptors. In addition to adenosine, protein kinase C (PKC) was recently found to activate the A2BR. The A2BR is coupled to both Gs and Gi, as well as Gq proteins in some cell types. Many primary cells and cell lines, such as bladder and breast cancer, bronchial smooth muscle, skeletal muscle, and fat cells, express the A2BR endogenously at high levels, suggesting its potentially important role in asthma, cancer, diabetes, and other conditions. The A2BR has been characterized as both pro- and anti-inflammatory, inducing cell type-dependent secretion of IL-6, IL-8, and IL-10. Theophylline and enprofylline have long been used for asthma treatment, although it is still not entirely clear if their A2BR antagonism contributes to their therapeutic effects or side effects. The A2BR is required in ischemic cardiac preconditioning by adenosine. Both A2BR and protein kinase C (PKC) contribute to cardioprotection, and both modes of A2BR signaling can be blocked by A2BR antagonists. Inhibitors of PKC and A2BR are in clinical cancer trials. Sulforaphane and other isothiocyanates from cruciferous vegetables such as broccoli and cauliflower have been reported to inhibit A2BR signaling via reaction with an intracellular A2BR cysteine residue (C210). A full, A2BR-selective agonist, critical to elucidate many controversial roles of the A2BR, is still not available, although agonist-bound A2BR structures have recently been reported.

Ligand Screening of Membrane Proteins Embedded in Nanodiscs: How to Manage Non-Specific Interactions in Weak Affinity Chromatography?

Miniaturized weak affinity chromatography is emerging as an interesting alternative to conventional biophysical tools for performing fragment-screening studies in the context of fragment-based drug discovery. In order to push back the analytical limits, it is necessary not only to control non-specific interactions with chromatographic support, but also to adapt this methodology by comparing the results obtained on an affinity column to a control column. The work presented in this study focused on fragment screening that targets a model membrane protein, the adenosine A2A receptor, embedded in nanodiscs (NDs) as biomimetic membranes. By studying the retention behavior of test fragment mixtures on supports modified with different types of NDs, we were able to determine the contribution of ND-related non-specific interactions, in particular the electrostatic effect of anionic phospholipids and the hydrophobic effect of neutral phospholipids. Different strategies for the preparation of control columns (empty NDs, orthosteric site blocking) were investigated and are presented for the first time. With these two types of control columns, the screening enabled the identification of two new fragments of AA2AR, which were confirmed by competition experiments and whose Kd values, estimated directly during the screening or after the competition experiments in frontal mode, were in good agreement.

Verification of In Vitro Anticancer Activity and Bioactive Compounds in Cordyceps Militaris-Infused Sweet Potato Shochu Spirits.

Many liqueurs, including spirits infused with botanicals, are crafted not only for their taste and flavor but also for potential medicinal benefits. However, the scientific evidence supporting their medicinal effects remains limited. This study aims to verify in vitro anticancer activity and bioactive compounds in shochu spirits infused with Cordyceps militaris, a Chinese medicine. The results revealed that a bioactive fraction was eluted from the spirit extract with 40% ethanol. The infusion time impacted the inhibitory effect of the spirit extract on the proliferation of colon cancer-derived cell line HCT-116 cells, and a 21-day infusion showed the strongest inhibitory effect. Furthermore, the spirit extract was separated into four fractions, A-D, by high-performance liquid chromatography (HPLC), and Fractions B, C, and D, but not A, exerted the effects of proliferation inhibition and apoptotic induction of HCT-116 cells and HL-60 cells. Furthermore, Fractions B, C, and D were, respectively, identified as adenosine, cordycepin, and N6-(2-hydroxyethyl)-adenosine (HEA) by comprehensive chemical analyses, including proton nuclear magnetic resonance (1H-NMR), Fourier transform infrared spectroscopy (FT-IR), and electrospray ionization mass spectrometry (ESI-MS). To better understand the bioactivity mechanisms of cordycepin and HEA, the agonist and antagonist tests of the A3 adenosine receptor (A3AR) were performed. Cell viability was suppressed by cordycepin, and HEA was restored by the A3AR antagonist MR1523, suggesting that cordycepin and HEA possibly acted as agonists to activate A3ARs to inhibit cell proliferation. Molecular docking simulations revealed that both adenosine and cordycepin bound to the same pocket site of A3ARs, while HEA exhibited a different binding pattern, supporting a possible explanation for the difference in their bioactivity. Taken together, the present study demonstrated that cordycepin and HEA were major bioactive ingredients in Cordyceps militaries-infused sweet potato shochu spirits, which contributed to the in vitro anticancer activity.

Astrocytes in preoptic area regulate acute nociception-induced hypothermia through adenosine receptors.

AIMS: The preoptic area (POA) of the hypothalamus, crucial in thermoregulation, has long been implicated in the pain process. However, whether nociceptive stimulation affects body temperature and its mechanism remains poorly studied. METHODS: We used capsaicin, formalin, and surgery to induce acute nociceptive stimulation and monitored rectal temperature. Optical fiber recording, chemical genetics, confocal imaging, and pharmacology assays were employed to confirm the role and interaction of POA astrocytes and extracellular adenosine. Immunofluorescence was utilized for further validation. RESULTS: Acute nociception could activate POA astrocytes and induce a decrease in body temperature. Manipulation of astrocytes allowed bidirectional control of body temperature. Furthermore, acute nociception and astrocyte activation led to increased extracellular adenosine concentration within the POA. Activation of adenosine A1 or A2A receptors contributed to decreased body temperature, while inhibition of these receptors mitigated the thermo-lowering effect of astrocytes. CONCLUSION: Our results elucidate the interplay between acute nociception and thermoregulation, specifically highlighting POA astrocyte activation. This enriches our understanding of physiological responses to painful stimuli and contributes to the analysis of the anatomical basis involved in the process.

1.8-cineole prevents platelet activation and aggregation by activating the cAMP pathway via the adenosine A2A receptor.

AIMS: Dysregulated platelet aggregation is a fatal condition in many bacterial- and virus-induced diseases. However, classical antithrombotics cannot completely prevent immunothrombosis, due to the unaddressed mechanisms towards inflammation. Thus, targeting platelet hyperactivation together with inflammation might provide new treatment options in diseases, characterized by immunothrombosis, such as COVID-19 and sepsis. The aim of this study was to investigate the antiaggregatory effect and mode of action of 1.8-cineole, a monoterpene derived from the essential oil of eucalyptus leaves, known for its anti-inflammatory proprieties. MAIN METHODS: Platelet activity was monitored by measuring the expression and release of platelet activation markers, i.e., P-selectin, CD63 and CCL5, as well as platelet aggregation, upon treatment with 1.8-cineole and stimulation with several classical stimuli and bacteria. A kinase activity assay was used to elucidate the mode of action, followed by a detailed analysis of the involvement of the adenylyl-cyclase (AC)-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) pathway by Western blot and ELISA. KEY FINDINGS: 1.8-cineole prevented the expression and release of platelet activation markers, as well as platelet aggregation, upon induction of aggregation with classical stimuli and immunological agonists. Mechanistically, 1.8- cineole influences the activation of the AC-cAMP-PKA pathway, leading to higher cAMP levels and vasodilator-stimulated phosphoprotein (VASP) phosphorylation. Finally, blocking the adenosine A2A receptor reversed the antithrombotic effect of 1.8-cineole. SIGNIFICANCE: Given the recognized anti-inflammatory attributes of 1.8-cineole, coupled with our findings, 1.8-cineole might emerge as a promising candidate for treating conditions marked by platelet activation and abnormal inflammation.

Exogenous Ketone Supplement Administration Abrogated Isoflurane-Anesthesia-Induced Increase in Blood Glucose Level in Female WAG/Rij Rats.

It has been demonstrated that isoflurane-induced anesthesia can increase the blood glucose level, leading to hyperglycemia and several adverse effects. The administration of a mix of ketone diester (KE) and medium-chain triglyceride (MCT) oil, named KEMCT, abolished the isoflurane-anesthesia-induced increase in blood glucose level and prolonged the recovery time from isoflurane anesthesia in a male preclinical rodent model, Wistar Albino Glaxo/Rijswijk (WAG/Rij) rats. While most preclinical studies use exclusively male animals, our previous study on blood glucose changes in response to KEMCT administration showed that the results can be sex-dependent. Thus, in this study, we investigated female WAG/Rij rats, whether KEMCT gavage (3 g/kg/day for 7 days) can change the isoflurane (3%)-anesthesia-induced increase in blood glucose level and the recovery time from isoflurane-evoked anesthesia using the righting reflex. Moreover, KEMCT-induced ketosis may enhance both the extracellular level of adenosine and the activity of adenosine A1 receptors (A1Rs). To obtain information on the putative A1R mechanism of action, the effects of an A1R antagonist, DPCPX (1,3-dipropyl-8-cyclopentylxanthine; intraperitoneal/i.p. 0.2 mg/kg), on KEMCT-generated influences were also investigated. Our results show that KEMCT supplementation abolished the isoflurane-anesthesia-induced increase in blood glucose level, and this was abrogated by the co-administration of DPCPX. Nevertheless, KEMCT gavage did not change the recovery time from isoflurane-induced anesthesia. We can conclude that intragastric gavage of exogenous ketone supplements (EKSs), such as KEMCT, can abolish the isoflurane-anesthesia-induced increase in blood glucose level in both sexes likely through A1Rs in WAG/Rij rats, while recovery time was not affected in females, unlike in males. These results suggest that the administration of EKSs as an adjuvant therapy may be effective in mitigating metabolic side effects of isoflurane, such as hyperglycemia, in both sexes.

The influence of the way of regression on the results obtained by the receptorial responsiveness method (RRM), a procedure to estimate a change in the concentration of a pharmacological agonist near the receptor.

The receptorial responsiveness method (RRM) enables the estimation of a change in concentration of an (even degradable) agonist, near its receptor, via curve fitting to (at least) two concentration-effect (E/c) curves of a stable agonist. One curve should be generated before this change, and the other afterwards, in the same system. It follows that RRM yields a surrogate parameter ("cx") as the concentration of the stable agonist being equieffective with the change in concentration of the other agonist. However, regression can be conducted several ways, which can affect the accuracy, precision and ease-of-use. This study utilized data of previous ex vivo investigations. Known concentrations of stable agonists were estimated with RRM by performing individual (local) or global fitting, this latter with one or two model(s), using a logarithmic (logcx) or a nonlogarithmic (cx) parameter (the latter in a complex or in a simplified equation), with ordinary least-squares or robust regression, and with an "all-at-once" or "pairwise" fitting manner. We found that the simplified model containing logcx was superior to all alternative models. The most complicated individual regression was the most accurate, followed closely by the moderately complicated two-model global regression and then by the easy-to-perform one-model global regression. The two-model global fitting was the most precise, followed by the individual fitting (closely) and by the one-model global fitting (from afar). Pairwise fitting (two E/c curves at once) improved the estimation. Thus, the two-model global fitting, performed pairwise, and the individual fitting are recommended for RRM, using the simplified model containing logcx.

A long­term complete response to namodenoson in liver cancer with Child­Pugh B cirrhosis: A case report.

Established treatments for advanced hepatocellular carcinoma (HCC) with Child-Pugh cirrhosis B (CPB, moderate hepatic dysfunction) are lacking. A recently published randomized phase 2 study in CPB HCC investigating the safety and efficacy of namodenoson (25 mg BID), an A3 adenosine-receptor agonist vs. placebo, suggested a favorable safety profile and a positive efficacy signal in patients with HCC with a CPB score of 7 (CPB7). The present study reports a 61-year-old woman with CPB7 HCC who received namodenoson for over 6 years through this study and its open-label extension. Computed tomography scans demonstrated partial and complete responses after 7 weeks and 4 years of treatment, respectively. Low albumin levels (31 g/l) and elevated baseline levels of alanine transaminase and aspartate aminotransferase (68 U/l and 44 U/l, respectively) were reported. After 4 weeks of treatment, these levels normalized and were stable for over 6 years. No treatment-emergent adverse events were noted. At the time of reporting, the response is ongoing as manifested by imaging studies and liver function evaluation.

Seizure suppression and neuroprotection in soman-exposed rats following delayed intramuscular treatment of adenosine A1 receptor agonist as an adjunct to standard medical treatment.

Soman produces excitotoxic effects by inhibiting acetylcholinesterase in the cholinergic synapses and neuromuscular junctions, resulting in soman-induced sustained status epilepticus (SSE). Our previous work showed delayed intramuscular (i.m.) treatment with A1 adenosine receptor agonist N-bicyclo-[2.2.1]-hept-2-yl-5'-chloro-5'-deoxyadenosine (ENBA) alone suppressed soman-induced SSE and prevented neuropathology. Using this same rat soman seizure model, we tested if delayed therapy with ENBA (60 mg/kg, i.m.) would terminate seizure, protect neuropathology, and aid in survival when given in conjunction with current standard medical countermeasures (MCMs): atropine sulfate, 2-PAM, and midazolam (MDZ). Either 15- or 30-min following soman-induced SSE onset, male rats received atropine and 2-PAM plus either MDZ or MDZ + ENBA. Electroencephalographic (EEG) activity, physiologic parameters, and motor function were recorded. Either 2- or 14-days following exposure surviving rats were euthanized and perfused for histology. All animals treated with MDZ + ENBA at both time points had 100% EEG seizure termination and reduced total neuropathology compared to animals treated with MDZ (2-day, p = 0.015 for 15-min, p = 0.002 for 30-min; 14-day, p < 0.001 for 15-min, p = 0.006 for 30-min), showing ENBA enhanced MDZ's anticonvulsant and neuroprotectant efficacy. However, combined MDZ + ENBA treatment, when compared to MDZ treatment groups, had a reduction in the 14-day survival rate regardless of treatment time, indicating possible enhancement of MDZ's neuronal inhibitory effects by ENBA. Based on our findings, ENBA shows promise as an anticonvulsant and neuroprotectant in a combined treatment regimen following soman exposure; when given as an adjunct to standard MCMs, the dose of ENBA needs to be adjusted.

Low Density Lipoprotein Cholesterol Decreases the Expression of Adenosine A2A Receptor and Lipid Rafts-Protein Flotillin-1: Insights on Cardiovascular Risk of Hypercholesterolemia.

High blood levels of low-density lipoprotein (LDL)-cholesterol (LDL-C) are associated with atherosclerosis, mainly by promoting foam cell accumulation in vessels. As cholesterol is an essential component of cell plasma membranes and a regulator of several signaling pathways, LDL-C excess may have wider cardiovascular toxicity. We examined, in untreated hypercholesterolemia (HC) patients, selected regardless of the cause of LDL-C accumulation, and in healthy participants (HP), the expression of the adenosine A2A receptor (A2AR), an anti-inflammatory and vasodilatory protein with cholesterol-dependent modulation, and Flotillin-1, protein marker of cholesterol-enriched plasma membrane domains. Blood cardiovascular risk and inflammatory biomarkers were measured. A2AR and Flotillin-1 expression in peripheral blood mononuclear cells (PBMC) was lower in patients compared to HP and negatively correlated to LDL-C blood levels. No other differences were observed between the two groups apart from transferrin and ferritin concentrations. A2AR and Flotillin-1 proteins levels were positively correlated in the whole study population. Incubation of HP PBMCs with LDL-C caused a similar reduction in A2AR and Flotillin-1 expression. We suggest that LDL-C affects A2AR expression by impacting cholesterol-enriched membrane microdomains. Our results provide new insights into the molecular mechanisms underlying cholesterol toxicity, and may have important clinical implication for assessment and treatment of cardiovascular risk in HC.

Adenosine receptors in breast cancer.

Adenosine receptors are important in the normal physiological function of cells and the pathogenesis of various cancer cells, including breast cancer cells. The activity of adenosine receptors in cancer cells is related to cell proliferation, angiogenesis, metastasis, immune system evasion, and interference with apoptosis. Considering the different roles of adenosine receptors in cancer cells, we intend to investigate the function of adenosine receptors and their biological pathways in breast cancer to improve understanding of therapeutically relevant signaling pathways.

Exploring Biginelli-based scaffolds as A2B adenosine receptor antagonists: Unveiling novel structure-activity relationship trends, lead compounds, and potent colorectal anticancer agents.

Antagonists of the A2B adenosine receptor have recently emerged as targeted anticancer agents and immune checkpoint inhibitors within the realm of cancer immunotherapy. This study presents a comprehensive evaluation of novel Biginelli-assembled pyrimidine chemotypes, including mono-, bi-, and tricyclic derivatives, as A2BAR antagonists. We conducted a comprehensive examination of the adenosinergic profile (both binding and functional) of a large compound library consisting of 168 compounds. This approach unveiled original lead compounds and enabled the identification of novel structure-activity relationship (SAR) trends, which were supported by extensive computational studies, including quantum mechanical calculations and free energy perturbation (FEP) analysis. In total, 25 molecules showed attractive affinity (Ki < 100 nM) and outstanding selectivity for A2BAR. From these, five molecules corresponding to the new benzothiazole scaffold were below the Ki < 10 nM threshold, in addition to a novel dual A2A/A2B antagonist. The most potent compounds, and the dual antagonist, showed enantiospecific recognition in the A2BAR. Two A2BAR selective antagonists and the dual A2AAR/A2BAR antagonist reported in this study were assessed for their impact on colorectal cancer cell lines. The results revealed a significant and dose-dependent reduction in cell proliferation. Notably, the A2BAR antagonists exhibited remarkable specificity, as they did not impede the proliferation of non-tumoral cell lines. These findings support the efficacy and potential that A2BAR antagonists as valuable candidates for cancer therapy, but also that they can effectively complement strategies involving A2AAR antagonism in the context of immune checkpoint inhibition.

Caffeine in Hepatocellular Carcinoma: Cellular Assays, Animal Experiments, and Epidemiological Investigation.

The use of caffeine in treating various liver diseases has made substantial progress in the past decade owing to advances in science, technology, and medicine. However, whether caffeine has a preventive effect on hepatocellular carcinoma (HCC) and its mechanism are still worth further investigation. In this review, we summarize and analyze the efficacy and safety of caffeine in the prevention of HCC. We conducted a review of articles published in PubMed and Web of Science in the past 2 decades until December 6, 2023, which were searched for using the terms "Caffeine" and "Hepatocellular Carcinoma." Studies have found that coffee intake is negatively correlated with HCC risk, especially caffeinated coffee. Recent studies have found that caffeine has beneficial effects on liver health, decreasing levels of enzymes responsible for liver damaging and slowing the progression of hepatic fibrosis and cirrhosis. Caffeine also acts against liver fibrosis through adenosine receptors (ARs), which promote tissue remodeling by inducing fibrin and collagen production. Additionally, new studies have found that moderate consumption of caffeinated beverages can decrease various the levels of various collagens in patients with chronic hepatitis C. Furthermore, polyphenolic compounds in coffee can improve fat homeostasis, reduce oxidative stress, and prevent liver steatosis and fibrosis. Moreover, many in vitro studies have shown that caffeine can protect liver cells and inhibit the activation and proliferation of hepatic stellate cells. Taken together, we describe the benefits of caffeine for liver health and highlight its potential values as a drug to prevent various hepatic diseases. As a protective agent of liver inflammation, non-selective AR inhibitor caffeine can inhibit the growth of HCC cells by inhibiting adenosine and AR binding to initiate immune response, providing a basis for the future development of caffeine as an adjuvant drug against HCC.

Blockade of endothelial adenosine receptor 2 A suppresses atherosclerosis in vivo through inhibiting CREB-ALK5-mediated endothelial to mesenchymal transition.

Cardiovascular diseases (CVDs) are the leading cause of death worldwide, and morbidity and mortality rates continue to rise. Atherosclerosis constitutes the principal etiology of CVDs. Endothelial injury, inflammation, and dysfunction are the initiating factors of atherosclerosis. Recently, we reported that endothelial adenosine receptor 2 A (ADORA2A), a G protein-coupled receptor (GPCR), plays critical roles in neovascularization disease and cerebrovascular disease. However, the precise role of endothelial ADORA2A in atherosclerosis is still not fully understood. Here, we showed that ADORA2A expression was markedly increased in the aortic endothelium of humans with atherosclerosis or Apoe-/- mice fed a high-cholesterol diet. In vivo studies unraveled that endothelial-specific Adora2a deficiency alleviated endothelial-to-mesenchymal transition (EndMT) and prevented the formation and instability of atherosclerotic plaque in Apoe-/- mice. Moreover, pharmacologic inhibition of ADORA2A with KW6002 recapitulated the anti-atherogenic phenotypes observed in genetically Adora2a-deficient mice. In cultured human aortic endothelial cells (HAECs), siRNA knockdown of ADORA2A or KW6002 inhibition of ADORA2A decreased EndMT, whereas adenoviral overexpression of ADORA2A induced EndMT. Mechanistically, ADORA2A upregulated ALK5 expression via a cAMP/PKA/CREB axis, leading to TGFß-Smad2/3 signaling activation, thereby promoting EndMT. In conclusion, these findings, for the first time, demonstrate that blockade of ADORA2A attenuated atherosclerosis via inhibition of EndMT induced by the CREB1-ALK5 axis. This study discloses a new link between endothelial ADORA2A and EndMT and indicates that inhibiting endothelial ADORA2A could be an effective novel strategy for the prevention and treatment of atherosclerotic CVDs.

Activation of adenosine A2B receptor alleviates myocardial ischemia-reperfusion injury by inhibiting endoplasmic reticulum stress and restoring autophagy flux.

Myocardial ischemia-reperfusion injury (MIRI) poses a significant threat to patients with coronary heart disease. Adenosine A2A receptors have been known as a protective role in MIRI by regulating autophagy, so we assumed that activation of adenosine A2B receptor (A2BAR) might exert a similar effect during MIRI and underlying mechanism be related to proteostasis maintenance as well. In situ hearts were subjected to 30 min of ischemia and 120 min of reperfusion (IR), while invitro cardiomyocytes from neonatal rats experienced 6 h of oxygen-glucose deprivation followed by 12 h of reoxygenation (OGDR). Initially, we observed that post-ischemia-reperfusion induced autophagy flux blockade and ERS both in vivo and in vitro, evident through the increased expression of p62, LC3II, and BIP, which indicated the deteriorated proteostasis. We used a selective A2BAR agonist, Bay 60-6583, to explore the positive effects of A2BAR on cardiomyocytes and found that A2BAR activation rescued damaged cardiac function and morphological changes in the IR group and improved frail cell viability in the OGDR group. The A2BAR agonist also alleviated the blockage of autophagic flux, coupled with augmented ERS in the IR/OGDR group, which was reassured by using an autophagy inhibitor chloroquine (CQ) and ERS inhibitor (4-PBA) in vitro. Additionally, considering cAMP/PKA as a well-known downstream effector of A2BAR, we utilized H89, a selective PKA inhibitor. We observed that the positive efficacy of Bay 60-6583 was inhibited by H89. Collectively, our findings demonstrate that the A2BAR/cAMP/PKA signaling pathway exerts a protective role in MIRI by mitigating impaired autophagic flux and excessive ERS.

Using caffeine as a chemical means to induce flow states.

Flow is an intrinsically rewarding state characterised by positive affect and total task absorption. Because cognitive and physical performance are optimal in flow, chemical means to facilitate this state are appealing. Caffeine, a non-selective adenosine receptor antagonist, has been emphasized as a potential flow-inducer. Thus, we review the psychological and biological effects of caffeine that, conceptually, enhance flow. Caffeine may facilitate flow through various effects, including: i) upregulation of dopamine D1/D2 receptor affinity in reward-associated brain areas, leading to greater energetic arousal and 'wanting'; ii) protection of dopaminergic neurons; iii) increases in norepinephrine release and alertness, which offset sleep-deprivation and hypoarousal; iv) heightening of parasympathetic high frequency heart rate variability, resulting in improved cortical stress appraisal, v) modification of striatal endocannabinoid-CB1 receptor-signalling, leading to enhanced stress tolerance; and vi) changes in brain network activity in favour of executive function and flow. We also discuss the application of caffeine to treat attention deficit hyperactivity disorder and caveats. We hope to inspire studies assessing the use of caffeine to induce flow.

Genetic and functional modulation by agonist MRS5698 and allosteric enhancer LUF6000 at the native A3 adenosine receptor in HL-60 cells.

The A3 adenosine receptor (AR) is an important inflammatory and immunological target. However, the underlying mechanisms are not fully understood. Here, we report the gene regulation in HL-60 cells treated acutely with highly selective A3AR agonist MRS5698, positive allosteric modulator (PAM) LUF6000, or both. Both pro- and anti-inflammatory genes, such as IL-1a, IL-1ß, and NFκBIZ, are significantly upregulated. During our observations, LUF6000 alone produced a lesser effect, while the MRS5698 + LUF6000 group demonstrated generally greater effects than MRS5698 alone, consistent with allosteric enhancement. The number of genes up- and down-regulated are similar. Pathway analysis highlighted the critical involvement of signaling molecules, including IL-6 and IL-17. Important upstream regulators include IL-1a, IL-1ß, TNF-α, NF-κB, etc. PPAR, which modulates eicosanoid metabolism, was highly downregulated by the A3AR agonist. Considering previous pharmacological results and mathematical modeling, LUF6000's small enhancement of genetic upregulation suggested that MRS5698 is a nearly full agonist, which we demonstrated in both cAMP and calcium assays. The smaller effect of LUF6000 on MRS5698 in comparison to its effect on Cl-IB-MECA was shown in both HL-60 cells endogenously expressing the human (h) A3AR and in recombinant hA3AR-expressing CHO cells, consistent with its HL-60 cell genetic regulation patterns. In summary, by using both selective agonists and PAM, we identified genes that are closely relevant to immunity and inflammation to be regulated by A3AR in differentiated HL-60 cells, a cell model of neutrophil function. In addition, we demonstrated the previously uncharacterized allosteric signaling-enhancing effect of LUF6000 in cells endogenously expressing the hA3AR.

Therapeutic potency of A1 adenosine receptor antagonists in the treatment of cardiovascular diseases, current status and perspectives.

BACKGROUND: Cardiomyocytes form, transport, and metabolize the omnipresent metabolite adenosine. Depending upon the adenosine concentrations and the pharmacological properties of receptor subtypes, adenosine exerts (patho)physiological responses in the cardiovascular system. The objective of this review is to present different protective mechanisms of A1-adenosine receptor inhibitors in cardiovascular diseases. METHODS AND RESULTS: Literature references were collected and sorted using relevant keywords and key phrases as search terms in scientific databases such as Web of Science, PubMed and Google Scholar. A1 adenosine receptor regulates free fatty acid metabolism, lipolysis, heart rate, blood pressure, and cardiovascular toxicity. The evidence clearly supporting the therapeutic potency of pharmacological A1 adenosine receptors agonists and antagonists in modulating cardiovascular risk factor parameters and treatment of cardiovascular diseases. CONCLUSION: This review summarizes the protective role of pharmacological A1-adenosine receptor regulators in the pathogenesis of cardiovascular diseases for a better management of cardiovascular diseases.

Clinically relevant therapeutic approaches against acetaminophen hepatotoxicity and acute liver failure.

Liver injury and acute liver failure caused by an acetaminophen (APAP) overdose is a significant clinical problem in western countries. With the introduction of the mouse model of APAP hepatotoxicity in the 1970 s, fundamental mechanisms of cell death were discovered. This included the recognition that part of the APAP dose is metabolized by cytochrome P450 generating a reactive metabolite that is detoxified by glutathione. After the partial depletion of glutathione, the reactive metabolite will covalently bind to sulfhydryl groups of proteins, which is the initiating event of the toxicity. This insight led to the introduction of N-acetyl-L-cysteine, a glutathione precursor, as antidote against APAP overdose in the clinic. Despite substantial progress in our understanding of the pathomechanisms over the last decades viable new antidotes only emerged recently. This review will discuss the background, mechanisms of action, and the clinical prospects of the existing FDA-approved antidote N-acetylcysteine, of several new drug candidates under clinical development [4-methylpyrazole (fomepizole), calmangafodipir] and examples of additional therapeutic targets (Nrf2 activators) and regeneration promoting agents (thrombopoietin mimetics, adenosine A2B receptor agonists, Wharton's Jelly mesenchymal stem cells). Although there are clear limitations of certain therapeutic approaches, there is reason to be optimistic. The substantial progress in the understanding of the pathophysiology of APAP hepatotoxicity led to the consideration of several drugs for development as clinical antidotes against APAP overdose in recent years. Based on the currently available information, it is likely that this will result in additional drugs that could be used as adjunct treatment for N-acetylcysteine.