Ennio Ongini

Pharmacology of adenosine A2A receptors

Adenosine A2A receptors, which have been cloned from several mammalian species, are activated by physiological concentrations of adenosine to stimulate the formation of cAMP and other mediators. The A2A receptors are found on neutrophil leukocytes, platelets, blood vessels and, very abundantly, on some cells in the CNS. In the caudate nucleus they coexist with dopamine D2 receptors, and stimulation of A2A receptors causes a decrease in D2 receptor-mediated neurotransmission. Thus, drugs that act on A2A receptors can be used to modify dopaminergic neurotransmission known to be important in neurological and psychiatric disorders. In this review, Ennio Ongini and Bertil Fredholm describe how recently developed potent and selective A2A receptor antagonists can be used to delineate the physiological and pathological processes regulated by A2A receptors. Results from in vitro and in vivo pharmacology studies strengthen the notion that A2A receptors are an interesting target for drug development.

Blockade of adenosine A2A receptors by SCH 58261 results in neuroprotective effects in cerebral ischaemia in rats.

BLOCKADE of adenosine receptors can reduce cerebral infarct size in the model of global ischaemia. Using the potent and selective A2A adenosine receptor antagonist, SCH 58261, we assessed whether A2A receptors are involved in the neuronal damage following focal cerebral ischaemia as induced by occluding the left middle cerebral artery. SCH 58261 (0.01 mg/kg either i.p. or i.v.) administered to normotensive rats 10min after ischaemia markedly reduced cortical infarct volume as measured 24 h later (30% vs controls, p < 0.05). Similar effects were observed when SCH 58261 (0.01 mg/kg, i.p.) was administered to hypertensive rats (28% infarct volume reduction vs controls, p < 0.05). Neuroprotective properties of SCH 58261 administered after ischaemia indicate that blockade of A2A adenosine receptors is a potentially useful biological target for the reduction of brain injury.

Non-steroidal anti-inflammatory drugs (NSAIDs) in Alzheimer's disease: old and new mechanisms of action

Alzheimers disease (AD) is characterized by cerebral deposits of β‐amyloid (Aβ) peptides and neurofibrillary tangles (NFT) which are surrounded by inflammatory cells. Epidemiological studies have shown that prolonged use of non‐steroidal anti‐inflammatory drugs (NSAIDs) reduces the risk of developing AD and delays the onset of the disease. It has been postulated that some NSAIDs target pathological hallmarks of AD by interacting with several pathways, including the inhibition of cyclooxygenases (COX) and activation of the peroxisome proliferator‐activated receptor γ. A variety of experimental studies indicate that a subset of NSAIDs such as ibuprofen, flurbiprofen, indomethacin and sulindac also possess Aβ‐lowering properties in both AD transgenic mice and cell cultures of peripheral, glial and neuronal origin. While COX inhibition occurs at low concentrations in vitro (nM‐low μm range), the Aβ‐lowering activity is observed at high concentrations (≤ 50 μm). Nonetheless, studies with flurbiprofen or ibuprofen in AD transgenic mice show that the effects on Aβ levels or deposition are attained at plasma levels similar to those achieved in humans at therapeutic dosage. Still, it remains to be assessed whether adequate concentrations are reached in the brain. This is a crucial aspect that will allow defining the dose‐window and the length of treatment in future clinical trials. Here, we will discuss how the combination of anti‐amyloidogenic and anti‐inflammatory activities of certain NSAIDs may produce a profile potentially relevant to their clinical use as disease‐modifying agents for the treatment of AD.

Nitric oxide release combined with nonsteroidal antiinflammatory activity prevents muscular dystrophy pathology and enhances stem cell therapy

Duchenne muscular dystrophy is a relatively common disease that affects skeletal muscle, leading to progressive paralysis and death. There is currently no resolutive therapy. We have developed a treatment in which we combined the effects of nitric oxide with nonsteroidal antiinflammatory activity by using HCT 1026, a nitric oxide-releasing derivative of flurbiprofen. Here, we report the results of long-term (1-year) oral treatment with HCT 1026 of two murine models for limb girdle and Duchenne muscular dystrophies (α-sarcoglycan-null and mdx mice). In both models, HCT 1026 significantly ameliorated the morphological, biochemical, and functional phenotype in the absence of secondary effects, efficiently slowing down disease progression. HCT 1026 acted by reducing inflammation, preventing muscle damage, and preserving the number and function of satellite cells. HCT 1026 was significantly more effective than the corticosteroid prednisolone, which was analyzed in parallel. As an additional beneficial effect, HCT 1026 enhanced the therapeutic efficacy of arterially delivered donor stem cells, by increasing 4-fold their ability to migrate and reconstitute muscle fibers. The therapeutic strategy we propose is not selective for a subset of mutations; it provides ground for immediate clinical experimentation with HCT 1026 alone, which is approved for use in humans; and it sets the stage for combined therapies with donor or autologous, genetically corrected stem cells.

Antagonism of adenosine A2A receptors underlies the behavioural activating effect of caffeine and is associated with reduced expression of messenger RNA for NGFI-A and NGFI-B in caudate–putamen and nucleus accumbens

Caffeine, the most widely consumed of all psychostimulant drugs, exerts its action by antagonizing adenosine receptors. To study the arousing properties of caffeine, we injected rats intraperitoneally with vehicle, caffeine (7.5, 15 or 30mg/kg), the selective adenosine A2A receptor antagonist, SCH 58261 (3.75 mg/kg) or the selective adenosine A1 receptor selective antagonist DPCPX (7.5 mg/kg). In a behavioural test it was found that administration of caffeine and SCH 58261 significantly increased locomotion and rearing, whereas DPCPX did not alter locomotion and reduced rearing. After the behavioural session the rats were killed, their brains were cut at several levels along a rostrocaudal axis and in situ hybridization against NGFI-A messenger RNA and NGFI-B messenger RNA was performed. A reduction of NGFI-A messenger RNA was found in several subregions of both caudate putamen and nucleus accumbens in caffeine-treated animals. Similarly, animals that had received SCH 58261 showed significant decreases of NGFI-A messenger RNA in the rostral part of caudate putamen and in the shell part of nucleus accumbens. By contrast, DPCPX treatment caused an increase in the expression of NGFI-A messenger RNA and a smaller increase in NGFI-B messenger RNA in the lateral parts of caudate putamen. In addition, it was found that caffeine, but not SCH 58261 or DPCPX, elevated the expression of NGFI-A and NGFI-B messenger RNA in the cerebral cortex, especially in its parietal part. Thus, these results provide evidence that endogenous adenosine, via adenosine A2A receptors, causes a tonic activation of striatopallidal neurons. By blocking this adenosine effect, caffeine causes behavioural activation.

Adenosine A2A receptor antagonism potentiates L-DOPA-induced turning behaviour and c-fos expression in 6-hydroxydopamine-lesioned rats

In order to investigate the role of adenosine A2A receptor blockade on dopamine-mediated motor responses, contralateral turning behaviour and expression of the early-gene c-fos was evaluated in rats with a unilateral 6-hydroxydopamine lesion of the dopaminergic nigrostriatal pathway. SCH 58261, (7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1 , 5-c]pyrimidine) a potent and selective antagonist of adenosine A2A receptors (5 mg/kg i.p.), induced a 70-fold increase in the contralateral turning behaviour induced by a low dose (2 mg/kg i.p.) of the dopamine precursor L-DOPA (L-3, 4-dihydroxyphenylalanine). Expression of c-fos as measured by Fos-like immunoreactivity after SCH 58261 plus L-DOPA was also potentiated as compared with L-DOPA alone, both in striatum and globus pallidus of the 6-hydroxydopamine-lesioned side of the brain. SCH 58261 induced a less marked potentiation (7-fold) of turning behaviour induced by dopamine D2 receptor stimulation with quinpirole, while Fos-like immunoreactivity in the striatum and globus pallidus was not affected. Previous studies have shown that SCH 58261 strongly potentiated dopamine D1 receptor-mediated responses. The results of the present study therefore indicate that the positive interaction between SCH 58261 and L-DOPA, in 6-hydroxydopamine-lesioned rats, is mainly due to an interaction with dopamine D1 receptors. The data also suggest that adenosine A2A receptor antagonists might be useful for potentiating the effects of L-DOPA in Parkinsons disease.

Interleukin‐10 modulates neuronal threshold of vulnerability to ischaemic damage

Interleukin‐10 (IL‐10) is a powerful suppressor of cellular immune responses, with a postulated role in brain inflammation. First, we have evaluated the role of this cytokine in ischaemic brain damage using IL‐10 knockout (IL‐10–/–) mice. The middle cerebral artery (MCA) was occluded in either IL‐10–/– or wild‐type animals of corresponding strain (C57Bl/6) and age. Infarct volume was assessed 24 h later in serial brain sections. Brain infarct produced by MCA occlusion was 30% larger in the IL‐10–/– than in wild‐type mice (21.8 ± 1.2 vs. 16.9 ± 1.0 mm3, respectively; P < 0.01; Students t‐test). To further characterize these findings, studies were extended to in vitro models. Primary neuronal cortical cultures derived from IL‐10–/– animals were more susceptible to both excitotoxicity and combined oxygen–glucose deprivation compared with cell cultures from wild‐type mice. Moreover, when added to the culture medium, recombinant murine IL‐10 (0.1–100 ng/mL) exerted a concentration‐dependent prevention of neuronal damage induced by excitotoxicity in both cortical and cerebellar granule cell cultures taken from either strain. The accordance of in vivo and in vitro data allows us to suggest a potential neuroprotective role of IL‐10 against cerebral ischaemia when administered exogenously or made available from endogenous sources.

Comparison of CGS 15943, ZM 241385 and SCH 58261 as antagonists at human adenosine receptors

Three structurally related non-xanthine compounds, CGS 15943, ZM 241385 and SCH 58261, are potent A2A adenosine receptor antagonists and have been used as tools in many pharmacological studies. We have now characterized their affinity and selectivity profile on human adenosine receptors stably transfected into either CHO cells (A1 and A2B receptors) or HEK-293 cells (A2A and A3 receptors). In binding studies using [3H]SCH 58261 as a radioligand, the three compounds were equally potent at A2A receptors, their Ki value being less than 1 nM. Affinity for A1 and A3 receptors was measured using [3H]DPCPX and [125I]AB-MECA as radioligands. Given the lack of selective ligands, interaction with A2B receptors was assessed using the cAMP accumulation assay following stimulation by the adenosine receptor agonist N-ethylcarboxamidoadenosine (NECA). CGS 15943 was almost as potent at A1 receptors (Ki 3.5 nM) as at A2A receptors, showed moderate affinity for A3 receptors (Ki 95 nM) and also interacted with A2B receptors (Ki 44 nM; pA2 7.5). ZM 241385 showed little affinity for A1 receptors (Ki 255 nM), and did not interact with A3 receptors (Ki>10 µM); however, it displayed moderate affinity for A2B receptors (Ki 50 nM; pA2 7.3). SCH 58261 had weak affinity for A1 receptors (Ki 287 nM), no interaction with A3 receptors (Ki>10 µM), and showed negligible interaction with A2B receptors (Ki 5 µM; pA2 6.0). These data indicate that SCH 58261 is the most selective A2A antagonist currently available. Moreover, the different receptor selectivity of these three chemically related compounds provides useful information to progress with structure-activity relationship studies.

Adenosine A2A receptor antagonists are potential antidepressants : evidence based on pharmacology and A2A receptor knockout mice

Adenosine, an ubiquitous neuromodulator, and its analogues have been shown to produce ‘depressant’ effects in animal models believed to be relevant to depressive disorders, while adenosine receptor antagonists have been found to reverse adenosine‐mediated ‘depressant’ effect. We have designed studies to assess whether adenosine A2A receptor antagonists, or genetic inactivation of the receptor would be effective in established screening procedures, such as tail suspension and forced swim tests, which are predictive of clinical antidepressant activity. Adenosine A2A receptor knockout mice were found to be less sensitive to ‘depressant’ challenges than their wildtype littermates. Consistently, the adenosine A2A receptor blockers SCH 58261 (1 – 10 mg kg−1, i.p.) and KW 6002 (0.1 – 10 mg kg−1, p.o.) reduced the total immobility time in the tail suspension test. The efficacy of adenosine A2A receptor antagonists in reducing immobility time in the tail suspension test was confirmed and extended in two groups of mice. Specifically, SCH 58261 (1 – 10 mg kg−1) and ZM 241385 (15 – 60 mg kg−1) were effective in mice previously screened for having high immobility time, while SCH 58261 at 10 mg kg−1 reduced immobility of mice that were selectively bred for their spontaneous ‘helplessness’ in this assay. Additional experiments were carried out using the forced swim test. SCH 58261 at 10 mg kg−1 reduced the immobility time by 61%, while KW 6002 decreased the total immobility time at the doses of 1 and 10 mg kg−1 by 75 and 79%, respectively. Administration of the dopamine D2 receptor antagonist haloperidol (50 – 200 μg kg−1 i.p.) prevented the antidepressant‐like effects elicited by SCH 58261 (10 mg kg−1 i.p.) in forced swim test whereas it left unaltered its stimulant motor effects. In conclusion, these data support the hypothesis that A2A receptor antagonists prolong escape‐directed behaviour in two screening tests for antidepressants. Altogether the results support the hypothesis that blockade of the adenosine A2A receptor might be an interesting target for the development of effective antidepressant agents.

Persistent Behavioral Sensitization to Chronic l-DOPA Requires A2A Adenosine Receptors

To investigate the role of A2A adenosine receptors in adaptive responses to chronic intermittent dopamine receptor stimulation, we compared the behavioral sensitization elicited by repeated l-DOPA treatment in hemiparkinsonian wild-type (WT) and A2A adenosine receptor knock-out (A2AKO) mice. Although the unilateral nigrostriatal lesion produced by intrastriatal injection of 6-hydroxydopamine was indistinguishable between WT and A2A KO mice, they developed strikingly different patterns of behavioral sensitization after daily treatment with low doses of l-DOPA for 3 weeks. WT mice initially displayed modest contralateral rotational responses and then developed progressively greater responses that reached a maximum within 1 week and persisted for the duration of the treatment. In contrast, any rotational behavioral sensitization in A2A KO mice was transient and completely reversed within 2 weeks. Similarly, the time to reach the peak rotation was progressively shortened in WT mice but remained unchanged in A2A KO mice. Furthermore, dailyl-DOPA treatment produced gradually sensitized grooming in WT mice but failed to induce any sensitized grooming in A2AKO mice. Finally, repeated l-DOPA treatment reversed the 6-OHDA-induced reduction of striatal dynorphin mRNA in WT but not A2A KO mice, raising the possibility that the A2A receptor may contribute to l-DOPA-induced behavioral sensitization by facilitating adaptations within the dynorphin-expressing striatonigral pathway. Together these results demonstrate that the A2A receptor plays a critical role in the development and particularly the persistence of behavioral sensitization to repeated l-DOPA treatment. Furthermore, they raise the possibility that the maladaptive dyskinetic responses to chronic l-DOPA treatment in Parkinsons disease may be attenuated by A2A receptor inactivation.