title: Quantitative pharmacological analysis of antagonist binding kinetics at CRF1 receptors in vitro and in vivo.
abstract: OBJECTIVE
  A series of novel non-peptide corticotropin releasing factor type-1 receptor (CRF(1)) antagonists were found to display varying degrees of insurmountable and non-competitive behaviour in functional in vitro assays. We describe how we attempted to relate this behaviour to ligand receptor-binding kinetics in a quantitative manner and how this resulted in the development and implementation of an efficient pharmacological screening method based on principles described by Motulsky and Mahan.
  
  
  METHODS
  A non-equilibrium binding kinetic assay was developed to determine the receptor binding kinetics of non-peptide CRF(1) antagonists. Nonlinear, mixed-effects modelling was used to obtain estimates of the compounds association and dissociation rates. We present an integrated pharmacokinetic-pharmacodynamic (PKPD) approach, whereby the time course of in vivo CRF(1) receptor binding of novel compounds can be predicted on the basis of in vitro assays.
  
  
  RESULTS
  The non-competitive antagonist behaviour appeared to be correlated to the CRF(1) receptor off-rate kinetics. The integrated PKPD model suggested that, at least in a qualitative manner, the in vitro assay can be used to triage and select compounds for further in vivo investigations.
  
  
  CONCLUSIONS
  This study provides evidence for a link between ligand offset kinetics and insurmountable/non-competitive antagonism at the CRF(1) receptor. The exact molecular pharmacological nature of this association remains to be determined. In addition, we have developed a quantitative framework to study and integrate in vitro and in vivo receptor binding kinetic behaviour of CRF(1) receptor antagonists in an efficient manner in a drug discovery setting.
authors: Discovery Biology, Pfizer, Kent, UK. simeon.ramsey@pfizer.com
authors: Simeon J
authors: SJ
authors: Ramsey
authors: Neil J
authors: NJ
authors: Attkins
authors: Rebecca
authors: R
authors: Fish
authors: Piet H
authors: PH
authors: van der Graaf
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D055808:Drug Discovery / Q000379:methods; D007700:Kinetics; D008024:Ligands; D008297:Male; D017711:Nonlinear Dynamics; D011485:Protein Binding; D011720:Pyrazoles / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D011869:Radioligand Assay / Q000379:methods; D051381:Rats; D017207:Rats, Sprague-Dawley; D018019:Receptors, Corticotropin-Releasing Hormone / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D014230:Triazoles / Q000494:pharmacology; D000099052:CRF Receptor, Type 1
chemical_list: C417190:4-(3-pentylamino)-2,7-dimethyl-8-(2-methyl-4-methoxyphenyl)pyrazolo(1,5-a)pyrimidine; D008024:Ligands; D011720:Pyrazoles; D011725:Pyridines; D011743:Pyrimidines; D018019:Receptors, Corticotropin-Releasing Hormone; C473245:SN003; D014230:Triazoles; D000099052:CRF Receptor, Type 1