Inhibition of ecto-5'-nucleotidase and adenosine deaminase is able to reverse long-term behavioural effects of early ethanol exposure in zebrafish (Danio rerio).

The behavioural impacts of prenatal exposure to ethanol include a lower IQ, learning problems, anxiety and conduct disorders. Several components of the neurochemical network could contribute to the long-lasting effects of ethanol embryonic exposure. Adenosine is an important neuromodulator, that has been indicated to be affected by acute and chronic exposure to ethanol. Here, embryos of zebrafish exposed to 1% ethanol during the developmental stages of gastrula/segmentation or pharyngula exhibited anxiolytic effect, increased aggressiveness, and decreased social interaction. The exposure during pharyngula stage was able to affect all behavioural parameters analysed at 3 months-post fertilization (mpf), while the treatment during gastrula stage affected the anxiety and social interaction parameters. The aggressiveness was the only behavioural effect of early ethanol exposure that lasted to 12 mpf. The use of a specific inhibitor of adenosine production, the inhibitor of ecto-5'-nucleotidase (AMPCP/150 mg/kg), and the specific inhibitor of adenosine degradation, the inhibitor of adenosine deaminase, EHNA (100 mg/kg) did not affect the effects over anxiety. However, AMPCP at 3 mpf, but not EHNA, reversed aggressive parameters. AMPCP also recovered the social interaction parameter at 3 mpf in animals treated in both stages, while EHNA recovered this parameter just in those animals treated with ethanol during the gastrula stage. These results suggest that long-lasting behavioural effects of ethanol can be modulated by intervention on ecto-5'-nucleotidase and adenosine deaminase activities.

Therapeutic Perspectives of Adenosine Deaminase Inhibition in Cardiovascular Diseases.

Adenosine deaminase (ADA) is an enzyme of purine metabolism that irreversibly converts adenosine to inosine or 2'deoxyadenosine to 2'deoxyinosine. ADA is active both inside the cell and on the cell surface where it was found to interact with membrane proteins, such as CD26 and adenosine receptors, forming ecto-ADA (eADA). In addition to adenosine uptake, the activity of eADA is an essential mechanism that terminates adenosine signaling. This is particularly important in cardiovascular system, where adenosine protects against endothelial dysfunction, vascular inflammation, or thrombosis. Besides enzymatic function, ADA protein mediates cell-to-cell interactions involved in lymphocyte co-stimulation or endothelial activation. Furthermore, alteration in ADA activity was demonstrated in many cardiovascular pathologies such as atherosclerosis, myocardial ischemia-reperfusion injury, hypertension, thrombosis, or diabetes. Modulation of ADA activity could be an important therapeutic target. This work provides a systematic review of ADA activity and anchoring inhibitors as well as summarizes the perspectives of their therapeutic use in cardiovascular pathologies associated with increased activity of ADA.

Adenosine deaminase inhibition.

Adenosine deaminase is a critical enzyme in purine metabolism that regulates intra and extracellular adenosine concentrations by converting it to inosine. Adenosine is an important purine that regulates numerous physiological functions by interacting with its receptors. Adenosine and consequently adenosine deaminase can have pro or anti-inflammatory effects on tissues depending on how much time has passed from the start of the injury. In addition, an increase in adenosine deaminase activity has been reported for various diseases and the significant effect of deaminase inhibition on the clinical course of different diseases has been reported. However, the use of inhibitors is limited to only a few medical indications. Data on the increase of adenosine deaminase activity in different diseases and the impact of its inhibition in various cases have been collected and are discussed in this review. Overall, the evidence shows that many studies have been done to introduce inhibitors, however, in vivo studies have been much less than in vitro, and often have not been expanded for clinical use.

Inhibition of adenosine deaminase and xanthine oxidase by valproic acid abates hepatic triglyceride accumulation independent of corticosteroids in female rats treated with estrogen-progestin.

Elevated circulating uric acid has been postulated to play an important pathophysiological role in estrogen-progestin combined oral contraceptive (COC)-induced hypertension and endothelial dysfunction. We hypothesized that disruption of glucoregulation and liver triglyceride (TG) accumulation induced by COC use would be abated by valproic acid (VPA) treatment through suppression of adenosine deaminase (ADA) and xanthine oxidase (XO) activities. Female Wistar rats aged 9-10 weeks were treated with a combination of estrogen-progestin COC steroids (1.0 µg ethinylestradiol and 5.0 µg levonorgestrel; p.o.) with or without VPA (100.0 mg/kg; p.o.) daily for 6 weeks. The result shows that the disrupted glucoregulation and associated elevated hepatic ADA activity, plasma and hepatic XO activity, uric acid (UA), TG/HDL-cholesterol, total cholesterol, and malondialdehyde induced by COC treatment were attenuated by VPA treatment. However, VPA did not have any effect on plasma aldosterone, corticosterone, ADA, circulating and hepatic free fatty acid. Our results demonstrate that suppression of plasma and hepatic XO activities, along with hepatic ADA activity and UA by VPA treatment, protects against disrupted glucoregulation and increased liver TG by COC independent of elevated corticosteroids. The findings imply that VPA would provide protection against the development of cardiometabolic disorder via inhibition of the ADA/XO/UA-mediated pathway.

Computational and experimental validation of morin as adenosine deaminase inhibitor.

Adenosine deaminase (ADA) is one of the major enzymes involved in purin metabolism, it has a significant role in cell growth and differentiation. Over-activity of ADA has been noticed in some pathology, like malignancy and inflammation and makes it an attractive target for the development of drugs for such diseases. In the present study, ADA inhibitory activity of morin, a bioactive flavonoid, was assessed through computational and biophysical methods. The enzyme kinetics data showed that morin is a competitive inhibitor of ADA. Binding energy calculated from ITC analysis was -7.11 kcal/mol. Interaction of morin with ADA was also studied using fluorescence quenching method. Molecular docking studies revealed the structural details of the interaction. Molecular dynamics study in explicit solvent was also conducted to assess the structural stability of protein ligand complex.

Curcumin improves episodic memory in cadmium induced memory impairment through inhibition of acetylcholinesterase and adenosine deaminase activities in a rat model.

Curcumin, the main polyphenolic component of turmeric (Curcuma longa) rhizomes has been reported to exert cognitive enhancing potential with limited scientific basis. Hence, this study sought to evaluate the effect of curcumin on cerebral cortex acetylcholinesterase (AChE) and adenosine deaminase (ADA) activities in cadmium (Cd)-induced memory impairment in rats. Animals were divided into six groups (n = 6): saline/vehicle, saline/curcumin 12.5 mg/kg, saline/curcumin 25 mg/kg, Cd/vehicle, Cd/curcumin 12.5 mg/kg, and Cd/curcumin 25 mg/kg. Rats received Cd (2.5 mg/kg) and curcumin (12.5 and 25 mg/kg, respectively) by gavage for 7 days. The results of this study revealed that cerebral cortex AChE and ADA activities were increased in Cd-poisoned rats, and curcumin co-treatment reversed these activities to the control levels. Furthermore, Cd intoxication increased the level of lipid peroxidation in cerebral cortex with a concomitant decreased in functional sulfuhydryl (-SH) group and nitric oxide (NO), a potent neurotransmitter and neuromodulatory agent. However, the co-treatment with curcumin at 12.5 and 25 mg/kg, respectively increased the non-enzymatic antioxidant status and NO in cerebral cortex with a decreased in malondialdehyde (MDA) level. Therefore, inhibition of AChE and ADA activities as well as increased antioxidant status by curcumin in Cd-induced memory dysfunction could suggest some possible mechanism of action for their cognitive enhancing properties.

Increased activity of vascular adenosine deaminase in atherosclerosis and therapeutic potential of its inhibition.

AIMS: Extracellular nucleotides and adenosine that are formed or degraded by membrane-bound ecto-enzymes could affect atherosclerosis by regulating the inflammation and thrombosis. This study aimed to evaluate a relation between ecto-enzymes that convert extracellular adenosine triphosphate to adenine dinucleotide phosphate, adenosine monophosphate, adenosine, and inosine on the surface of the vessel wall with the severity or progression of experimental and clinical atherosclerosis. Furthermore, we tested whether the inhibition of adenosine deaminase will block the development of experimental atherosclerosis. METHODS AND RESULTS: Vascular activities of ecto-nucleoside triphosphate diphosphohydrolase 1, ecto-5'-nucleotidase, and ecto-adenosine deaminase (eADA) were measured in aortas of apolipoprotein E-/- low density lipoprotein receptor (ApoE-/-LDLR-/-) and wild-type mice as well as in human aortas. Plaques were analysed in the entire aorta, aortic root, and brachiocephalic artery by Oil-Red O and Orcein Martius Scarlet Blue staining and vascular accumulation of macrophages. The cellular location of ecto-enzymes was analysed by immunofluorescence. The effect of eADA inhibition on atherosclerosis progression was studied by a 2-month deoxycoformycin treatment of ApoE-/-LDLR-/- mice. The vascular eADA activity prominently increased in ApoE-/-LDLR-/- mice when compared with wild type already at the age of 1 month and progressed along atherosclerosis development, reaching a 10-fold difference at 10 months. The activity of eADA correlated with atherosclerotic changes in human aortas. High abundance of eADA in atherosclerotic vessels originated from activated endothelial cells and macrophages. There were no changes in ecto-nucleoside triphosphate diphosphohydrolase 1 activity, whereas ecto-5'-nucleotidase was moderately decreased in ApoE-/-LDLR-/- mice. Deoxycoformycin treatment attenuated plaque development in aortic root and brachiocephalic artery of ApoE-/-LDLR-/- mice, suppressed vascular inflammation and improved endothelial function. CONCLUSIONS: This study highlights the importance of extracellular nucleotides and adenosine metabolism in the atherosclerotic vessel in both experimental and clinical setting. The increased eADA activity marks an early stage of atherosclerosis, contributes to its progression and could represent a novel target for therapy.

Adenosina desaminase: uma enzima extraordinária e onipresente Adenosina desaminase: uma enzima extraordinária e onipresente / Adenosine deaminase: an extraordinary and omnipresent enzyme

Depois de introduzir os conceitos básicos da enzima adenosina desaminase (ADA), uma breve discussão sobre a estrutura, o mecanismo enzimático, terapia genética e potencial utilização terapêutica de inibidores de ADA são apresentados. O estudo da ADA é muito mais complexo do que simplesmente seu papel como biomarcador diagnóstico para tuberculose pleural que veio revolucionar o setor de diagnóstico na medicina clínica nos últimos anos. O aumento de sua atividade no líquido pleural, e em outros líquidos orgânicos, impede que o paciente na maioria dos casos com síndrome do derrame pleural por tuberculose seja submetido a procedimentos cirúrgicos invasivos com possíveis complicações potencialmente fatais AU.

After introducing the basic concepts of ADA, a brief discussion on the structure, enzymatic mechanism, gene therapy and potential therapeutic use of ADA inhibitors are presented. The study of the ADA is much more complex than simply its role as a biomarker for pleural tuberculosis that has revolutionized the diagnostic in clinical medicine in recent years. The increase in its activity in the pleural fluid, and other body fluids, prevents the patient in most cases with pleural effusion tuberculosis is subjected to invasive surgical procedures with possible life-threatening complications. AU

Advanced Glycation End Products: A Molecular Target for Vascular Complications in Diabetes.

A nonenzymatic reaction between reducing sugars and amino groups of proteins, lipids and nucleic acids contributes to the aging of macromolecules and subsequently alters their structural integrity and function. This process has been known to progress at an accelerated rate under hyperglycemic and/or oxidative stress conditions. Over a course of days to weeks, early glycation products undergo further reactions such as rearrangements and dehydration to become irreversibly cross-linked, fluorescent and senescent macroprotein derivatives termed advanced glycation end products (AGEs). There is a growing body of evidence indicating that interaction of AGEs with their receptor (RAGE) elicits oxidative stress generation and as a result evokes proliferative, inflammatory, thrombotic and fibrotic reactions in a variety of cells. This evidence supports AGEs' involvement in diabetes- and aging-associated disorders such as diabetic vascular complications, cancer, Alzheimer's disease and osteoporosis. Therefore, inhibition of AGE formation could be a novel molecular target for organ protection in diabetes. This report summarizes the pathophysiological role of AGEs in vascular complications in diabetes and discusses the potential clinical utility of measurement of serum levels of AGEs for evaluating organ damage in diabetes.

Adenosine deaminase inhibitor EHNA exhibits a potent anticancer effect against malignant pleural mesothelioma.

BACKGROUND/AIMS: Malignant pleural mesothelioma (MPM) is an aggressive malignant tumor and an effective therapy has been little provided as yet. The present study investigated the possibility for the adenosine deaminase (ADA) inhibitor EHNA as a target of MPM treatment. METHODS: MTT assay, TUNEL staining, monitoring of intracellular adenosine concentrations, and Western blotting were carried out in cultured human MPM cell lines without and with knocking-down ADA. The in vivo effect of EHNA was assessed in mice inoculated with NCI-H2052 MPM cells. RESULTS: EHNA induced apoptosis of human MPM cell lines in a concentration (0.01-1 mM)- and treatment time (24-48 h)-dependent manner, but such effect was not obtained with another ADA inhibitor pentostatin. EHNA increased intracellular adenosine concentrations in a treatment time (3-9 h)-dependent manner. EHNA-induced apoptosis of MPM cells was mimicked by knocking-down ADA, and the effect was neutralized by the adenosine kinase inhibitor ABT-702. EHNA clearly suppressed tumor growth in mice inoculated with NCI-H2052 MPM cells. CONCLUSION: The results of the present study show that EHNA induces apoptosis of MPM cells by increasing intracellular adenosine concentrations, to convert to AMP, and effectively prevents MPM cell proliferation. This suggests that EHNA may be useful for treatment of the tragic neoplasm MPM.

Influence of treatment with 3'-deoxyadenosine associated deoxycoformycin on hematological parameters and activity of adenosine deaminase in infected mice with Trypanosoma evansi.

This study aimed to verify the effect of 3'-deoxyadenosine and deoxycoformycin on hematologic parameters and adenosine deaminase (ADA) activity in plasma and brain of mice infected with Trypanosoma evansi. Seventy animals were divided into seven groups, which were divided into two subgroups each for sampling on days 4 and 8 post-infection (PI). The groups were composed of three uninfected groups (A-C), namely, not-treated (A), treated with 3'-deoxyadenosine (B), and treated with deoxycoformycin (C) and four infected groups, mice with T. evansi (D-G), namely, not-treated (D), treated with 3'-deoxyadenosine (E), treated with deoxycoformycin (F), and treated with a combination 3'-deoxyadenosine and deoxycoformycin (G). Hematological parameters and ADA activity were evaluated in plasma and brain. Animals in groups B and C exhibited a reduction in the levels of plasma total protein compared group A. Animals in groups D and F showed changes in the hematological parameters. The ADA activity significantly reduced in the animals of groups C, D, F and G. Mice in the group E presented increased ADA activity in plasma. Therefore, we conclude that the treatment interferes significantly in the hematologic parameters in mice infected with T. evansi. On the other hand, when the ADA inhibitor was used we observed a significant decrease in the values of hematocrit, total erythrocytes, and hemoglobin concentration. The deoxycoformycin was able to inhibit the ADA activity of parasite thus it may be one of the mechanisms of efficacy of this treatment.

Acute effect of ß-sitosterol on calcium uptake mediates anti-inflammatory effect in murine activated neutrophils.

OBJECTIVES: To evaluate the effect of ß-sitosterol on 45Ca²âº uptake in activated murine neutrophils, and upon myeloperoxidase and adenosine deaminase activity, and interleukin-1ß (IL-1ß) and tumour necrosis factor-α (TNF-α) levels, in carrageenan-induced inflammation in the mouse air pouch model. METHODS: Dried Esenbeckia leiocarpa bark was macerated and extracted resulting in a crude hydroalcoholic extract (CHE) that was partitioned to obtain an alkaloid fraction. The alkaloid was then partitioned in polar and nonpolar subfractions. ß-Sitosterol was isolated from the nonpolar subfraction and identified by comparison with the literature. The effect of ß-sitosterol on 45Ca²âº uptake in activated murine neutrophils, and upon myeloperoxidase and adenosine deaminase activity, IL-1ß and TNF-α levels in carrageenan-induced inflammation in mice were evaluated. KEY FINDINGS: ß-Sitosterol promoted a time- and dose-dependent increase of the calcium uptake in activated neutrophils that was promptly reversed by nifedipine, BAPTA-AM, LY294002, and colchicine. ß-Sitosterol inhibited myeloperoxidase and adenosine deaminase activity, and IL-1ß and TNF-α levels. CONCLUSIONS: ß-Sitosterol inhibited either myeloperoxidase and adenosine deaminase activity or IL-1ß and TNF-α levels. This effect seemed to be mediated by the calcium uptake in activated neutrophils in a time- and concentration-dependent manner through L-type voltage dependent calcium channels, intracellular calcium, phosphoinositide kinase-3, and microtubule modulation.

Adenosine deaminase in the modulation of immune system and its potential as a novel target for treatment of inflammatory disorders.

The adenosine pathway is a powerful evolutionarily selected mechanism aimed at a fine modulation of inflammatory responses and protection of tissues from injuries. Adenosine exerts its modulatory effects via interaction with G protein-coupled receptors, designated as A(1), A(2A), A(2B) and A(3). In this regard, extracellular adenosine concentrations are critical in determining its ability of regulating several biological functions. The levels achieved by adenosine in close proximity of its receptors are strictly regulated by a variety of dynamic mechanisms, including intracellular and extracellular biosynthesis, transport and metabolism, based on tissue energy status. In this context, the catabolic enzyme adenosine deaminase (ADA) represents a critical checkpoint in the regulation of extracellular adenosine levels and, consequently, in the control of receptor stimulation, thus playing a pivotal role in the modulation of purinergic responses to several pathophysiological events, such as chronic pulmonary diseases, rheumatoid arthritis, inflammatory bowel diseases and sepsis. This article reviews current data on the role played by ADA in the regulation of immune system activity through its modulation of adenosine pathways. Particular attention has been paid to the involvement of ADA in the pathophysiology of relevant inflammatory diseases. In addition, the interest in designing and developing novel ADA inhibitors, as new tools potentially useful for the therapeutic management of inflammatory disorders, has been discussed.

Long-term results for pentostatin and cladribine treatment of hairy cell leukemia.

Over the past 25 years we have collected data at our institution from 242 patients with hairy cell leukemia (HCL), treated with pentostatin (n = 188) or cladribine (n = 54), with a median follow-up of 16 years. From this we have been able to conclude that there is no significant difference in outcome between the two agents either at first or subsequent lines of therapy. Overall, the complete response (CR) rate is 81% and the median disease-free survival (DFS) is 16 years. After relapse or non-response patients can be successfully retreated with pentostatin or cladribine achieving a lower rate of CRs with each line of therapy, although these remain equally durable. Complete response and pretreatment counts of hemoglobin >10 g/dL together with platelets >100 × 10(9)/L are associated with the longest DFS. Importantly, for patients achieving a CR the DFS is five times as long as for those achieving a partial response (PR). Patients still in CR at 5 years have only a 25% risk of relapse by 15 years. Outcomes for patients with recurrent disease have improved with the addition of rituximab to either purine analog. Overall, only eight patients have died of HCL-related causes. Patients with HCL who achieve a CR can expect a normal lifespan.

Hairy cell leukemia: a successful model for experimental therapeutics--pentostatin and new ideas.

Hairy cell leukemia (HCL) was once considered an untreatable form of chronic lymphoid malignancy. Based upon the recognition of the importance of adenosine deaminase to the normal B cell survival and proliferation, a hypothesis was developed that temporary inhibition of this enzyme might be therapeutically successful in treating chronic B cell leukemias. Pentostatin was initially explored in patients with refractory chronic lymphocytic leukemia (CLL). Both pentostatin and cladribine, purine nucleoside analogs, have been utilized to successfully treat HCL. The high degree of complete and durable remission observed with either agent resulted in many believing that the treatment of this rare disease had been fully optimized. However, a considerable number of patients will relapse. While tremendous progress has been made in initial management, the issues related to optimal therapy, timing of initiation of treatment, and discovery of novel agents that may be effective in those who have relapsed are important. Investigational agents currently being explored in chronic lymphocytic leukemia may also have benefit for those patients who have relapsed or are resistant to therapy of hairy cell leukemia. Many important questions remain (e.g. importance of minimal residual disease) and will require international collaboration to fully address these unanswered questions. The Hairy Cell Leukemia Consortium was established to address these unanswered questions.

Pentostatin as rescue therapy for glucocorticoid-refractory acute and chronic graft-versus-host disease.

BACKGROUND: Despite scientific advances in hematopoietic cell allografting, glucocorticoid-refractory acute (aGVHD) and chronic graft-versus-host disease (cGVHD) represent major sources of transplant-related morbidity and mortality. We aimed to characterize the activity of pentostatin as rescue therapy for refractory GVHD. MATERIAL/METHODS: In a retrospective analysis, we examined the activity of pentostatin as rescue therapy of glucocorticoid-refractory acute and chronic GVHD. RESULTS: In 12 patients with advanced (overall aGVHD grade III/IV in 8/12) refractory aGVHD, overall response (ORR) was achieved in 6/12, and complete remission (CR) of aGVHD in 4/12 allowing additional rescue immunosuppressive agents. Median overall survival (OS) was 1.4 months (95% CI: 0.26-2.4). Causes of death included refractory aGVHD and infection. In 18 patients with refractory cGVHD (12/18 with severe cGVHD), pentostatin induced CR in 1/18, and partial response (PR) in 9/18. Activity was observed in all affected organs. The median decrease in glucocorticoid therapy over 24 months after pentostatin initiation for refractory cGVHD was 38% (range=0-100%). Median OS was 5 months (95% CI: 1.6 - NR). CONCLUSIONS: Allowing for the utilization of additional immune suppressive agents, this series suggests the activity of pentostatin as rescue therapy of refractory GVHD.