AB-MECA

Pharmacological Study of A3 Adenosine Receptor agonist (AB Meca) in Xenograft Lung Cancer Model in Mice through In Silico and In Vivo Approach: Targeting TNF-α

Background: Lung cancer is the leading cause of mortality in India. Adenosine Receptor (AR) has emerged as a novel cancer-specific target. A3AR levels are upregulated in various tumor cells, which may mean that the specific AR may act as a biological marker and target specific ligands leading to cell growth inhibition. Aim: Our aim was to study the efficacy of the adenosine receptor agonist, AB MECA, by in silico (molecular docking) and in vitro (human cancer cells in xenografted mice) studies. Methods: Molecular docking on the AB-meca and TNF-α was performed using AutoDock. A549 Human lung cancer 2 ×106 cells per microliter per mouse injected via intrabronchial route. Rat TNF-α level was assessed by ELISA method. Results: AB Meca's predicted binding energy (beng) with TNF-α was 97.13 kcal/mol, and the compatible docking result of a small molecular inhibitor with TNF-α native ligand beng was 85.76 kcal/mol. In vivo, a single dose of lung cancer cell A549 is being researched to potentiate tumor development. Doxorubicin and A3AR agonist therapies have lowered TNF-alpha levels that were associated with in silico function. The A3AR Agonist showed myeloprotective effects in the groups treated along with doxorubicin. Conclusion: AB MECA's higher binding energy (beng) with TNF-α mediated reduction of tumor growth in our lung cancer in vivo model suggested that it may be an effective therapy for lung cancer.

[125I]4-aminobenzyl-5'-N-methylcarboxamidoadenosine (125I)AB-MECA) labels multiple adenosine receptor subtypes in rat brain

Adenosine modulates neuronal activity and neurotransmitter release through interaction with cell surface receptors. Four adenosine receptor subtypes, A1, A2A, A2B, and A3 receptors, have been cloned and characterized. The agonist ligand, [125I]AB-MECA ([125I]4-aminobenzyl-5'N-methylcarboxamidoadenosine) has high affinity for recombinant A1 and A3 receptors [Olah et al., Mol. Pharmacol, 45 (1994) 978-982]. Rodent A3 receptors are relatively insensitive to xanthines; inhibition of A1 receptors with xanthines allows selective detection of A3 receptors despite the lack of selectivity of the ligand. We studied whether [125I]AB-MECA is useful for localization and characterization of A3 receptors in rat brain. The autoradiographic distribution of total [125I]AB-MECA (400 pM) binding closely resembled the pattern of A1 receptor binding, with highest levels in cerebellum, hippocampus, and thalamus, and moderate levels in cortex and striatum. Drug competition studies confirmed that almost all [125I]AB-MECA binding could be attributed to labeling of A1 receptors. Xanthine amine congener (1 microM) reduced specific [125I]AB-MECA binding by > 95%, indicating that xanthine-resistant A3 receptors represent a quantitatively minor subtype. Despite the use of a radioligand with high affinity and high specific activity, the low density of A3 receptors in rat brain appears insufficient to allow localization, or even consistent detection, of this receptor subtype. In the presence of DPCPX (50 nM, to block A1 receptors), residual [125I]AB-MECA binding to A2A receptors was observed in the striatum. Thus [125I]AB-MECA labels primarily A1 and A2A adenosine receptors in rat brain.

The role of the adenosinergic system in lung fibrosis

Adenosine (ADO) is a retaliatory metabolite that is expressed in conditions of injury or stress. During these conditions ATP is released at the extracellular level and is metabolized to adenosine. For this reason, adenosine is defined as a "danger signal" for cells and organs, in addition to its important role as homeostatic regulator. Its physiological functions are mediated through interaction with four specific transmembrane receptors called ADORA1, ADORA2A, ADORA2B and ADORA3. In the lungs of mice and humans all four adenosine receptors are expressed with different roles, having pro- and anti-inflammatory roles, determining bronchoconstriction and regulating lung inflammation and airway remodeling. Adenosine receptors can also promote differentiation of lung fibroblasts into myofibroblasts, typical of the fibrotic event. This last function suggests a potential involvement of adenosine in the fibrotic lung disease processes, which are characterized by different degrees of inflammation and fibrosis. Idiopathic pulmonary fibrosis (IPF) is the pathology with the highest degree of fibrosis and is of unknown etiology and burdened by lack of effective treatments in humans.

A3 adenosine receptor agonist IB-MECA reduces myocardial ischemia-reperfusion injury in dogs

We examined the effect of the A3 adenosine receptor (AR) agonist IB-MECA on infarct size in an open-chest anesthetized dog model of myocardial ischemia-reperfusion injury. Dogs were subjected to 60 min of left anterior descending (LAD) coronary artery occlusion and 3 h of reperfusion. Infarct size and regional myocardial blood flow were assessed by macrohistochemical staining with triphenyltetrazolium chloride and radioactive microspheres, respectively. Four experimental groups were studied: vehicle control (50% DMSO in normal saline), IB-MECA (100 microg/kg iv bolus) given 10 min before the coronary occlusion, IB-MECA (100 microg/kg iv bolus) given 5 min before initiation of reperfusion, and IB-MECA (100 microg/kg iv bolus) given 10 min before coronary occlusion in dogs pretreated 15 min earlier with the ATP-dependent potassium channel antagonist glibenclamide (0.3 mg/kg iv bolus). Administration of IB-MECA had no effect on any hemodynamic parameter measured including heart rate, first derivative of left ventricular pressure, aortic pressure, LAD coronary blood flow, or coronary collateral blood flow. Nevertheless, pretreatment with IB-MECA before coronary occlusion produced a marked reduction in infarct size ( approximately 40% reduction) compared with the control group (13.0 +/- 3.2% vs. 25.2 +/- 3.7% of the area at risk, respectively). This effect of IB-MECA was blocked completely in dogs pretreated with glibenclamide. An equivalent reduction in infarct size was observed when IB-MECA was administered immediately before reperfusion (13.1 +/- 3.9%). These results are the first to demonstrate efficacy of an A3AR agonist in a large animal model of myocardial infarction by mechanisms that are unrelated to changes in hemodynamic parameters and coronary blood flow. These data also demonstrate in an in vivo model that IB-MECA is effective as a cardioprotective agent when administered at the time of reperfusion.

125I-4-aminobenzyl-5'-N-methylcarboxamidoadenosine, a high affinity radioligand for the rat A3 adenosine receptor

The rat A3 adenosine receptor (AR) is a recently characterized AR subtype cloned from testis and brain cDNA libraries. N6-2-(4-Amino-3-[125I]iodophenyl)ethyladenosine, a high affinity A1AR agonist, has served as the only radioligand available for study of the A3AR. The relatively low affinity of N6-2-(4-amino-3-[125I] iodophenyl)ethyladenosine for the A3AR and its greater A1AR selectivity necessitate the development of more appropriate radioligands for A3AR analysis. This report characterizes 125I-4-aminobenzyl-5'-N-methylcarboxamidoadenosine (125I-AB-MECA), a high affinity radioligand for the A3AR, in two cell lines that express this AR subtype. Membranes from Chinese hamster ovary (CHO) cells expressing the rat A3AR and from the rat mast cell line RBL-2H3 bound 125I-AB-MECA with Kd values of 1.48 +/- 0.33 nM and 3.61 +/- 0.30 nM, respectively. As determined by 125I-AB-MECA binding, levels of A3AR expression in the A3AR-CHO cell line and RBL-2H3 cells were 3.06 +/- 0.21 pmol/mg and 1.02 +/- 0.13 pmol/mg, respectively. Binding of 125I-AB-MECA was characterized in competition assays. In the A3AR-CHO cell line a potency order of cyclohexyl-5'-N-ethylcarboxamidoadenosine (cyclohexyl-NECA) = benzyl-NECA > (-)-N6-[(R)-phenylisopropyl]adenosine = NECA was observed, and in RBL-2H3 cells (-)-N6-[(R)-phenylisopropyl]adenosine and NECA were equipotent. Xanthine amine congener (XAC) and 8-cyclopentyl-1,3-dipropylxanthine did not significantly inhibit 125I-AB-MECA binding. The parent compound, AB-MECA, dose-dependently inhibited forskolin-stimulated adenylyl cyclase activity in A3AR-CHO cell membranes. 125I-AB-MECA bound to the rat A1AR and canine A2aAR expressed in COS-7 cells with Kd values of 3.42 +/- 0.43 nM and 25.1 +/- 12.6 nM, respectively. This binding was significantly reduced in the presence of 1 microM XAC. In RBL-2H3 cells, XAC had no effect on 125I-AB-MECA affinity and reduced the level of radioligand binding by approximately 5%.