Pharmacological Characterization of P626, a Novel Dual Adenosine A2A/A2B Receptor Antagonist, on Synaptic Plasticity and during an Ischemic-like Insult in CA1 Rat Hippocampus

In recent years, the use of multi-target compounds has become an increasingly pursued strategy to treat complex pathologies, including cerebral ischemia. Adenosine and its receptors (A1AR, A2AAR, A2BAR, A3AR) are known to play a crucial role in synaptic transmission either in normoxic or ischemic-like conditions. Previous data demonstrate that the selective antagonism of A2AAR or A2BAR delays anoxic depolarization (AD) appearance, an unequivocal sign of neuronal injury induced by a severe oxygen-glucose deprivation (OGD) insult in the hippocampus. Furthermore, the stimulation of A2AARs or A2BARs by respective selective agonists, CGS21680 and BAY60-6583, increases pre-synaptic neurotransmitter release, as shown by the decrease in paired-pulse facilitation (PPF) at Schaffer collateral-CA1 synapses. In the present research, we investigated the effect/s of the newly synthesized dual A2AAR/A2BAR antagonist, P626, in preventing A2AAR- and/or A2BAR-mediated effects by extracellular recordings of synaptic potentials in the CA1 rat hippocampal slices. We demonstrated that P626 prevented PPF reduction induced by CGS21680 or BAY60-6583 and delayed, in a concentration-dependent manner, AD appearance during a severe OGD. In conclusion, P626 may represent a putative neuroprotective compound for stroke treatment with the possible translational advantage of reducing side effects and bypassing differences in pharmacokinetics due to combined treatment.


Introduction
In recent years, the use of multi-target compounds has gained the interest of the scientific community considering their several advantages (i.e., eliminating the risk of drugdrug interactions, reduction of possible side effects, pharmacokinetics and metabolism) in the treatment of various pathological conditions, such as cerebral ischemia [1,2]. Ischemic stroke is a leading cause of permanent disability and death worldwide today [3]. Although much pharmacological progress has been made in the field, current treatments are still limited by a very narrow therapeutic time window, i.e., within 4 h from the insult for the thrombolytic enzyme tissue plasminogen activator (tPA), and several side effects. Therefore, the study of new possible treatments for cerebral ischemia is extremely necessary, especially effects. In this study, we provide the first functional characterization of a newly synthesized dual A 2A AR/A 2B AR antagonist, P626, on PPF and OGD conditions in the CA1 area of the rat hippocampus as an advantageous therapeutic approach to dampen neurodegeneration after energy failure in the brain.

Materials and Methods
All animal experiments were carried out according to the Italian Law on Animal Welfare (DL 26/2014). The document was approved by the Italian Ministry of Health (authorization code: 301/2021) and by the Institutional Animal Care and Use Committee of the University of Florence. To minimize animal suffering for our experiments we used only the number of animals necessary to obtain consistent scientific results. Male Wistar rats (Envigo, Italy, 100-150 g body weight for PPF experiments; 150-180 g body weight for OGD experiments, 6-8 weeks old) were used. All animals were located in a temperaturecontrolled room (22 ± 1 • C) in groups of two-five per cage, with food and water ad libitum, and with a 12 h light/dark cycle.

Preparation of Acute Hippocampal Slices
All the experiments were performed on hippocampal slices, acutely isolated from rat brains, as already described [31,39]. Rats were anesthetized with isoflurane (Baxter, Rome, Italy) and then sacrificed by decapitation. The hippocampi were quickly removed and placed in ice-cold oxygenated (95% O 2 -5% CO 2 ) artificial cerebrospinal fluid (aCSF) of the following composition (mM): NaCl 125, KCl 3, NaH 2 PO 4 1.25, MgSO 4 1, CaCl 2 2, NaHCO 3 25, and D-glucose 10. Transverse slices (400 µm nominal thickness) were cut using a McIlwain Tissue Chopper (Mickle Laboratory Engineering Co. Ltd., Gomshall, UK) and kept in oxygenated aCSF at room temperature to recover their functionality for at least 1 h. Once this time lapsed, a slice was transferred on a nylon mesh, completely submerged in a small chamber (0.8 mL), and superfused with oxygenated aCSF (31)(32) • C) at a constant flow rate of 2 mL/min. The treated solutions reached the preparation in 60 s and this delay was considered in our calculations.

Extracellular Recordings
A bipolar nichrome electrode was located in the CA1 stratum radiatum to stimulate the Schaffer collateral-commissural fibers with test pulses (80 µs, 0.066 Hz) delivered every 15 s. Evoked potentials were extracellularly recorded with borosilicate microelectrodes (2-10 M Ω, Harvard Apparatus Ltd., Edenbridge, UK) filled with 150 mM NaCl. The recording electrode was situated at the CA1 dendritic level to record field excitatory postsynaptic potentials (fEPSPs, Figure 1A). Data were amplified (200×, BM 622, Mangoni, Pisa, Italy), digitized (sample rate, 33.33 kHz), and stored for later analysis with LTP (version 2.30D) program [40]. Synaptic potentials were expressed as the initial slope (calculated between 20 and 80% of maximal amplitude). Input-output curves were constructed by gradual increases in stimulus strength at the beginning of each experiment. To generate a synaptic response of about 40% of the maximum, we adjusted the test stimulus strength, and it was kept constant throughout the experiment. The onset of each experiment was established after recording a stable baseline for 30 min.

Paired-Pulse Facilitation
Paired-pulse facilitation (PPF) was obtained by stimulation of Schaffer collateralcommissural fibers twice (40 ms inter-stimuli interval). We chose the 40 ms interstimulus interval because, as also reported in the literature [18], this value is particularly useful to underline the eventual effect, of a given compound, on presynaptic neurotransmitter release as it induces a robust potentiation of the second fEPSP over the first. After steady control baseline responses were established (basal synaptic neurotransmission: BSN), the PPF protocol was applied, still once every 15 s, for 5 min either before or after 20 min application of the selected compounds (see Figure 1B,C). The degree of facilitation was

Paired-Pulse Facilitation
Paired-pulse facilitation (PPF) was obtained by stimulation of Schaffer collateralcommissural fibers twice (40 ms inter-stimuli interval). We chose the 40 ms interstimulus interval because, as also reported in the literature [18], this value is particularly useful to underline the eventual effect, of a given compound, on presynaptic neurotransmitter release as it induces a robust potentiation of the second fEPSP over the first. After steady control baseline responses were established (basal synaptic neurotransmission: BSN), the PPF protocol was applied, still once every 15 s, for 5 min either before or after 20 min application of the selected compounds (see Figure 1B,C). The degree of facilitation was calculated as the PPF ratio (PPR) between the slope of the second (P2) and the first (P1) fEPSPs (P2/P1; Figure 1B,C).

Oxygen-Glucose Deprivation
OGD insults in vitro were realized by superfusing the slice with aCSF without glucose and oxygen, and gassed with nitrogen (95% N2-5% CO2) [30,41] for 30 min. This OGD-time duration does not allow the recovery of fEPSPs as it always induces irreversible synaptic failure, as previously demonstrated by us [30,42]. After the OGD insult, each slice was again superfused with normal, glucose-containing, oxygenated aCSF. The new mixed A2AAR/A2BAR antagonist, P626, was applied 15 min before, during, and 5 min after OGD. In all conditions, fEPSPs were continuously monitored and never recovered their amplitude after a 30 min OGD, in line with our previous results [30,42]. In some experiments, both the amplitude and initial fEPSP slope were quantified, but since no appreciable differences between these two parameters were observed in drug effects nor during OGD, we calculated only the amplitude measurement (data not shown). AD was recorded as negative extracellular direct current (d.c.) shifts induced by OGD. This phenomenon is considered a sign that the cells around the tip of the glass electrode were depolarized [43]. AD latency was calculated from the beginning of OGD insult and was expressed in min; while AD amplitude was calculated at the maximal negativity peak and expressed in mV. In this work, AD amplitude values were expressed as positive values.

Oxygen-Glucose Deprivation
OGD insults in vitro were realized by superfusing the slice with aCSF without glucose and oxygen, and gassed with nitrogen (95% N 2 -5% CO 2 ) [30,41] for 30 min. This OGDtime duration does not allow the recovery of fEPSPs as it always induces irreversible synaptic failure, as previously demonstrated by us [30,42]. After the OGD insult, each slice was again superfused with normal, glucose-containing, oxygenated aCSF. The new mixed A 2A AR/A 2B AR antagonist, P626, was applied 15 min before, during, and 5 min after OGD. In all conditions, fEPSPs were continuously monitored and never recovered their amplitude after a 30 min OGD, in line with our previous results [30,42]. In some experiments, both the amplitude and initial fEPSP slope were quantified, but since no appreciable differences between these two parameters were observed in drug effects nor during OGD, we calculated only the amplitude measurement (data not shown). AD was recorded as negative extracellular direct current (d.c.) shifts induced by OGD. This phenomenon is considered a sign that the cells around the tip of the glass electrode were depolarized [43]. AD latency was calculated from the beginning of OGD insult and was expressed in min; while AD amplitude was calculated at the maximal negativity peak and expressed in mV. In this work, AD amplitude values were expressed as positive values. nist, the 7-amino-2-(2-furanyl)-thiazolo [5,4-d] pyrimidine derivative (P626, Figure 2 [44]). P626 showed high potency at both hA2AAR and hA2BAR (IC50 = 5.20 nM and 34 nM, respectively, cAMP assay). P626 showed a Ki = 1326 nM at hA1AR and a Ki = 1874 nM at hA3AR. All drugs were dissolved in dimethyl sulphoxide (DMSO). Stock solutions of 1000-10,000 times the desired final concentration were stored at −20 °C. The final concentration of DMSO (0.05% and 0.1% in aCSF) used in our experiments did not affect either fEPSP slope or amplitude in all different protocols applied.

Statistical Analysis
Data were expressed as mean ± SEM (standard error of the mean). Kolmogorov-Smirnov normality test was performed to check data distribution: all data reported in the present research are normally distributed. Two-tailed Student s paired or unpaired t-tests or one-way ANOVA followed by Bonferroni post-test analysis were performed, as appropriated, in order to determine statistical significance (set at p < 0.05) between groups. Data were analyzed using "GraphPad Prism" (GraphPad Software, San Diego, CA, USA) software.

Statistical Analysis
Data were expressed as mean ± SEM (standard error of the mean). Kolmogorov-Smirnov normality test was performed to check data distribution: all data reported in the present research are normally distributed. Two-tailed Student's paired or unpaired t-tests or one-way ANOVA followed by Bonferroni post-test analysis were performed, as appropriated, in order to determine statistical significance (set at p < 0.05) between groups. Data were analyzed using "GraphPad Prism" (GraphPad Software, San Diego, CA, USA) software.

Results
In this study, we functionally characterized the new mixed A 2A AR/A 2B AR antagonist, P626, in the CA1 region of rat hippocampus, a brain area involved in synaptic plasticity phenomena and particularly susceptible to hypoxic-ischemic injuries. All data were obtained by an extracellular recording of fEPSP from 85 slices isolated from 37 rats. In a first series of experiments, we tested the effects of the selective A 2B AR agonist BAY60-6583 on basal synaptic transmission in the CA1 rat hippocampal slices. According to our previous results [15,38] BAY60-6583 (200 nM) did not significantly modify fEPSP slope during basal Schaffer collateral fiber stimulation (once every 15 s) in the CA1 rat hippocampus ( Figure 3A,B, n = 11). fEPSP slope values were from 0.39 ± 0.03 mV/ms before to 0.40 ± 0.04 mV/ms after 20 min of applying the selective A 2B AR agonist (see Table 1). Conversely, we demonstrated that the selective A 2A AR agonist CGS21680 (50 nM) induced a modest, but significant, increase in fEPSPs slope ( Figure 3C,D, n = 6) measured at the end of 20 min application. fEPSP slope values were from 0.49 ± 0.03 mV/ms before to 0.53 ± 0.03 mV/ms after 20 min of applying the selective A 2A AR agonist (see Table 1). This result confirmed the involvement of A 2A ARs in the CA1 basal synaptic transmission in accord to Lopes et al. (2002) [16]. The enhancement in basal synaptic transmission was prevented by the new dual A 2A AR/A 2B AR antagonist, P626 (200 nM, n = 7, Figure 3C,D). In particular, the fEPSP slope values were 0.48 ± 0.02 mV/ms in P626 alone and 0.48 ± 0.03 mV/ms in combination with CGS21680. When applied alone P626 did not modify, per se, basal synaptic transmission (see Table 1) nor PPF ratio ( Figure S1). In addition, we evaluated the effects of P626 in the absence or presence of BAY60-6583 or CGS21680 during the application of PPF, a paradigm of short-term synaptic plasticity. Following previous results [15,16] we confirmed that either BAY60-6583 or CGS21680 (Figure 4), significantly decreased the PPF ratio in CA1 rat hippocampal slices. Indeed, the P2/P1 ratio of fEPSP slope values, measured at the end of a 20 min application versus respective pre-drug baseline values, was reduced from 1.58 ± 0.05 in the absence to 1.52 ± 0.04 in the presence of 200 nM BAY60-6583 ( Figure 4A, n = 11). Regarding the A 2A AR agonist, P2/P1 ratio was from 1.70 ± 0.01 in the absence to 1.64 ± 0.02 in the presence of 50 nM CGS21680 ( Figure 4B, n = 6). The inhibitory effects induced by both "A 2 ARs" agonists on PPF were completely prevented in the presence of 200 nM P626 ( Figure 4A,B). Globally these results suggest that P626, antagonizing the reduction in PPF induced by the selective stimulation of "A 2 ARs", may counteract the increase in neurotransmitter release elicited by either of the two receptor agonists.  Note that CGS21680 significantly enhanced basal synaptic transmission and that this effect was antagonized in the presence of P626. Paired columns refer to data collected from the same slice,

P626 Delayed AD onset Induced by Irreversible OGD in the CA1 Rat Hippocampus
The early phases of a hypoxic-ischemic insult are characterized by a significant increase in extracellular glutamate and adenosine levels [45]. The enhancement in glutamate release under pathological conditions contributes to excitotoxic damage [46].
In these experiments, we tested the new dual compound, P626, on neurotransmission before and after applying an irreversible, 30 min-long, OGD. This experimental protocol always elicited the appearance of AD, an unequivocal sign of tissue damage, and the irreversible failure of neurotransmission [30,38]. The experiments were conducted in the absence or the presence of different concentrations of P626 applied before, during, and 5

P626 Delayed AD Onset Induced by Irreversible OGD in the CA1 Rat Hippocampus
The early phases of a hypoxic-ischemic insult are characterized by a significant increase in extracellular glutamate and adenosine levels [45]. The enhancement in glutamate release under pathological conditions contributes to excitotoxic damage [46]. In these experiments, we tested the new dual compound, P626, on neurotransmission before and after applying an irreversible, 30 min-long, OGD. This experimental protocol always elicited the appearance of AD, an unequivocal sign of tissue damage, and the irreversible failure of neurotransmission [30,38]. The experiments were conducted in the absence or the presence of different concentrations of P626 applied before, during, and 5 min after an ischemic-like episode. As illustrated in Figure 5, the appearance of AD in untreated OGD slices was recorded ( Figure 5A,C), with a mean latency of 6.22 ± 0.21 min and a mean peak amplitude Biomolecules 2023, 13, 894 9 of 14 of 7.27 ± 0.28 mV (n = 19). The application of P626 was ineffective in modifying AD latency at the concentration of 10 nM (from 6.22 ± 0.21 min before to 6.83 ± 0.27 after drug application, Figure 5C, n = 6), while a significant AD delay started from the concentration of 100 nM (from 6.22 ± 0.21 min before to 7.72 ± 0.37 after drug application, Figure 5B,C, n = 8). When the OGD was applied in the presence of 400 nM or 1 µM P626, the d.c shift was also significantly delayed. The AD latency values were postponed to 7.98 ± 0.26 min in the presence of 400 nM P626 ( Figure 5C, n = 9) and to 9.33 ± 0.72 min in the presence of 1 µM P626 ( Figure 5C n = 6). Based on P626 affinity for all adenosine receptors, concentrations of P626 higher than 1 µM were not used since the compound could exert its effects by blocking all the adenosine receptor subtypes [44]. Finally, no difference in AD amplitude among all experimental groups was found ( Figure 5D).
Biomolecules 2023, 13, x FOR PEER REVIEW 10 of 16 min after an ischemic-like episode. As illustrated in Figure 5, the appearance of AD in untreated OGD slices was recorded ( Figure 5A,C), with a mean latency of 6.22 ± 0.21 min and a mean peak amplitude of 7.27 ± 0.28 mV (n = 19). The application of P626 was ineffective in modifying AD latency at the concentration of 10 nM (from 6.22 ± 0.21 min before to 6.83 ± 0.27 after drug application, Figure 5C, n = 6), while a significant AD delay started from the concentration of 100 nM (from 6.22 ± 0.21 min before to 7.72 ± 0.37 after drug application, Figure 5B,C, n = 8). When the OGD was applied in the presence of 400 nM or 1 µM P626, the d.c shift was also significantly delayed. The AD latency values were postponed to 7.98 ± 0.26 min in the presence of 400 nM P626 ( Figure 5C, n = 9) and to 9.33 ± 0.72 min in the presence of 1 µM P626 ( Figure 5C n = 6). Based on P626 affinity for all adenosine receptors, concentrations of P626 higher than 1 µM were not used since the compound could exert its effects by blocking all the adenosine receptor subtypes [44]. Finally, no difference in AD amplitude among all experimental groups was found ( Figure 5D). . Dotted lines and respective filled areas represent when the d.c shifts were recorded in the two conditions (grey-filled lines for the untreated OGD slices; green-filled lines for the P626-treated OGD slices). (C) Each column represents the mean ± SEM of AD latency recorded in CA1 hippocampal slices during 30 min OGD in different experimental groups. AD was measured from the beginning of the OGD insult. Note that 100 nM, 400 nM, and 1 µM P626 significantly delayed AD development. ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. OGD, one-way ANOVA followed by Bonferroni multiple comparison test. (D) Each column represents the mean ± SEM of AD amplitude recorded in the CA1 region during 30 min OGD. P626 10 nM: n = 6 slices taken from 5 animals; 400 nM: n = 9 slices taken from 8 animals; 1 µM: n = 6 slices taken from 5 animals.

Discussion
In the present work, we provided the first evidence of the functional effects of the newly synthesized dual A 2A AR/A 2B AR antagonist, P626. This compound prevented the effects of A 2A AR and/or A 2B AR stimulation on short-term synaptic plasticity and during an ischemic-like insult in the CA1 region of rat hippocampal slices.
Multi-target compounds are designed to activate more than one cellular target simultaneously. Their use has increased in recent years, as these molecules offer the possibility to allow better pharmacokinetic and symptomatology control in various pathological conditions, by reducing side effects due to the administration of two different compounds [47].
To characterize the action/s of this innovative antagonist, we firstly demonstrated that P626 prevented the effects elicited by the selective A 2A AR or A 2B AR agonists, CGS21680 and BAY60-6583, respectively, on hippocampal neurotransmission either under basal condition or during PPF stimulation at Schaffer collateral-CA1 synapses. In particular, consistent with the literature (respectively: [15,16]), we confirmed, that CGS21680 significantly increased basal synaptic transmission, while BAY60-6583 did not show any effect. Of note, endogenous extracellular adenosine levels in acute hippocampal slices are estimated to be between 50 and 200 nM [8,45]. Hence, as the affinity of A 2A ARs for the endogenous agonist is reported to be 20-300 nM, a submaximal A 2A AR activation is already achieved before the CGS21680 application. Conversely, no activation of A 2B ARs is expected to occur under physiological-like conditions because the affinity of this adenosine receptor for the endogenous ligand is over 20-30 µM [10]. Notably, the effect of CGS21680 on basal neurotransmission at CA1 synapses was antagonized by P626, thus demonstrating that this compound prevents A 2A AR activation in the CA1 hippocampus.
The application of CGS21680 or BAY60-6583, during the PPF protocol, reduced P2/P1 ratio, which reflects a presynaptic increase in glutamate release at the hippocampal level [48,49]. These effects can be explained by the "residual Ca 2+ hypothesis" of neurotransmitter release for inter-stimulus intervals lower than 500 ms (for review see: Zucker and Regehr 2002 [50]; Regher, 2012 [18]). In addition, the newly synthesized compound, P626, prevented the effects of the selective A 2A AR or A 2B AR agonist on PPF, proving once again that Gs-coupled adenosine receptors are involved in synaptic plasticity phenomena in the CA1 region, following data from Lopes et al., (2002) and Fusco et al., (2019) [15,16]. It is worth noting that one mechanism common to both A 2A AR and A 2B AR in the hippocampus is the downregulation of A 1 AR-mediated inhibition of synaptic transmission since PPF reduction by either A 2A AR or A 2B AR agonists is prevented by the selective A 1 AR antagonist DPCPX [15,20].
A 2A ARs are known to be expressed on astrocytes [51,52], as well as on pre-and postsynaptic glutamatergic terminals of hippocampal neurons [12], where they can regulate synaptic plasticity [53,54] and neurotransmitter release [16]. Concerning the A 2B AR, their expression in the central nervous system on glia and neurons is scarce but widespread if compared to A 2A ARs [37,55] (for a review see: Coppi et al., 2020 [56]), and up to now evidence about their localization on hippocampal neurons is limited to presynaptic glutamatergic sites, where their activation is involved in the control of glutamate release [19]. The different localization of "A 2 ARs", as well as the higher expression level of A 2A ARs vs. A 2B ARs [12,57], might explain the sole involvement of the A 2A ARs in basal synaptic transmission, aside from the well-known inhibitory role of the A 1 AR in neurotransmission [58]. The facilitatory role of "A 2 ARs" is worthy of note as modification in neurotransmitter release probability, by affecting the filtering role of the hippocampus, influences the information-processing capabilities of the brain circuitry [49,59].
It is known that an increase in glutamate release plays different roles under physiological conditions; it facilitates neuronal excitability, synaptic plasticity, and coordination of neural networks. However, under pathological conditions (such as cerebral ischemia), this increase contributes to excitotoxic damage [46]. The early phases of a hypoxic-ischemic insult are characterized by a significant increase in extracellular glutamate levels, which triggers a hyper-activation of glutamate receptors, particularly NMDA subtype, production of reactive oxygen species, pathological increase in intracellular Ca 2+ , rapid decrease in ATP reserves and activation of various proteolytic enzymes [60][61][62]. Contemporarily to the glutamate increase, also the extracellular adenosine concentration significantly rises, as demonstrated both in vivo and in vitro experiments (for a review see: [45,63,64]). In these conditions, it is important to underline that A 2B AR has a lower affinity for its endogenous ligand compared to the other adenosine receptor subtypes [65] which highlighted its selective involvement only in pathological conditions when extracellular adenosine concentrations reach micromolar levels. Therefore, A 2B AR may represent a specific sensor of damage.
An OGD episode, which is an experimental condition that mimics the most common consequences of cerebral ischemia (embolic vessel occlusion), allows us to obtain highly valuable information in terms of the time course of the electrophysiological events, changes in membrane potential (i.e., AD) and synaptic transmission impairment [31,38,66]. As stated above, a pharmacological treatment that postpones the onset of AD could protect the penumbra, a brain region potentially salvageable after an ischemic-like insult [23,28,29,31,32]. The selective antagonism of A 2A AR or A 2B AR prevents or delays the AD onset induced by severe OGD in the CA1 region of the rat hippocampus. This mechanism reduces neuronal damage and astrocytic over-activation and stimulates survival pathways [30,38]. In this work, we tested the effects of the dual antagonist, P626, during 30 min OGD and we demonstrated that it could delay the AD onset induced by a severe OGD in the CA1 rat hippocampus. During the first minutes (2-3 min) of an OGD insult, adenosine concentration gradually increases activating principally the higher affinity A 1 AR and A 2A AR subtypes. Then (af-ter~4 min, see [6,8]), when the adenosine concentration reaches micromolar levels (between 10 and 30 micromolar), it is also able to activate the A 2B AR subtype. Following these events, Fusco et al. (2019) demonstrated that the selective A 2B AR antagonist, PSB603, did not modify OGD-induced fEPSP depression during the first 2 min of the ischemic-like insult, indicating that A 2B ARs are not involved in the first phases of an ischemic episode [15]. This is also consistent with the extracellular adenosine levels measured over such a period, less than 5 µM [6,8,63,67], which are insufficient to activate the A 2B ARs [10]. Therefore, during the first minutes after OGD, we presume that the new dual antagonist, P626, could exert its effect in delaying AD only by antagonizing the A 2A AR subtype. Then, in the min following the OGD, when adenosine reaches a higher concentration, the overall effect of P626 could also be due to the block of A 2B ARs. This condition is strengthened by the fact that, based on IC 50 reported by Varano and colleagues [44], P626 apparently favors the block of A 2A AR over A 2B AR by about a factor of six. Thus, P626 could represent a favorable strategy for neuroprotection by a concurrent block of "A 2 ARs" subtypes during an acute ischemic insult. Moreover, given a translational clinical approach, we can speculate that the advantage of using the dual antagonist could be to bypass eventual differences in pharmacokinetic and side effects due to the administration of two different compounds. Finally, the simultaneous "A 2 ARs" blockade could have the presumed advantage of widening the therapeutic time window for efficacious post-stroke treatment.

Conclusions
In conclusion, the use of the novel dual A 2A AR/A 2B AR antagonist, P626, could represent a favorable strategy to achieve neuroprotection by a simultaneous block of "A 2 ARs" subtypes during an acute ischemic insult to prevent glutamate overload and expand the therapeutic time window in stroke patients.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/biom13060894/s1, Figure S1: The new dual "A 2 ARs" antagonist, P626, did not affect basal synaptic transmission nor PPF ratio in the CA1 region of rat hippocampus.