human P2X3 (pIC50 = 8.0); rat P2X3 (pIC50 = 8.0); human P2X2/3 (pIC50 = 7.3)

AF-353 (Ro-4) exhibits great potency in suppressing intracellular calcium flux caused by α,β-meATP in cell lines that express recombinant rat and human P2X3 and human P2X2/3 channels [1]. With a p50 of 7.3, AF-353 (Ro-4) likewise inhibits human P2X2/3 channel activity, but with somewhat less potency [1].

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The antagonistic potencies (pIC(50)) of AF-353 for rat and human P2X3 and human P2X2/3 receptors were determined using methods of radioligand binding, intracellular calcium flux and whole cell voltage-clamp electrophysiology. Key results: The pIC(50) estimates for these receptors ranged from 7.3 to 8.5, while concentrations 300-fold higher had little or no effect on other P2X channels or on an assortment of receptors, enzymes and transporter proteins. In contrast to A-317491 and TNP-ATP, competition binding and intracellular calcium flux experiments suggested that AF-353 inhibits activation by ATP in a non-competitive fashion. Conclusions and implications: The combination of a favourable pharmacokinetic profile with the antagonist potency and selectivity for P2X3 and P2X2/3 receptors suggests that AF-353 is an excellent in vivo tool compound for study of these channels in animal models and demonstrates the feasibility of identifying and optimizing molecules into potential clinical candidates, and, ultimately, into a novel class of therapeutics for the treatment of pain-related disorders [1].

AF-353 (Ro-4) (10 mg/kg, 20 mg/kg; intravenously; for 4-6 hours) inhibits purinergic responses in both normal and spinal cord injured (SCI) rats [2]. It also shortens the systolic interval in normal rats but not in SCI rats [2]. Nevertheless, the frequency of non-voiding (NVC) is significantly reduced in SCI rats. AF-353 (Ro-4) does not impair oxygen levels or heart function [2].

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Spinal cord-injured (SCI) was induced in female rats by complete transection at T8-T9 and experiments were performed 4 weeks later, when bladder overactivity developed. Non-transected rats were used as controls (normal rats). Neural activity was recorded in the dorsal horn of the spinal cord and field potentials were acquired in response to intravesical pressure steps via a suprapubic catheter. Field potentials were recorded under control conditions, after stimulation of bladder mucosal purinergic receptors with intravesical ATP (1 mm), and after intravenous injection of the P2X3/P2X2/3 antagonist AF-353 (10 mg/kg and 20 mg/kg). Cystometry was performed in urethane-anaesthetised rats intravesically infused with saline. AF-353 (10 mg/kg) was systemically applied after baseline recordings; the rats also received a second dose of AF-353 (20 mg/kg). Changes in the frequency of voiding (VC) and non-voiding (NVC) contractions were evaluated. Results: SCI rats had significantly higher frequencies for field potentials and NVC than NL rats. Intravesical ATP increased field potential frequency in control but not SCI rats, while systemic AF-353 significantly reduced this parameter in both groups. AF-353 also reduced the inter-contractile interval in control but not in SCI rats; however, the frequency of NVC in SCI rats was significantly reduced. Conclusion: The P2X3/P2X2/3 receptors on bladder afferent nerves positively regulate sensory activity and NVCs in overactive bladders [2].

Pharmacological selectivity [1]
The selectivity of AF-353 for P2X3 and P2X2/3 channels over other homomeric P2X channels was established by measuring the potency of antagonism by AF-353 of agonist-evoked intracellular calcium flux in cell lines expressing recombinant P2X channels (see above). Additionally, AF-353 was examined in two broad commercially available panels of selectivity, one covering 75 receptors, channels, enzymes and transporters), and a second one covering more than 100 kinases (Ambit).

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Radioligand binding [1]
Radioligand binding experiments were conducted in membranes derived from Chinese hamster ovary cells (CHO) expressing the rat P2X3 (CHO-rP2X3) ion channel using a tetracycline-off expression vector (Lachnit et al., 2000) or 1321N1 human astrocytoma cells expressing hP2X3 or hP2X2/3. Cells were harvested in 1X Versene and homogenized by a Polytron in ice-cold 50 mM Tris pH 7.4 with 1X Complete™ protease inhibitor cocktail. Plasma membranes were isolated by a two-step centrifugation. Homogenized membranes were centrifuged at 1000×g for 15 min at 4°C. The 1000×g pellet was discarded, and the supernatant was centrifuged at 43 000×g for 30 min at 4°C. The 43 000×g supernatant was discarded, and the pellet was stored at −70°C until assayed. Tritium-labelled AF-353 (81.2 Ci·mmol−1) was synthesized by the Radiochemistry Department at Roche; purity was confirmed by HPLC to be >97%. The ligand affinities at P2X3 and P2X2/3 membranes were determined under equilibrium-binding conditions in 50 mM Tris pH 7.4. [3H]-AF-353 (1.7–140 nM for homomeric and 1.3–660 nM for heteromeric ion channels) was incubated with membranes (200–350 µg·mL−1) in the absence or presence of 10 µM of an unlabelled AF-353 analogue, AF-010 (to define non-specific binding) for 2–5 h at 22°C to determine its equilibrium dissociation affinity constant (Kd), as well as the receptor expression level (Bmax) of the membranes. For unlabelled molecules, dissociation affinity constants (KB) were determined by co-incubating unlabelled molecules (serially diluted over a million-fold concentration range) with [3H] AF-353 (1–5 nM) and CHO-rP2X3 membranes. In all cases, incubation was ended by filtration with ice-cold 50 mM Tris (pH 7.4) on GF/B filters. Filters were soaked in MicroScint-20 scintillation cocktail for at least 3 h prior to quantification of filter-trapped radioactivity using a Perkin Elmer TopCount plate reader. Competition binding data were analyzed by non-linear regression using a four-parameter hyperbolic function to estimate curve maxima, curve minima, Hill slope and IC50; KB estimates were calculated from observed IC50 values using the Cheng–Prusoff equation (Cheng and Prusoff, 1973). Affinities are presented as the mean and standard deviations determined over two to four repeat experiments.
Assessment of competition in the binding modes between [3H] AF-353 and other unlabelled test ligands was based on the expectations of the Cheng–Prusoff relationship that describes binding of two ligands in a mutually exclusive manner (Cheng and Prusoff, 1973). This relationship is described by the equation Inline graphic, where [A*] represents the radioligand concentration, Kd the equilibrium dissociation constant of A*, KB the equilibrium dissociation constant of the unlabelled test compound, and IC50 is the test compound concentration that displaces 50% of the binding of A*. Radioligand binding studies were conducted using the conditions described above by incubating CHO-rP2X3 membranes with [3H]-AF-353 in the absence or presence of competing agents. For this analysis, IC50 values were determined for each unlabelled test compound over a range of 5–8 different radioligand concentrations ([3H]-AF-353 0.1–60 nM). The observed IC50 values were plotted as a ratio of IC50/KB versus [A*]/Kd for all concentrations of radioligand (A*) tested. From the Cheng–Prusoff equation, a competitive interaction between unlabelled ligand and [3H] AF-353 will plot as a line with a slope of unity, and a Y-axis intercept of 1.

Whole cell voltage clamp electrophysiology [1]
Standard giga-seal patch clamp technique was employed to study the P2X2/3 channel for these experiments. The patch clamp rig consisted of the following components: Anti-vibration table, microscope, micromanipulator, patch-clamp amplifier, digitizer, drug perfusion system, and acquisition software. For recordings, the bath solution consisted of (in mM) 155 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 d-glucose, 10 HEPES, pH 7.4 with NaOH, 310 mOsM, and the pipette intracellular solution consisted of (in mM) 130 CsF, 10 NaCl, 10 EGTA, 1 MgCl2, 10 HEPES, pH 7.2 with CsOH, 290mOsM. Standard wall borosilicate glass electrodes (OD 1.50 mm, ID 0.87 mm, with filament) were pulled with a Sutter Instruments P-87 pipette puller. The average resistance of the electrodes used was 3.5 MOhm. To activate the P2X2/3 heteromeric channel, 10 µM α,β-meATP solution (pH adjusted with NaOH) was used. This value is approximately the EC50 for the channel under the conditions of the experiment. The channel was activated for 2 s, at regular intervals of approximately 30 s. Test compound was added when the current from the channel was consistent for at least three agonist applications (about 90 s). The block due to the compound was monitored until equilibrium was achieved, and then compound was washed out to determine the off-rate kinetics.

Animal/Disease Models: Female SD (SD (Sprague-Dawley)) rats with SCI (250-300 g) [2]
Doses: 10 mg/kg, 20 mg/kg
Route of Administration: intravenous (iv) (iv)injection; intravenous (iv) (iv)injection; intravenous (iv) (iv)injection. 90 minutes apart, lasting 4 hrs (hrs (hours)) to 6 hrs (hrs (hours))
Experimental Results: Purinergic responses were Dramatically diminished in both normal and SCI rats. STIMULATION PROTOCOLS [2]
Spinal cord field potentials were evaluated during intravesical pressure steps from 0 to 60 cmH2O. For stimulation of intravesical purinergic receptors each experiment was divided into three sections. First, bladders were infused with saline solution; second, bladders were filled with a saline solution containing 1 mm ATP; third, bladders were filled with 1 mm ATP during two (10 or 20 mg/kg) consecutive i.v. injections of AF-353 (i.e. 5-[5-iodo-4-methoxy-2-(1-methylethyl)phenoxy]-2,4-pyrimidinediamine hydrochloride, RO-4 hydrochloride). In each case, bladder pressure stimulation was maintained for 1 min followed by 3 min of recovery without pressure. This procedure was repeated twice. The total time of the experiment varied from 4 to 6 h and the interval between AF-353 applications was set at 90 min.

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SURGICAL PREPARATION FOR CYSTOMETRY [2]
Control or SCI rats were anaesthetised with 1.0 g/kg urethane (s.c.). A suprapubic catheter was placed through the bladder dome and another catheter was inserted into the jugular vein for drug delivery.

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CYSTOMETRIC EVALUATION [2]
The bladder catheter was connected to a syringe pump and saline solution was infused at 0.12 mL/min for 2 h, while the bladder contractions were measured with a pressure transducer (World Precision Instruments) connected to a data acquisition system. Bladder contractions were designated as voiding (VC) or non-voiding (NVC) contractions depending on whether saline was expelled during a bladder contraction. After baseline recordings, AF-353 was administered at a dose of 10 mg/kg, and 2 h later rats received a second application of 20 mg/kg. The frequency of the contractions was calculated during the last 60 min of each condition using the WINDAQ playback program

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Blood collection [1]
Blood was collected at pre-determined time points, using lithium heparin as anticoagulant from the jugular vein. After centrifugation at 3000×g for 5 min, plasma was obtained and stored at −80°C until analysis. [1]

Plasma protein binding [1]
Heparinized rat plasma was obtained from Pel-Freez® Biologicals and stored at −80°C until use. Centrifree Micropartition Devices were used to separate unbound from protein-bound material. AF-353 was added to heparinized ultrafiltered plasma (n = 3) to yield a final concentration between 200 and 5000 ng·mL−1. One millilitre of the plasma solutions and 0.3 mL of the ultrafiltrate solution were added to the filtration device and centrifuged (fixed angle) for 20 min at 2000×g.

To assess the utility of AF-353 as a tool to investigate antagonism of P2X3 and P2X2/3 receptors in vivo, rats were dosed with 2 mg·kg−1 of AF-353 i.v. and orally as a suspension. The relevant pharmacokinetic parameters that were determined are shown in Table 3. AF-353 is orally bioavailable (F = 32.9%) with a Tmax of ∼30 min and plasma half-life of 1.63 h. CNS penetration was determined by measuring the brain to plasma ratio (B/P); AF-353 is highly CNS penetrant, with a B/P ratio of 6 (total brain extracted concentration/total plasma concentration). In addition; the in vitro protein binding was determined to be 98.2% in rat plasma. [1]
Favourable pharmacokinetic parameters were observed in rat, with good oral bioavailability (%F = 32.9), reasonable half-life (t(1/2) = 1.63 h) and plasma-free fraction (98.2% protein bound).

[1]. AF-353, a novel, potent and orally bioavailable P2X3/P2X2/3 receptor antagonist. Br J Pharmacol. 2010 Jul;160(6):1387-1398.

[2]. Modulation of bladder afferent signals in normal and spinal cord-injured rats by purinergic P2X3 and P2X2/3receptors. BJU Int. 2012 Oct;110(8 Pt B):E409-414.

Background and purpose: Purinoceptors containing the P2X3 subunit (P2X3 homotrimeric and P2X2/3 heterotrimeric) are members of the P2X family of ion channels gated by ATP and may participate in primary afferent sensitization in a variety of pain-related diseases. The current work describes the in vitro pharmacological characteristics of AF-353, a novel, orally bioavailable, highly potent and selective P2X3/P2X2/3 receptor antagonist. Experimental approach: The antagonistic potencies (pIC(50)) of AF-353 for rat and human P2X3 and human P2X2/3 receptors were determined using methods of radioligand binding, intracellular calcium flux and whole cell voltage-clamp electrophysiology. Key results: The pIC(50) estimates for these receptors ranged from 7.3 to 8.5, while concentrations 300-fold higher had little or no effect on other P2X channels or on an assortment of receptors, enzymes and transporter proteins. In contrast to A-317491 and TNP-ATP, competition binding and intracellular calcium flux experiments suggested that AF-353 inhibits activation by ATP in a non-competitive fashion. Favourable pharmacokinetic parameters were observed in rat, with good oral bioavailability (%F = 32.9), reasonable half-life (t(1/2) = 1.63 h) and plasma-free fraction (98.2% protein bound). Conclusions and implications: The combination of a favourable pharmacokinetic profile with the antagonist potency and selectivity for P2X3 and P2X2/3 receptors suggests that AF-353 is an excellent in vivo tool compound for study of these channels in animal models and demonstrates the feasibility of identifying and optimizing molecules into potential clinical candidates, and, ultimately, into a novel class of therapeutics for the treatment of pain-related disorders.[1]

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We used in vivo electrophysiological and cystometric methods to evaluate the effect of the selective P2X3/P2X2/3 antagonist AF-353 on bladder sensory pathways in normal and SCI rats. The present results showed: (i) basal spinal neural activity was amplified in SCI rats compared with normal rats; (ii) in contrast to normal rats, spinal neural activity in SCI rats in response to noxious pressure was not augmented by activation of purinergic receptors with intravesical ATP; (iii) spinal neural activity in response to chemical (ATP) and noxious pressure stimulation was significantly reduced in normal and SCI rats by systemic application of the selective P2X3/P2X2/3 antagonist AF-353; and (iv) in agreement with the electrophysiological results, reflex bladder contractions were markedly decreased in both normal and SCI rats by AF-353. [2]

C14H17N4O2I

400.21438

400.04

C, 42.02; H, 4.28; I, 31.71; N, 14.00; O, 8.00

865305-30-2

927887-18-1 (HCl)

15953802

Light brown to brown solid powder

3.03

2

6

4

21

334

0

IC1C(OC)=CC(C(C)C)=C(OC2C(N)=NC(N)=NC=2)C=1

AATPYXMXFBBKFO-UHFFFAOYSA-N

InChI=1S/C14H17IN4O2/c1-7(2)8-4-11(20-3)9(15)5-10(8)21-12-6-18-14(17)19-13(12)16/h4-7H,1-3H3,(H4,16,17,18,19)

5-(5-iodo-4-methoxy-2-propan-2-ylphenoxy)pyrimidine-2,4-diamine

Ro-4; Ro 4; Ro4; AF353; 865305-30-2; 2,4-Pyrimidinediamine, 5-[5-iodo-4-methoxy-2-(1-methylethyl)phenoxy]-; 5-(5-IODO-2-ISOPROPYL-4-METHOXYPHENOXY)PYRIMIDINE-2,4-DIAMINE; 5-(5-iodo-4-methoxy-2-propan-2-ylphenoxy)pyrimidine-2,4-diamine; CHEMBL526307; AF-353; AF 353.

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 : ~100 mg/mL (~249.87 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.25 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 25.0 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: ≥ 2.5 mg/mL (6.25 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.25 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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