A Novel A1 Adenosine Receptor Antagonist, L-97-1 [3-[2-(4-Aminophenyl)-ethyl]-8-benzyl-7-{2-ethyl-(2-hydroxy-ethyl)-amino]-ethyl}-1-propyl-3,7-dihydro-purine-2,6-dione], Reduces Allergic Responses to House Dust Mite in an Allergic Rabbit Model of Asthma

Adenosine, an important signaling molecule in asthma, produces bronchoconstriction in asthmatics. Adenosine produces bronchoconstriction in allergic rabbits, primates, and humans by activating A1 adenosine receptors (ARs). Effects of L-97-1 [3-[2-(4-aminophenyl)-ethyl]-8-benzyl-7-{2-ethyl- (2-hydroxy-ethyl)-amino]-ethyl}-1-propyl-3,7-dihydro-purine-2,6-dione] a water-soluble, small molecule A1 AR antagonist were investigated on early and late phase allergic responses (EAR and LAR) in a hyper-responsive rabbit model of asthma. Rabbits were made allergic by intraperitoneal injections of house dust mite [HDM; 312 allergen units (AU)] extract within 24 h of their birth. Booster HDM injections were given weekly for 1 month, biweekly for 4 months, and continued monthly thereafter. Hyper-responsiveness was monitored by measuring lung dynamic compliance (Cdyn), after histamine or adenosine aerosol challenge in allergic rabbits. Hyper-responsive rabbits were subjected to aerosol of HDM (2500 AU), 1 h after intragastric administration of L-97-1 (10 mg/kg) solution or an equivalent volume of saline. Cdyn was significantly higher after treatment with L-97-1 compared with untreated controls (p < 0.05 n = 5). Histamine PC30 was significantly higher (p < 0.05; n = 5) after L-97-1 at 24 h compared with histamine PC30 at 24 h after HDM. Adenosine PC30 was significantly higher at 15 min and 6 h after L-97-1 compared with control (p < 0.05; n = 5). L-97-1 showed strong affinity for human A1 ARs in radioligand binding studies and no inhibition toward human phosphodiesterase II, III, IV, and V enzymes. These data suggest that L-97-1 produces a significant reduction of histamine or adenosine-induced hyper-responsiveness and HDMinduced EAR and LAR in allergic rabbits by blocking A1 ARs and may be beneficial as an oral therapy for human asthma. Originally published Journal of Pharmacology and Experimental Therapies, Vol. 315, No. 1, Oct 200

Respirable antisense oligonucleotides: a new drug class for respiratory disease

Respirable antisense oligonucleotides (RASONs), which attenuate specific disease-associated mRNAs, represent a new class of respiratory therapeutics with considerable potential. RASONs overcome previous obstacles that have impeded the development of antisense therapeutics targeting diseases in other organ systems. RASONs are delivered directly to the target tissue via inhalation; their uptake seems to be enhanced by cationic properties inherent in pulmonary surfactant, and, because of the markedly different target properties of mRNA and proteins, they can have very long durations of effect compared with traditional drugs targeting the protein of the same gene. RASONs contain chemical modifications that decrease their degradation by cellular nucleases. However, total insensitivity to nucleases is probably not an optimal design criterion for RASONs, because moderate nuclease sensitivity can prevent their systemic delivery, decreasing the potential for systemic toxicity. EPI-2010 is a 21-mer phosphorothioate RASON that attenuates bronchoconstriction, inflammation and surfactant depletion in preclinical models of human asthma, has a duration of effect of seven days, and seems to undergo minimal systemic delivery

Modulating effect of adenosine deaminase on function of adenosine A 1 receptors 1

Aim : To study the modulating effect of adenosine deaminase (ADA) on yhe adenosine A 1 receptor (A 1 R) in HEK293 cells stably expressing the human A 1 R. Methods : cDNA was amplified by RT-PCR using total RNA from human embryo brain tissue as the template. The PCR products were subcloned into the plasmid pcDNA3 and cloned into the plasmid pcDNA3.1. The cloned A 1 R cDNA was sequenced and stably expressed in HEK293 cells. The modulating effect of adenosine deaminase on A 1 R was studied by using [ 3 H]DPCPX binding assay and an intracellular calcium assay. Results : HEK293 cells stably expressing human A 1 R were obtained. Saturation studies showed that the K D value and B max value of [ 3 H]DPCPX were 1.6±0.2 nmol/L and 1.819±0.215 nmol/g of protein respectively, in the absence of ecto-ADA respectively, and 1.3±0.2 nmol/L and 1.992±0.130 nmol/g of protein in the presence of ecto-ADA respectively, suggesting that the K D value and B max value of [ 3 H]DPCPX were unaffected by ecto-ADA. In the case of [ 3 H]DPCPX competition curves obtained from intact cells or membranes, A 1 R agonist CCPA/[ 3 H]DPCPX competition curve could be fitted well to a one-site model in the absence of ecto-ADA and a two-site model in the presence of ecto-ADA with a K H value of 0.74 (0.11–4.8) nmol/L (intact cells) or 1.8 (0.25–10) nmol/L (membrane) and a K L value of 0.94 (0.62–1.41) Μmol/L (intact cells) or 0.77 (0.29–0.99) Μmol/L (membrane). The K L value is not significantly different from the EC 50 value of 0.84(0.57–1.23) Μmol/L (intact cells) or 0.84 (0.63–1.12) Μmol/L (membrane) obtained in the absence of ecto-ADA. Similar results were obtained from the CPA/[ 3 H]DPCPX competition curve in the absence or presence of ecto-ADA on intact cells or membranes. Intracellular calcium assay demonstrated that the EC 50 value of CPA were 10 (5–29) nmol/L and 94 (38–229) nmol/L in the presence or absence of ecto-ADA, respectively. Conclusion : A 1 R stably expressed in the HEK293 cells display a low affinity for agonists in the absence of ADA and high and low affinities for agonists in the presence of ADA. The presence of ADA may promote the signaling through the adenosine A 1 receptor in HEK293 cells.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73693/1/j.1745-7254.2005.00524.x.pd

In vivo antisense targeting on vasopressin mRNA in rats

Die tatsächlichen molekularen und zellulären Abläufe, die der Technik des Antisense Targetings zugrunde liegen sind bis heute noch weitgehend unbekannt. In der vorliegenden Arbeit injizierten wir in Ratten intracerebroventrikulär eine synthetische Oligonukleotid-Sonde, die komplementär zur Startcodon Region des Arginin-Vasopressin Precursors war. Bei zwei aufeinanderfolgenden Injektionen von geringen Dosen der Oligonukleotid-Sonde wurden die Plasma-Vasopressin-Spiegel aber nicht verändert, die Wasseraufnahme dieser Tiere war sogar leicht reduziert. Mit Immunzytochemie beobachteten wir in den magnozellulären hypothalamischen Kerngebieten und im Hypophysen-Hinterlappen eine Koexistenz von Vasopressin und Oxytocin

Perinatal Stress, Brain Inflammation and Risk of Autism-Review and Proposal

Background: Autism Spectrum Disorders (ASD) are neurodevelopmental disorders characterized by varying deficits in social interactions, communication, and learning, as well as stereotypic behaviors. Despite the significant increase in ASD, there are few if any clues for its pathogenesis, hampering early detection or treatment. Premature babies are also more vulnerable to infections and inflammation leading to neurodevelopmental problems and higher risk of developing ASD. Many autism “susceptibility” genes have been identified, but “environmental” factors appear to play a significant role. Increasing evidence suggests that there are different ASD endophenotypes. Discussion: We review relevant literature suggesting in utero inflammation can lead to preterm labor, while insufficient development of the gut-blood–brain barriers could permit exposure to potential neurotoxins. This risk apparently may increase in parents with “allergic” or autoimmune problems during gestation, or if they had been exposed to stressors. The presence of circulating auto-antibodies against fetal brain proteins in mothers is associated with higher risk of autism and suggests disruption of the blood–brain-barrier (BBB). A number of papers have reported increased brain expression or cerebrospinal fluid (CSF) levels of pro-inflammatory cytokines, especially TNF, which is preformed in mast cells. Recent evidence also indicates increased serum levels of the pro-inflammatory mast cell trigger neurotensin (NT), and of extracellular mitochondrial DNA (mtDNA), which is immunogenic. Gene mutations of phosphatase and tensin homolog (PTEN), the negative regulator of the mammalian target of rapamycin (mTOR), have been linked to higher risk of autism, but also to increased proliferation and function of mast cells. Summary: Premature birth and susceptibility genes may make infants more vulnerable to allergic, environmental, infectious, or stress-related triggers that could stimulate mast cell release of pro-inflammatory and neurotoxic molecules, thus contributing to brain inflammation and ASD pathogenesis, at least in an endophenotype of ASD patients

Involvement of adenosine A1 receptors in systemic inflammation and altered vascular reactivity in allergic mice

Epidemiological studies have established that people who have lung diseases such as asthma and COPD are likely to develop adverse cardiovascular events. While the reason for this has not been elucidated, it is believed that inflammation could be the key component linking the two conditions. Adenosine, an endogenous purine nucleoside, has been strongly implicated in asthma pathophysiology and also has potent effects on the cardiovascular system. This work was undertaken to gain a better understanding of the relationship between asthma and its cardiovascular effects, and more specifically the possible role adenosine plays under these conditions.;We hypothesize that asthmatic lung inflammation translates into systemic inflammation and alters vascular responses where adenosine plays an important role. Therefore, this study investigated the effects of aerosolized adenosine, used to elevate lung adenosine levels, on vascular reactivity and inflammation in a well established mouse model of allergic asthma established in our lab. We found that allergic mice had poor vasorelaxation to adenosine and systemic inflammation, in addition to lung inflammation, and aerosolized adenosine exacerbated these effects. Data indicated a possible role for A1 adenosine receptor (AR) in altered vascular reactivity and inflammation. Based on these findings, we next studied the effects of adenosine using genetically modified mice in which the A1 AR gene was deleted (A1KO) and corresponding A1 wild-type (A1WT) mice, in which the A1 receptor was present.;Allergic A1KO mice had no systemic inflammation, and the adenosine-mediated vasorelaxation responses obtained were comparable to non-allergic mice. Aerosolized adenosine also did not have any effect in A1KO mice. On the other hand, allergic A1WT mice had lower aortic relaxation response to non-selective adenosine analog NECA, presence of significantly higher levels of inflammatory cytokines in plasma and higher airway responsiveness to NECA, compared to non-allergic A1WT controls and all groups of A 1KO mice, with aerosolized adenosine exacerbating all these responses. From these data, it can be concluded that asthmatic mice had poor vascular responses and inflammation, possibly mediated through the A1 AR