title: AF-353, a novel, potent and orally bioavailable P2X3/P2X2/3 receptor antagonist.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  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
  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.
authors.affiliation: Department of Inflammation Discovery, Roche Palo Alto, Palo Alto, CA, USA.
authors.forename: Joel R
authors.initials: JR
authors.lastname: Gever
authors.forename: Rothschild
authors.initials: R
authors.lastname: Soto
authors.forename: Robert A
authors.initials: RA
authors.lastname: Henningsen
authors.forename: Renee S
authors.initials: RS
authors.lastname: Martin
authors.forename: David H
authors.initials: DH
authors.lastname: Hackos
authors.forename: Sandip
authors.initials: S
authors.lastname: Panicker
authors.forename: Werner
authors.initials: W
authors.lastname: Rubas
authors.forename: Ian B
authors.initials: IB
authors.lastname: Oglesby
authors.forename: Michael P
authors.initials: MP
authors.lastname: Dillon
authors.forename: Marcos E
authors.initials: ME
authors.lastname: Milla
authors.forename: Geoffrey
authors.initials: G
authors.lastname: Burnstock
authors.forename: Anthony P D W
authors.initials: AP
authors.lastname: Ford
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000284:Administration, Oral; D000818:Animals; D001682:Biological Availability; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D006207:Half-Life; D006801:Humans; D020128:Inhibitory Concentration 50; D008297:Male; D018408:Patch-Clamp Techniques; D010647:Phenyl Ethers / Q000008:administration & dosage / Q000493:pharmacokinetics / Q000494:pharmacology*; D058919:Purinergic P2 Receptor Antagonists; D011743:Pyrimidines / Q000008:administration & dosage / Q000493:pharmacokinetics / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D058476:Receptors, Purinergic P2X2; D058477:Receptors, Purinergic P2X3
chemical_list: C552885:5-(5-iodo-2-isopropyl-4-methoxyphenoxy)pyrimidine-2,4-diamine; C546401:P2RX2 protein, human; C546407:P2RX3 protein, human; C546403:P2rx2 protein, rat; C546410:P2rx3 protein, rat; D010647:Phenyl Ethers; D058919:Purinergic P2 Receptor Antagonists; D011743:Pyrimidines; D058476:Receptors, Purinergic P2X2; D058477:Receptors, Purinergic P2X3; D000255:Adenosine Triphosphate; D002118:Calcium