P2X4 Receptor

5-BDBD, with an IC50 of 0.75 μM, concentration-dependently suppresses the current produced by 10 μM ATP in HEK293 cells expressing rP2X4R [1]. 5-BDBD causes an EC50 increase from 4.7 to 15.9 μM, which causes the ATP concentration response curve to move to the right [1]. P2X1R, P2X2aR, P2X2bR, P2X3R, P2X4R, and P2X7R may all be distinguished using 5-BDBD [1].

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P2X receptors (P2XRs) are a family of ATP-gated ionic channels that are expressed in numerous excitable and non-excitable cells. Despite the great advance on the structure and function of these receptors in the last decades, there is still lack of specific and potent antagonists for P2XRs subtypes, especially for the P2X4R. Here, researchers studied in detail the effect of the P2X4R antagonist 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD) on ATP-induced currents mediated by the rat P2X4R and compared its specificity among another rat P2XRs. Researchers found that 5-BDBD is a potent P2X4R antagonist, with an IC50 of 0.75 μM when applied for 2 min prior and during ATP stimulation. Moreover, at 10 μM concentration, 5-BDBD did not affect the ATP-induced P2X2aR, P2X2bR, and P2X7R current amplitude or the pattern of receptor desensitization. However, at 10 μM concentration but not 0.75 μM 5-BDBD inhibited the P2X1R and P2X3R-gated currents by 13 and 35% respectively. Moreover, researchers studied the effects of 5-BDBD in long-term potentiation experiments performed in rat hippocampal slices, finding this antagonist can partially decrease LTP, a response that is believed to be mediated in part by endogenous P2X4Rs. These results indicate that 5-BDBD could be used to study the endogenous effects of the P2X4R in the central nervous system and this antagonist can discriminate between P2X4R and other P2XRs, when they are co-expressed in the same tissue [1].

The entire eruptive response to recurrent NTG injection-induced basal hyperalgesia can be achieved with 5-BDBD (28 mg/kg; intraperitoneally; once daily for 9 days) [2].

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Researchers used an animal model with recurrent intermittent administration of nitroglycerin (NTG), which closely mimics CM. NTG-induced basal and acute mechanical hypersensitivity were evaluated using the von Frey filament test. Then, Researchers detected Iba1 immunoreactivity (Iba1-IR) and P2X4R expression in the trigeminal nucleus caudalis (TNC). To understand the effect of microglia and P2X4R on central sensitization of CM, Researchers examined whether minocycline, an inhibitor of microglia activation, and 5-BDBD, a P2X4R antagonist, altered NTG-induced mechanical hyperalgesia. In addition, Researchers also evaluated the effect of 5-BDBD on c-Fos and calcitonin gene-related peptide (CGRP) expression within the TNC. Results: Chronic intermittent administration of NTG resulted in acute and chronic basal mechanical hyperalgesia, accompanied with microglia activation and upregulation of P2X4R expression. Minocycline significantly decreased basal pain hypersensitivity but did not alter acute NTG-induced hyperalgesia. Minocycline also reduced microglia activation. 5-BDBD completely blocked the basal and acute hyperalgesia induced by NTG. This effect was associated with a significant inhibition of the NTG-induced increase in c-Fos protein and CGRP release in the TNC. Conclusions: Our results indicate that blocking microglia activation may have an effect on the prevention of migraine chronification. Moreover, Researchers speculate that the P2X4R may be implicated in the microglia-neuronal signal in the TNC, which contributes to the central sensitization of CM [2].

Receptor transfection and current measurements. [1]
Experiments were done with rat P2XRs. HEK293 cells were routinely maintained in DMEM containing 10% (v/v) fetal bovine serum and 100 µg/ml gentamicin. Cells were plated at a density of 500,000 cells per 35 mm culture dish. The transient transfection was conducted 24 h after plating the cells using 2 µg of DNA and 5 µl of LipofectAMINE 2000 reagent in 2 ml of serum-free Opti-MEM. After 4.5 h of incubation, the transfection mixture was replaced with normal culture medium. The experiments were performed 24–48 h after transfection. Before recordings, transfected cells were mechanically dispersed and re-cultured on 35-mm dishes for 2–10 h. Electrophysiological experiments were performed on cells at room temperature using whole-cell patch-clamp recording techniques. The currents were recorded using an Axopatch 200B patch-clamp amplifier and were filtered at 2 kHz using a low-pass Bessel filter. Patch electrodes, fabricated from borosilicate glass, using a Flaming Brown horizontal puller, were heat polished to a final tip resistance of 2–4 MΩ. All current records were captured and stored using the pClamp 9 software packages in conjunction with the Digidata 1322A analog-to-digital converter. Patch electrodes were filled with a solution containing the following (in mM): 142 NaCl, 1 MgCl2, 10 EGTA, and 10 HEPES, pH adjusted to 7.35 with 10 M NaOH. The osmolarity of the internal solutions was 306 mOsm. The bath solution contained the following (in mM): 142 NaCl, 3 KCl, 1 MgCl2, 2 CaCl2, 10 glucose, and 10 HEPES, pH adjusted to 7.35 with 10 M NaOH. The osmolarity of this solution was 295–305 mOsm. ATP was daily prepared in bath buffer and applied using a rapid solution changer system. Stock solutions of 5-BDBD were prepared in dimethylsulfoxide, and aliquots were stored at –20 °C. The current responses were recorded from single cells clamped at –60 mV. Concentration–response data were collected from recordings of a range of ATP concentration applied to single cells with a washout interval of 1–5 min between each application and normalized to the highest current amplitude.

Animal/Disease Models: Male C57BL/6 mice [2]
Doses: 28 mg/kg
Route of Administration: IP; one time/day for 9 days
Experimental Results: Prevention of NTG-induced mechanical allergy.

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Hippocampal slice preparation. [1]
After decapitation, brains were rapidly immersed in ice-cold dissection buffer containing (in mM) 212.7 sucrose; 5 KCl; 1.25 NaH2PO4; 3 MgSO4; 1 CaCl2; 26 NaHCO3; and 10 glucose; pH 7.4. Hippocampi were dissected and transverse slices (400 µm thick) were obtained from the middle third portion using a vibratome. Slices were transferred to an interface storage chamber containing artificial cerebrospinal fluid (aCSF) saturated with 95% O2 / 5% CO2 and were left at least 1 hour at 37 ºC. After this procedure, slices were maintained in aCSF or incubated 1 hour with 5-BDBD, PPADS or AF-353 before recording. aCSF contained (in mM) 124 NaCl; 5 K l; 1.25 NaH2PO4; 1.0 MgCl2; 2.0 CaCl2; 26 NaHCO3; and 10 glucose; pH 7.4. Single slices were then transferred to a recording chamber where they were kept completely submerged in ACSF and continually perfused (2 ml/min).

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Long-term potentiation (LTP) experiments. [1]
Field responses from control and 5-BDBD-incubated hippocampi were evoked by electrical stimulation (biphasic, constant current, 200 μs stimuli) delivered every 15 s at the Schaffer collateral pathway using bipolar electrodes connected to a stimulus isolator unit and recorded in the stratum radiatum, in order to visualize field Extracellular Postsynaptic Potentials (fEPSP) at the CA1 hippocampal area, using glass micropipettes (1–2 MΩ) filled with ACSF as recording electrodes. At the beginning of each experiment, stimulus/response curves were done by increasing the intensity of the stimulus in order to adjust it to elicit 50% of the maximum response. LTP was elicited after 20 min of a stable baseline by theta burst stimulation (TBS) consisting on 5 trains of stimulus with an inter-train interval of 10 s. Each train consisted of 10 bursts at 5 Hz, each burst having 4 pulses at 100 Hz. After TBS, data acquisition lasted for 1 h. Data were acquired using an extracellular amplifier and a data acquisition board (National Instruments, Austin, TX) controlled through Igor Pro software. LTP values were constructed by normalizing the fEPSP using baseline as 100%. Magnitude of LTP was observed between 40 and 60 min following TBS.

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To understand the effect of microglia and P2X4R on NTG-induced hyperalgesia, we treated the animals with the microglia inhibitor minocycline or the P2X4R-selective antagonist 5-BDBD. Minocycline and 5-BDBD were dissolved in DMSO solution, which was used as the vehicle control. Minocycline/vehicle or 5-BDBD/vehicle was administered immediately prior to the administration of NTG/saline [14, 15]. The treatment of the mice on testing days was as follows: mice were habituated to the test room, followed by a baseline measurement of mechanical thresholds 15–20 min later and an administration of minocycline (30 mg/kg, i.p.), 5-BDBD (28 mg/kg, i.p.), or vehicle; then, an injection of NTG/saline was given, and 2 h later, acute mechanical responses were tested. This procedure was repeated every other day for 9 days. [2]

[1]. Characterization of the antagonist actions of 5-BDBD at the rat P2X4 receptor. Neurosci Lett. 2019;690:219-224.

[2]. Microglia P2X4 receptor contributes to central sensitization following recurrent nitroglycerin stimulation. J Neuroinflammation. 2018;15(1):245. Published 2018 Aug 30.

In summary, we have characterized 5-BDBD as a specific P2X4R antagonist, the development of such molecules will help to clearly establish to contributions of P2XRs to physiological processes and will also help to design specific drugs to be used in pathologies in which these receptors are involved. [1]

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In our studies, CGRP-immunoreactive fibers were mainly distributed in the superficial of TNC, which are associated primarily with processing nociceptive information. 5-BDBD pretreatment mitigated the NTG-induced CGRP immunoreactivity changes in the TNC, modulating the trigeminal system activation. The cellular mechanism underlying the P2X4R-induced CGRP release may be related to brain-derived neurotrophic factor (BDNF). Previous studies demonstrated that BDNF is released from ATP-stimulated microglia via the P2X4R signaling pathway in vitro and vivo. In addition, BDNF, acting via trkB receptors, can regulate the expression and release of CGRP from sensory neurons.
Chronic migraine occurs with central sensitization of trigeminal sensory pathways that involve microglial activation and increased expression of P2X4R. Inhibition of microglia may be effective in preventing migraine chronification but be ineffective in aborting an acute migraine attack. In addition, we speculated that P2X4R may be implicated in microglia-neuronal signaling in the TNC. Inhibition of P2X4R function might be a potential therapeutic option for CM. [2]

C17H11BRN2O2

355.1854

354

C, 57.49; H, 3.12; Br, 22.50; N, 7.89; O, 9.01

768404-03-1

9841560

White to light yellow solid powder

3.505

1

3

1

22

481

0

BrC1=C([H])C([H])=C([H])C(=C1[H])C1C2=C(C3=C([H])C([H])=C([H])C([H])=C3O2)N([H])C(C([H])([H])N=1)=O

NKYMVQPXXTZHSF-UHFFFAOYSA-N

InChI=1S/C17H11BrN2O2/c18-11-5-3-4-10(8-11)15-17-16(20-14(21)9-19-15)12-6-1-2-7-13(12)22-17/h1-8H,9H2,(H,20,21)

5-(3-bromophenyl)-1,3-dihydro-[1]benzofuro[3,2-e][1,4]diazepin-2-one

5-BDBD; 768404-03-1; 5-(3-Bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one; 5 BDBD;5BDBD; 5-(3-bromophenyl)-1,3-dihydro-[1]benzofuro[3,2-e][1,4]diazepin-2-one; CHEMBL2180179; DTXSID30431707; 5-(3-bromophenyl)-1,3-dihydro-(1)benzofuro(3,2-e)(1,4)diazepin-2-one;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~62.5 mg/mL (~175.96 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.86 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (5.86 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 9.8 mg/mL (27.59 mM) in 15% Solutol HS 15 10% Cremophor EL 35% PEG 400 40% water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)