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 utility of the TNF-α agonist, AB MECA, by in silico (molecular docking) and in vitro (human cancer cells in xenografted mice) studies. Method: 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 myeloprotective effect was also found in groups treated 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 suggests that it may be an effective therapy for lung cancer.

Namodenoson in Advanced Hepatocellular Carcinoma and Child–Pugh B Cirrhosis: Randomized Placebo-Controlled Clinical Trial

Namodenoson, an A3 adenosine-receptor agonist, showed promising results in advanced hepatocellular carcinoma (HCC) and moderate hepatic dysfunction (Child–Pugh B; CPB) in a phase I/II clinical study. This phase II study investigated namodenoson as second-line therapy in such patients. Patients were randomized 2:1 to twice a day (BID) namodenoson (25 mg; n = 50) or placebo (n = 28). The primary endpoint (overall survival [OS]) was not met. Median OS was 4.1/4.3 months for namodenoson/placebo (hazard ratio [HR], 0.82; 95% confidence interval [CI] 0.49–1.38; p = 0.46). Pre-planned subgroup analysis of CPB7 patients (34 namodenoson-treated, 22 placebo-treated) showed a nonsignificant improvement in OS/progression-free survival (PFS). OS: 6.9 versus 4.3 months; HR, 0.81; 95% CI: 0.45–1.43, p = 0.46. PFS: 3.5 versus 1.9 months; HR, 0.89; 95% CI: 0.51–1.55, p = 0.67 (log-rank test). The difference in 12-month OS was significant (44% versus 18%, p = 0.028). Response rates were determined in patients for whom ≥ 1 assessment post-baseline was available (34 namodenoson-treated, 21 placebo-treated). Partial response was achieved by 3/34 (8.8%) and 0/21 (0%) patients, respectively. Namodenoson was well-tolerated, with a safety profile comparable to that of the placebo group. No treatment-related deaths were reported; no patients withdrew due to toxicity. In conclusion, namodenoson demonstrated a favorable safety profile and a preliminary efficacy signal in HCC CPB.

Synthesis and Characterization of Potential and Degraded Impurities of Regadenoson

Background: Regadenoson is an A2A adenosine receptor agonist that is a coronary vasodilator and commonly used as a pharmacologic cardiac stressing agents. Methods: HPLC method was used for the analysis of related substances. The degraded impurities during the process were isolated and characterized by IR, Mass and NMR spectral analysis. Results: Forced degradation study of regadenoson under conditions of hydrolysis (neutral, acidic and alkaline) and oxidations suggested in the ICH Q1A(R2) was accomplished. The drug showed significant degradation under all the above conditions. On the whole, five novel degradation products were found under diverse conditions along with process related impurities which were not reported earlier. Conclusion: All the degradation products were well characterized by using advanced spectroscopic techniques like IR, 1H NMR, 13C NMR and Mass spectra. The identification of these impurities will be productive for the quality control during the production and stability behavior of the regadenoson drug substance.

P069 CD73 promotes colitis-associated tumourigenesis in mice

Background Patients with inflammatory bowel disease (IBD) are at a higher risk of developing colitis-associated colorectal cancer. The aim of the present study was to investigate the role of CD73 in IBD-associated tumourigenesis. Methods A mouse model of colitis-associated tumourigenesis (CAT) induced by azoxymethane and dextran sulphate sodium (AOM/DSS) was successfully constructed. Model mice were injected with CD73 inhibitor or adenosine receptor agonist. Colon length, body weight loss and tumour formation were assessed macroscopically. Measurement of inflammatory cytokines and RNA sequencing on colon tissues were performed. Results Inhibition of CD73 by adenosine 5′-(α,β-methylene) diphosphate (APCP) suppressed the severity of CAT with attenuated weight loss, longer colons, lower tumour number and smaller tumour size when compared with the model group. On the other hand, activation of adenosine receptors using 1-(6-amino-9H-purin-9-yl)-1-deoxy-N-ethyl-β-d-ribofuranuronamide (NECA) exacerbated CAT. Histological assessment indicated that inhibition of CD73 reduced while activation of adenosine receptors exacerbated the histological damage of the colon compared with the model group. Increased expression of pro-inflammatory cytokines (tumour necrosis factor-α and interleukin-6) in colonic tissue was detected in the NECA group. According to the results of RNA sequencing, potential oncogenes such as ALOX15, Bcl2l15 and Nat8l were found to be downregulated in the APCP group and upregulated in the NECA group compared with the model group. Conclusion Therefore, inhibition of CD73 attenuated IBD-associated tumourigenesis, while activation of adenosine receptors exacerbated tumourigenesis in a C57BL/6J mouse model. This effect may be associated with the expression of pro-inflammatory cytokines and the regulation of ALOX15, Bcl2l15 and Nat8l.

P327 Concordance between regadenoson stress echocardiography and spect-MIBI (99mTc) in patients with suspected myocardial ischemia

Background Regadenoson is a selective A2A adenosine receptor agonist approved for the detection of myocardial with SPECT-MIBI. To date, few studies have appraised the utility of this drug using transthoracic echocardiography with the same purpose. We designed this prospective study to evaluate the diagnostic agreement between these two techniques used simultaneously to detect myocardial ischemia. We report our first results evaluating the concordance between both techniques. Methods Patients enrolled were referred to our clinic for the evaluation of chest pain. Myocardial perfusion imaging was performed with regadenoson (400 µg ) which was injected before 99mTc-MIBI. Stress and rest sets of images were evaluated for relative uptake of the radiotracer in order to detect and characterize perfusion defects. The echocardiogram was acquired 60-90 seconds after the administration of the drug using a standardized technique. Two independent observers (one cardiologist and one specialist in nuclear medicine) interpreted the images, both unaware of the result of the complementary technique. Results One hundred twenty five patients were included, 69 (55%) of them males. Median age was 73 years (IQR 65-79). One hundred seventeen patients had both studies interpretable. Thirty-nine (32.5%) patients had had a diagnosis of ischemic heart disease before the study (either a myocardial infarction or a coronary revascularization). The median pre-test probability of coronary artery disease in those without a true previous diagnosis was 30.5% (16.0-50.8), being 50% in the intermediate risk (15-85%) and 49% in the low risk stratum (less than 15%). Significant reductions is systolic and diastolic blood pressure were detected with regadenoson [systolic 134 (21) mmHg Vs 131 (23) mmHg, p < 0.001; diastolic: 78 (12) mmHg Vs 73 (13) mmHg, p < 0.001], with a striking increase in heart rate: 67 (13) bpm Vs 91 (17) bpm, p < 0.001. More patients had a SPECT test showing myocardial ischemia (28% Vs 16%; p < 0.001). Agreement between both tests were 84%. The kappa index obtained from this sample was 0.34 (CI95% 0.15-0.53). Segregating those patients without a history of coronary artery disease (n = 81), the positive test rate were 14% for echo and 20% for SPECT-MIBI (p = 0.019). Kappa index was even lower: 0.29 (CI95% 0.02-0.56). Conclusions. SPECT-MIBI provides more positive tests than stress echocardiography when regadenoson is used as the stressor agent. The concordance between tests is low.

Expression of Adenosine Receptors in Rodent Pancreas

Adenosine regulates exocrine and endocrine secretions in the pancreas. Adenosine is considered to play a role in acini-to-duct signaling in the exocrine pancreas. To identify the molecular basis of functional adenosine receptors in the exocrine pancreas, immunohistochemical analysis was performed in the rat, mouse, and guinea pig pancreas, and the secretory rate and concentration of HCO3− in pancreatic juice from the rat pancreas were measured. The A2A adenosine receptor colocalized with ezrin, an A-kinase anchoring protein, in the luminal membrane of duct cells in the mouse and guinea pig pancreas. However, a strong signal ascribed to A2B adenosine receptors was detected in insulin-positive β cells in islets of Langerhans. The A2A adenosine receptor agonist 4-[2-[[6-Amino-9-(N-ethyl-β-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid (CGS 21680) stimulated HCO3−-rich fluid secretion from the rat pancreas. These results indicate that A2A adenosine receptors may be, at least in part, involved in the exocrine secretion of pancreatic duct cells via acini-to-duct signaling. The adenosine receptors may be a potential therapeutic target for cancer as well as exocrine dysfunctions of the pancreas.

A novel small molecule A2A adenosine receptor agonist, indirubin-3′-monoxime, alleviates lipid-induced inflammation and insulin resistance in 3T3-L1 adipocytes

Saturated free fatty acid-induced adipocyte inflammation plays a pivotal role in implementing insulin resistance and type 2 diabetes. Recent reports suggest A2A adenosine receptor (A2AAR) could be an attractive choice to counteract adipocyte inflammation and insulin resistance. Thus, an effective A2AAR agonist devoid of any toxicity is highly appealing. Here, we report that indirubin-3′-monoxime (I3M), a derivative of the bisindole alkaloid indirubin, efficiently binds and activates A2AAR which leads to the attenuation of lipid-induced adipocyte inflammation and insulin resistance. Using a combination of in silico virtual screening of potential anti-diabetic candidates and in vitro study on insulin-resistant model of 3T3-L1 adipocytes, we determined I3M through A2AAR activation markedly prevents lipid-induced impairment of the insulin signaling pathway in adipocytes without any toxic effects. While I3M restrains lipid-induced adipocyte inflammation by inhibiting NF-κB dependent pro-inflammatory cytokines expression, it also augments cAMP-mediated CREB activation and anti-inflammatory state in adipocytes. However, these attributes were compromised when cells were pretreated with the A2AAR antagonist, SCH 58261 or siRNA mediated knockdown of A2AAR. I3M, therefore, could be a valuable option to intervene adipocyte inflammation and thus showing promise for the management of insulin resistance and type 2 diabetes.

Polarized Entamoeba: A model for stable bleb driven motility

Protozoan parasites Entamoeba histolytica and Entamoeba invadens formed a polarized phenotype, an elongated shape with a single leading edge and a trailing edge when treated with pentoxifylline. The leading edge of the polarized morphology was a spherical protrusion devoid of F-actin but with occasional F-actin scars, indicating the presence of bleb. The polarized form was stable bleb driven since the blebbing was limited to the leading edge. Pentoxifylline induced chemokinesis in Entamoeba as it switched the motility pattern from slow and random to fast and directionally persistent. Pentoxifylline speeded up the cell aggregation in E. invadens during growth and encystation due to enhanced chemotaxis of the polarized form. The transformation of non-polarized adherent trophozoites to nonadherent stable bleb driven form occurred via lamellipodial and bleb driven adherent intermediate phenotypes. The nonadherent polarized phenotype was highly motile under confinement and moved by rearward plasma membrane flow. In contrast to pentoxifylline, adenosine, the adenosine receptor agonist, stimulated the formation of multiple protrusions leading to random motility. Thus pentoxifylline might prevent lateral protrusions by inhibiting adenosine receptor, producing the monopodial polarized morphology.Summary statementPentoxifylline, the adenosine receptor antagonist induced a stable bleb driven polarized morphology in Entamoeba characterized by fast, directionally persistent and highly chemotactic motility.