Silvio Dionisotti

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.

Binding of the radioligand [3H]-SCH 58261, a new non-xanthine A2A adenosine receptor antagonist, to rat striatal membranes

1 The present study describes the binding to rat striatal A2A adenosine receptors of the new potent and selective antagonist radioligand, [3H]‐5‐amino‐7‐(2‐phenylethyl)‐2‐(2‐furyl)‐pyrazolo[4,3‐e]‐1,2,4‐triazolo [1,5‐c] pyrimidine, [3H]‐SCH 58261. 2 [3H]‐SCH 58261 specific binding to rat striatal membranes (> 90%) was saturable, reversible and dependent upon protein concentration. Saturation experiments revealed that [3H]‐SCH 58261 labelled a single class of recognition sites with high affinity (Kd = 0.70 nM) and limited capacity (apparent Bmax = 971 fmol mg−1 of protein). The presence of 100 μm GTP in the incubation mixture did not modify [3H]‐SCH 58261 binding parameters. 3 Competition experiments showed that [3H]‐SCH 58261 binding is consistent with the labelling of A2A striatal receptors. Adenosine receptor agonists competed with the binding of 0.2 nM [3H]‐SCH 58261 with the following order of potency: 2‐hexynyl‐5′‐N‐ethyl carboxamidoadenosine (2HE‐NECA) > 5′‐N‐ ethylcarboxamidoadenosine (NECA)>2‐[4‐(2‐carboxyethyl)‐phenethylamino]‐5′‐N‐ethylcarboxamido‐ adenosine (CGS 21680) > 2‐phenylaminoadenosine (CV 1808)> R‐N6‐phenylisopropyladenosine (R‐ PIA) > N6‐cyclohexyladenosine (CHA) = 2‐chloro‐N6‐cyclopentyladenosine (CCPA) > S‐N6‐phenylisopro‐ pyladenosine (S‐PIA). 4 Adenosine antagonists inhibited [3H]‐SCH 58261 binding with the following order: 5‐amino‐9‐chloro‐ 2‐(2‐furyl)‐[1,2,4]‐triazolo[1,5‐c] quinazoline (CGS 15943) > 5‐amino‐8‐(4‐fluorobenzyl)‐2‐(2‐furyl)‐pyra‐ zolo [4,3‐e]‐1,2,4‐triazolo [1,5‐c] pyrimidine (8FB‐PTP) = SCH 58261 > xanthine amine congener (XAC) = (E, 18%‐Z,82%)7‐methyl‐8‐(3,4‐dimethoxystyryl)‐1,3‐dipropylxanthine (KF 17837S) > 8‐cyclo‐ pentyl‐1,3‐dipropylxanthine (DPCPX) ≥ 8‐phenyltheophylline (8‐PT). 5 The Ki values for adenosine antagonists were similar to those labelled with the A2A agonist [3H]‐CGS 21680. Affinities of agonists were generally lower. The Ai‐selective agonist, R‐PIA, was found to be about 9 fold more potent than its stereoisomer, S‐PIA, thus showing the stereoselectivity of [3H]‐SCH 58261 binding. Except for 8‐PT, the adenosine agonists and antagonists examined inhibited [3H]‐SCH 58261 binding with Hill coefficients not significantly different from unity. 6 The present results indicate that [3H]‐SCH 58261 is the first non‐xanthine adenosine antagonist radioligand which directly labels A2A striatal receptors. High receptor affinity, good selectivity and very low non‐specific binding make [3H]‐SCH 58261 an excellent probe for studying the A2A adenosine receptor subtype in mammalian brain.

Synthesis of new pyrazolo[4,3-e]1,2,4-triazolo[1,5-c] pyrimidine and 1,2,3-triazolo[4,5-e]1,2,4-triazolo[1,5-c] pyrimidine displaying potent and selective activity as A2a adenosine receptor antagonists.

A series of pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidines and 1,2,3-triazolo[4,5-e]1,2,4-triazolo[1,5-c]pyrimidines were prepared and evaluated for their activity as adenosine A 2a receptor antagonists. In the present study, 5-amino-7-(phenylethyl)-2-(2-furyl)pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine 7d ( SCH 58261 ) was identified as potent and selective adenosine A 2a antagonist in binding assays (Ki = 2.3 nM, Ki ratio: A 1 /A 2a = 52.6).

[3H]‐SCH 58261 labelling of functional A2A adenosine receptors in human neutrophil membranes

The present study describes the direct labelling of A2A adenosine receptors in human neutrophil membranes with the potent and selective antagonist radioligand, [3H]‐5‐amino‐7‐(2‐phenylethyl)‐2‐(2‐furyl)‐pyrazolo[4,3‐e]‐1,2,4 triazolo[1,5‐c]pyrimidine, ([3H]‐SCH 58261). In addition, both receptor affinity and potency of a number of adenosine receptor agonists and antagonists were determined in binding, adenylyl cyclase and superoxide anion production assays. Saturation experiments revealed a single class of binding sites with Kd and Bmax values of 1.34 nM and 75 fmol mg−1 protein, respectively. Adenosine receptor ligands competed for the binding of 1 nM [3H]‐SCH 58261 to human neutrophil membranes, with a rank order of potency consistent with that typically found for interactions with the A2A adenosine receptors. In the adenylyl cyclase and in the superoxide anion production assays the same compounds exhibited a rank order of potency identical to that observed in binding experiments. Thermodynamic data indicated that [3H]‐SCH 58261 binding to human neutrophils is entropy and enthalpy‐driven. This finding is in agreement with the thermodynamic behaviour of antagonists binding to rat striatal A2A adenosine receptors. It was concluded that in human neutrophil membranes, [3H]‐SCH 58261 directly labels binding sites with pharmacological properties similar to those of A2A adenosine receptors of other tissues. The receptors labelled by [3H]‐SCH 58261 mediated the effects of adenosine and adenosine receptor agonists to stimulate cyclic AMP accumulation and inhibition of superoxide anion production in human neutrophils.

[3H]SCH 58261, a Selective Adenosine A2A Receptor Antagonist, Is a Useful Ligand in Autoradiographic Studies

Abstract: We have characterized the new potent and selective nonxanthine adenosine A2A receptor antagonist SCH 58261 as a new radioligand for receptor autoradiography. In autoradiographic studies using agonist radioligands for A2A receptors ([3H]CGS 21680) or A1 receptors (N6‐[3H]cyclohexyladenosine), it was found that SCH 58261 is close to 800‐fold selective for rat brain A2A versus A1 receptors (Ki values of 1.2 nM versus 0.8 µM). Moreover, receptor autoradiography showed that [3H]SCH 58261, in concentrations below 2 nM, binds only to the dopamine‐rich regions of the rat brain, with a KD value of 1.4 (0.8–1.8) nM. The maximal number of binding sites was 310 fmol/mg of protein in the striatum. Below concentrations of 3 nM, the nonspecific binding was <15%. Three adenosine analogues displaced all specific binding of [3H]SCH 58261 with the following estimated Ki values (nM): 2‐hex‐1‐ynyl‐5′‐N‐ethylcarboxamidoadenosine, 3.9 (1.8–8.4); CGS 21680, 130 (42–405); N6‐cyclohexyladenosine, 9,985 (3,169–31,462). The binding of low concentrations of SCH 58261 was not influenced by either GTP (100 µM) or Mg2+ (10 mM). The present results show that in its tritium‐labeled form, SCH 58261 appears to be a good radioligand for autoradiographic studies, because it does not suffer from some of the problems encountered with the currently used agonist radioligand [3H]CGS 21680.

Characterization of human A2A adenosine receptors with the antagonist radioligand [3H]-SCH 58261

We have characterized the binding of the new potent and selective antagonist radioligand [3H]‐5‐amino‐7‐(2‐phenylethyl)‐2‐(2‐furyl)‐pyrazolo[4,3‐e]‐1,2,4‐triazolo[1,5‐c]pyrimidine, [3H]‐SCH 58261, to human cloned A2A adenosine receptors. In Chinese hamster ovary (CHO) cells transfected with the human cloned A2A receptor, [3H]‐SCH 58261 specific binding (about 70%) was rapid, saturable, reversible and proportional to protein concentration. The kinetic KD value was 0.75 nM. Saturation experiments showed that [3H]‐SCH 58261 labelled a single class of recognition sites with high affinity (KD=2.3 nM) and limited capacity (apparent Bmax=526 fmol mg−1 protein). Competition experiments revealed that binding of 0.5 nM [3H]‐SCH 58261 was displaced by adenosine receptor agonists with the following order of potency: 2‐hexynyl‐5′‐N‐ethylcarboxamido‐adenosine (2HE‐NECA)>5′‐N‐ethylcarboxamidoadenosine (NECA)=2‐phenylaminoadenosine (CV 1808)>2‐[4‐(2‐carboxyethyl)‐phenethylamino]‐5′‐N‐ethylcarboxamidoadenosine (CGS 21680)>R‐N6‐phenylisopropyladenosine (R‐PIA)N6‐cyclohexyladenosine (CHA)>S‐N6‐phenylisopropyladenosine (S‐PIA). Adenosine receptor antagonists inhibited [3H]‐SCH 58261 binding with the following order: 5‐amino‐9‐chloro‐2‐(2‐furyl)‐[1,2,4]‐triazolo[1,5‐c] quinazoline (CGS 15943)>SCH 58261>xanthine amine congener (XAC)>(E,18%‐Z,82%)7‐methyl‐8‐(3,4‐dimethoxystyryl)‐1,3‐dipropylxanthine (KF 17837S)> 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX)>theophylline. Affinity values and rank order of potency of both receptor agonists and antagonists were similar to those previously obtained in human platelet and rat striatal membranes, except for CV 1808 which was more potent than CGS 21680. SCH 58261 was a competitive antagonist at inhibiting NECA‐induced adenosine 3′ : 5′‐cyclic monophosphate (cyclic AMP) accumulation in CHO cells transfected with human A2A receptors. Good agreement was found between binding and functional data. Thus, the new antagonist radioligand is preferable to the receptor agonist radioligand [3H]‐CGS 21680 hitherto used to examine the pharmacology of human cloned A2A adenosine receptors.

Effects of the new A2 adenosine receptor antagonist 8FB-PTP, an 8 substituted pyrazolo-triazolo-pyrimidine, on in vitro functional models.

1 We have characterized the in vitro pharmacological profile of putative A2 adenosine antagonists, two non‐xanthine compounds, 5‐amino‐8‐(4‐fluorobenzyl)‐2‐(2‐furyl)‐pyrazolo [4,3‐e]‐1,2,4‐triazolo[1,5‐c] pyrimidine (8FB‐PTP) and 5‐amino‐9‐chloro‐2‐(2‐furyl 1,2,4‐triazolo [1,5‐c] quinazoline (CGS 15943), and the xanthine derivative (E)7‐methyl‐8‐(3,4‐dimethoxystyryl)‐1,3‐dipropyl‐xanthine (KF 17837). 2 In binding studies on bovine brain, 8FB‐PTP was the most potent (Ki = 0.074 nm) and selective (28 fold) drug on A2 receptors, whereas CGS 15943 and KF 17837 exhibited affinity in the low and high nanomolar range, respectively, and showed little selectivity. 3 In functional studies, 8FB‐PTP antagonized 5′‐N‐ethyl‐carboxamidoadenosine (NECA)‐induced vasorelaxation of bovine coronary artery (pA2 = 7.98) and NECA‐induced inhibition of rabbit platelet aggregation (pA2 = 8.20). CGS 15943 snowed weak activity in the platelet aggregation model (pA2 = 7.43) and failed to antagonize NECA‐induced vasodilatation. KF 17837 was ineffective in both models up to micromolar concentrations. 4 Antagonism of A1‐mediated responses was tested versus 2‐chloro‐N6‐cyclopentyladenosine (CCPA) in rat atria. 8FB‐PTP and CGS 15943 also antagonized competitively the negative chronotropic response induced by CCPA. Conversely, KF 17837 was unable to reverse A1‐mediated responses. 5 8FB‐PTP is a potent and competitive antagonist of responses mediated by A2 adenosine receptors. The data provided a basis to reduce, by further chemical modifications, the affinity at A1 receptor and therefore enhance A2 receptor selectivity.

Effects of adenosine derivatives on human and rabbit platelet aggregation

SummaryThe inhibitory effects of several adenosine analogues, including the new A2-selective agonists 2-[p-(2-carboxyethyl)phenethylamino]-5′-N-ethylcarboxamido-adenosine (CGS 21680) and 2-hexynyl-5′-N-ethylcarbox-amidoadenosine (2-hexynyl-NECA), were investigated in vitro on human and rabbit platelet aggregation. The compounds examined inhibited ADP-induced platelet aggregation over a wide range of potency. The rank order of activity was similar between the two species thus showing that the rabbit is a useful animal model for studying the effects of adenosine derivatives on platelet aggregation. 2-Hexynyl-NECA was found to be the most potent adenosine compound of those currently available, having IC50 values of 0.10 and 0.07 μM in human and rabbit platelets, respectively. Conversely, the A1 agonists R(−)-N-6-(2-phenylisopropyl) adenosine (R-PIA), S(+)-N6-(2phenylisopropyl) adenosine (S-PIA) and 2-chloro-N6-cyclopentyl-adenosine (CCPA) were the least potent compounds with IC50 values in the micromolar range. The potency of the compounds in inhibiting platelet aggregation was found to be highly correlated with their affinity for A2 receptors as measured using 3H-CGS 21680 binding in rat brain striatum.

Neuropharmacology of the adenosine A2A receptors

Studies done over the last 20 years have clearly shown that the adenosine A2A receptors are abundant in the striatum of several animal species. A2A receptors have also been found in the cerebral cortex and hippocampus. The distribution of A2A receptors closely matches that of dopamine D2 receptors, being expressed in striatopallidal GABAergic neurons that also express enkephalin. A variety of functional and behavioural studies have shown that antagonistic interactions exist between the A2A and D2 receptors. Thus, blockade of A2A receptors mimics the action of dopamine D2 receptor agonists. More recent studies have indicated that A2A receptors interact more broadly with dopaminergic pathways, D1 receptors are also involved in such interactions. Altogether, a variety of data support the suggestion that A2A receptor antagonists have a potential for treatment of Parkinsons disease, whereas A2A receptor agonists, which inhibit motor behaviour, may possess neuroleptic properties. Great progress is being made thanks to the development of potent and selective A2A receptor antagonists, notably the xanthines KF 17837 and CSC, and the non‐xanthine heterocycle SCH 58261. These compounds and their radiolabelled forms make it possible to elucidate the role of brain A2A receptors further and open the way to the development of new agents for treatment of central nervous system disorders. Drug Dev. Res. 39:450–460, 1996.

Relation of Dosing Regimen to Aminoglycoside Ototoxicity: Evaluation of Auditory Damage in the Guinea Pig

Groups of guinea pigs were injected intramuscularly for 21 days with netilmicin or amikacin 150 mg/kg/day by one or three daily injections. Amikacin was also tested at 225 mg/kg/day with each dosing regimen. Auditory function was evaluated during the experiment by reflexological and electrophysiological tests. Morphological damage to the inner ear was also evaluated. Netilmicin had no effect on the auditory function nor did it damage the organ of Corti. Conversely, amikacin impaired the auditory function and produced loss of hair cells in a dose-related manner. The effect was equally marked with both dosing regimens of 225 mg/kg/day, whereas a slight decrease of auditory impairment was observed with 150 mg/kg administered once a day. The data suggest that administration of a single daily dose of aminoglycosides does not increase the risk of ototoxicity specifically associated with each compound.