title: Solubility assessment and on-line exposure confirmation in a patch-clamp assay for hERG (human ether-a-go-go-related gene) potassium channel inhibition.
abstract: BACKGROUND
  The hERG (human ether-a-go-go-related gene) potassium channel (KV11.1) is an important anti-target in drug discovery as its inhibition by small molecules has considerable promiscuity and is linked to an increased risk of the potentially fatal ventricular arrhythmia torsade de pointes. Therefore, great efforts are taken in the pharmaceutical industry to early on screen out compounds that block the channel. Early screening activities most often include compounds with sub-optimal physicochemical properties such as limited solubility. Therefore, careful monitoring of achieved compound concentration importantly supports the validity of experimental data.
  
  
  METHODS
  A novel principle of exposure confirmation in a constant flow patch-clamp assay for hERG interaction is presented. Quantification is based on-real time UV absorption spectroscopy of the perfusion solution using long light path fiber optic flow cells. Calibration is performed using solutions which are confirmed by turbidometry to be free of precipitates.
  
  
  RESULTS
  Turbidometry is shown to be sensitive enough to ensure valid calibration of the UV spectroscopic measurement. For a typical drug-like small molecule (verapamil) it is shown that even 30 nM can be accurately quantified using a 100 cm fiber optic flow cell.
  
  
  CONCLUSIONS
  The combination of turbidometry and long light path fiber optic UV spectroscopy offers accurate, almost real-time exposure determination in a wide range of concentrations with little effort, affordable instrumentation, and no delay for data reporting. For research compounds with challenging physicochemical properties this method provides valuable data to support the validity of the measurements.
authors.affiliation: Boehringer Ingelheim Pharma GmbH & Co. KG, Dept. of Drug Discovery Support, Birkendorfer Strasse 65, 88397 Biberach, Germany. Electronic address: Georg.Rast@boehringer-ingelheim.com.
authors.forename: Georg
authors.initials: G
authors.lastname: Rast
authors.affiliation: Boehringer Ingelheim Pharma GmbH & Co. KG, Dept. of Drug Discovery Support, Birkendorfer Strasse 65, 88397 Biberach, Germany.
authors.forename: Brian D
authors.initials: BD
authors.lastname: Guth
mesh_terms: D002138:Calibration; D051638:Ether-A-Go-Go Potassium Channels / Q000037:antagonists & inhibitors* / Q000235:genetics; D006801:Humans; D018408:Patch-Clamp Techniques; D026902:Potassium Channel Blockers / Q000494:pharmacology*; D012995:Solubility; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship
chemical_list: D051638:Ether-A-Go-Go Potassium Channels; D026902:Potassium Channel Blockers
keywords: Exposure; Flow cell; ICH S7B; Methods; Patch-clamp; Screening; Solubility; Turbidometry; UV spectroscopy; hERG