Fabrizio Vincenzi

A2A adenosine receptor overexpression and functionality, as well as TNF-α levels, correlate with motor symptoms in Parkinson’s disease

The antagonistic interaction between adenosine and dopamine receptors could have important pathophysiological and therapeutic implications in Parkinsons disease (PD). The primary aim of this study was to investigate the expression, affinity, and density of A1, A2A, A2B, and A3 adenosine receptors (ARs) and D2 dopamine receptors (D2Rs) in PD. An increase in A2AAR density in putamen was found. The presence and functionality of ARs in human lymphocyte and neutrophil membranes from patients with PD revealed a specific A2AAR alteration compared with healthy subjects. A statistically significant linear correlation among the A2AAR density, functionality, or tumor necrosis factor‐α (TNF‐α) levels and Unified Parkinsons Disease Rating Scale (UPDRS) motor score was reported. Adenosine concentration and TNF‐α levels were increased in plasma of patients with PD. In rat adrenal pheochromocytoma (PC12) cells, a widely useful model, adenosine antagonists decreased dopamine uptake, and an opposite effect was mediated by A2A agonists. This is the first report showing the presence of an A2AAR alteration in putamen in PD that mirrors a similar up‐regulation in human peripheral blood cells. Moreover, the correlation found between A2AAR density or A2A agonist potency and UPDRS motor score highlights the central role of A2AARs in the pharmacological treatment of PD.—Varani, K., Vincenzi, F., Tosi, A., Gessi, S., Casetta, I., Granieri, G., Fazio, P., Leung, E., MacLennan, S., Granieri, E., Borea, P. A. A2A adenosine receptor overexpression and functionality, as well as TNF‐α levels, correlate with motor symptoms in Parkinsons disease. FASEB J. 24, 587–598 (2010). www.fasebj.org

The A3 Adenosine Receptor: History and Perspectives

By general consensus, the omnipresent purine nucleoside adenosine is considered a major regulator of local tissue function, especially when energy supply fails to meet cellular energy demand. Adenosine mediation involves activation of a family of four G protein–coupled adenosine receptors (ARs): A1, A2A, A2B, and A3. The A3 adenosine receptor (A3AR) is the only adenosine subtype to be overexpressed in inflammatory and cancer cells, thus making it a potential target for therapy. Originally isolated as an orphan receptor, A3AR presented a twofold nature under different pathophysiologic conditions: it appeared to be protective/harmful under ischemic conditions, pro/anti-inflammatory, and pro/antitumoral depending on the systems investigated. Until recently, the greatest and most intriguing challenge has been to understand whether, and in which cases, selective A3 agonists or antagonists would be the best choice. Today, the choice has been made and A3AR agonists are now under clinical development for some disorders including rheumatoid arthritis, psoriasis, glaucoma, and hepatocellular carcinoma. More specifically, the interest and relevance of these new agents derives from clinical data demonstrating that A3AR agonists are both effective and safe. Thus, it will become apparent in the present review that purine scientists do seem to be getting closer to their goal: the incorporation of adenosine ligands into drugs with the ability to save lives and improve human health.

A2A and A3 adenosine receptor expression in rheumatoid arthritis: upregulation, inverse correlation with disease activity score and suppression of inflammatory cytokine and metalloproteinase release

IntroductionThe reduction of the inflammatory status represents one of the most important targets in rheumatoid arthritis (RA). A central role of A2A and A3 adenosine receptors (ARs) in mechanisms of inflammation has been reported in different pathologies. The primary aim of this study was to investigate the A2A and A3ARs and their involvement in RA progression measured by Disease Activity Score in 28 or 44 joints (DAS28 or DAS).MethodsARs were analyzed by saturation binding assays, mRNA and Western blotting analysis in lymphocytes from early and established RA patients. The effect of A2A and A3AR agonists in nuclear factor kB (NF-kB) pathway was evaluated. Tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) release was carried out by A2A and A3AR activation. AR pharmacological regulation in matrix metalloproteinase-1 (MMP-1) and metalloproteinase-3 (MMP-3) release was also studied.ResultsIn lymphocytes obtained from RA patients, A2A and A3ARs were up-regulated if compared with healthy controls. A2A and A3AR activation inhibited the NF-kB pathway and diminished inflammatory cytokines such as TNF-α, IL-1β and IL-6. A2A and A3AR agonists mediated a reduction of MMP-1 and MMP-3 release. A2A and A3AR density inversely correlated with DAS28 and DAS suggesting a direct role of the endogenous activation of these receptors in the control of RA joint inflammation.ConclusionsTaken together these data demonstrate that the inflammatory and clinical responses in RA are regulated by A2A and A3ARs and support the use of A2A and/or A3AR agonists as novel and effective pharmacological treatment in RA patients.

Pulsed Electromagnetic Fields Increased the Anti-Inflammatory Effect of A2A and A3 Adenosine Receptors in Human T/C-28a2 Chondrocytes and hFOB 1.19 Osteoblasts

Adenosine receptors (ARs) have an important role in the regulation of inflammation and their activation is involved in the inhibition of pro-inflammatory cytokine release. The effects of pulsed electromagnetic fields (PEMFs) on inflammation have been reported and we have demonstrated that PEMFs increased A2A and A3AR density and functionality in different cell lines. Chondrocytes and osteoblasts are two key cell types in the skeletal system that play important role in cartilage and bone metabolism representing an interesting target to study the effect of PEMFs. The primary aim of the present study was to evaluate if PEMF exposure potentiated the anti-inflammatory effect of A2A and/or A3ARs in T/C-28a2 chondrocytes and hFOB 1.19 osteoblasts. Immunofluorescence, mRNA analysis and saturation binding assays revealed that PEMF exposure up-regulated A2A and A3AR expression. A2A and A3ARs were able to modulate cAMP production and cell proliferation. The activation of A2A and A3ARs resulted in the decrease of some of the most relevant pro-inflammatory cytokine release such as interleukin (IL)-6 and IL-8, following the treatment with IL-1β as an inflammatory stimuli. In human chondrocyte and osteoblast cell lines, the inhibitory effect of A2A and A3AR stimulation on the release of prostaglandin E2 (PGE2), an important lipid inflammatory mediator, was observed. In addition, in T/C-28a2 cells, the activation of A2A or A3ARs elicited an inhibition of vascular endothelial growth factor (VEGF) secretion. In hFOB 1.19 osteoblasts, PEMF exposure determined an increase of osteoprotegerin (OPG) production. The effect of the A2A or A3AR agonists in the examined cells was enhanced in the presence of PEMFs and completely blocked by using well-known selective antagonists. These results demonstrated that PEMF exposure significantly increase the anti-inflammatory effect of A2A or A3ARs suggesting their potential therapeutic use in the therapy of inflammatory bone and joint disorders.

Normalization of A2A and A3 adenosine receptor up-regulation in rheumatoid arthritis patients by treatment with anti–tumor necrosis factor α but not methotrexate

OBJECTIVE To investigate A(1), A(2A), A(2B), and A(3) adenosine receptors in lymphocytes and neutrophils from patients with early rheumatoid arthritis (ERA) as well as from RA patients treated with methotrexate (MTX) or anti-tumor necrosis factor alpha (anti-TNFalpha), as compared with those in age-matched healthy controls, and to examine correlations between the status and functionality of adenosine receptors and TNFalpha release and NF-kappaB activation. METHODS Adenosine receptors were analyzed by saturation binding assays and Western blot analyses. We investigated the potency of typical A(2A) and A(3) agonists in the production of cAMP in control subjects, ERA patients, and RA patients treated with MTX or anti-TNFalpha. In a separate cohort of RA patients, TNFalpha release and NF-kappaB activation were evaluated in plasma and nuclear extracts, respectively. RESULTS In ERA patients, we found a high density and altered functionality of A(2A) and A(3) receptors. The binding and functional parameters of A(2A) and A(3) receptors normalized after anti-TNFalpha, but not MTX, treatment. TNFalpha release was increased in ERA patients and in MTX-treated RA patients, whereas in anti-TNFalpha-treated RA patients, release was comparable to that in the controls. NF-kappaB activation was elevated in ERA patients and in MTX-treated RA patients. Anti-TNFalpha treatment mediated decreased levels of NF-kappaB activation. CONCLUSION A(2A) and A(3) receptor up-regulation in ERA patients and in MTX-treated RA patients was associated with high levels of TNFalpha and NF-kappaB activation. Treatment with anti-TNFalpha normalized A(2A) and A(3) receptor expression and functionality. This new evidence of A(2A) and A(3) receptor involvement opens the possibility of exploiting their potential role in human diseases characterized by a marked inflammatory component.

Electromagnetic fields (EMFs) and adenosine receptors modulate prostaglandin E(2) and cytokine release in human osteoarthritic synovial fibroblasts.

Synovial fibroblasts (SFs) contribute to the development of osteoarthritis (OA) by the secretion of a wide range of pro‐inflammatory mediators, including cytokines and lipid mediators of inflammation. Previous studies suggest that electromagnetic fields (EMFs) may represent a potential therapeutic approach to limit cartilage degradation and control inflammation associated to OA, and that they may act through the adenosine pathway. Therefore, we investigated whether EMFs might modulate inflammatory activities of human SFs from OA patients (OASFs) treated with interleukin‐1β (IL‐1β), and the possible involvement of adenosine receptors (ARs) in mediating EMF effects. EMF exposure induced a selective increase in A2A and A3 ARs. These increases were associated to changes in cAMP levels, indicating that ARs were functionally active also in EMF‐exposed cells. Functional data obtained in the presence of selective A2A and A3 adenosine agonists and antagonists showed that EMFs inhibit the release of prostaglandin E2 (PGE2) and the proinflammatory cytokines interleukin‐6 (IL‐6) and interleukin‐8 (IL‐8), while stimulating the release of interleukin‐10 (IL‐10), an antinflammatory cytokine. These effects seem to be mediated by the EMF‐induced upregulation of A2A and A3 ARs. No effects of EMFs or ARs have been observed on matrix degrading enzyme production. In conclusion, this study shows that EMFs display anti‐inflammatory effects in human OASFs, and that these EMF‐induced effects are in part mediated by the adenosine pathway, specifically by the A2A and A3 AR activation. Taken together, these results open new clinical perspectives to the control of inflammation associated to joint diseases. J. Cell. Physiol. 227: 2461–2469, 2012.

2-Phenylpyrazolo[4,3-d]pyrimidin-7-one as a New Scaffold To Obtain Potent and Selective Human A3 Adenosine Receptor Antagonists: New Insights into the Receptor-Antagonist Recognition

A molecular simplification approach of previously reported 2-arylpyrazolo[3,4-c]quinolin-4-ones was applied to design 2-arylpyrazolo[4,3-d]pyrimidin-7-one derivatives as new human A(3) adenosine receptor antagonists. Substituents with different lipophilicity and steric hindrance were introduced at the 5-position of the bicyclic scaffold (R(5) = H, Me, Et, Ph, CH(2)Ph) and on the 2-phenyl ring (OMe, Me). Most of the synthesized derivatives were highly potent hA(3) adenosine receptor antagonists, the best being the 2-(4-methoxyphenyl)pyrazolo[4,3-d]pyrimidin-7-one (K(i) = 1.2 nM). The new compounds were also highly selective, being completely devoid of affinity toward hA(1), hA(2A), and hA(2B) adenosine receptors. On the basis of the recently published human A(2A) receptor crystallographic information, we propose a novel receptor-driven hypothesis to explain both A(3) AR affinity and A(3) versus A(2A) selectivity profiles of these new antagonists.

Expression and functional role of adenosine receptors in regulating inflammatory responses in human synoviocytes

Background and purpose:  Adenosine is an endogenous modulator, interacting with four G‐protein coupled receptors (A1, A2A, A2B and A3) and acts as a potent inhibitor of inflammatory processes in several tissues. So far, the functional effects modulated by adenosine receptors on human synoviocytes have not been investigated in detail. We evaluated mRNA, the protein levels, the functional role of adenosine receptors and their pharmacological modulation in human synoviocytes.

Antinociceptive effects of the selective CB2 agonist MT178 in inflammatory and chronic rodent pain models

&NA; The selective CB2 agonist MT178 is effective in inflammatory and chronic pain models and reduces d‐aspartate and substance P release as well as NF‐κB activation. &NA; Cannabinoid CB2 receptor activation by selective agonists has been shown to produce analgesic effects in preclinical models of inflammatory, neuropathic, and bone cancer pain. In this study the effect of a novel CB2 agonist (MT178) was evaluated in different animal models of pain. First of all, in vitro competition binding experiments performed on rat, mouse, or human CB receptors revealed a high affinity, selectivity, and potency of MT178. The analgesic properties of the novel CB2 agonist were evaluated in various in vivo experiments, such as writhing and formalin assays, showing a good efficacy comparable with that produced by the nonselective CB agonist WIN 55,212‐2. A dose‐dependent antiallodynic effect of the novel CB2 compound in the streptozotocin‐induced diabetic neuropathy was found. In a bone cancer pain model and in the acid‐induced muscle pain model, MT178 was able to significantly reduce mechanical hyperalgesia in a dose‐related manner. Notably, MT178 failed to provoke locomotor disturbance and catalepsy, which were observed following the administration of WIN 55,212‐2. CB2 receptor mechanism of action was investigated in dorsal root ganglia where MT178 mediated a reduction of [3H]‐d‐aspartate release. MT178 was also able to inhibit capsaicin‐induced substance P release and NF‐κB activation. These results demonstrate that systemic administration of MT178 produced a robust analgesia in different pain models via CB2 receptors, providing an interesting approach to analgesic therapy in inflammatory and chronic pain without CB1‐mediated central side effects.

Synthesis and Biological Evaluation of 2-Amino-3-(4-Chlorobenzoyl)-4-[N-(Substituted) Piperazin-1-yl]Thiophenes as Potent Allosteric Enhancers of the A1 Adenosine Receptor

The synthesis and evaluation of a series of 2-amino-3-(4-chlorobenzoyl)-4-[4-(alkyl/aryl)piperazin-yl]thiophene derivatives as allosteric enhancers of the A 1-adenosine receptor are described. The nature of substituents on the phenyl ring tethered to the piperazine seem to exert a fundamental influence on the allosteric enhancer activity, with the 4-chlorophenyl 8f and 4-trifluoromethyl 8j derivatives being the most active compounds in binding (saturation and displacement experiments) and functional cAMP studies.