title: Recommendations on the Development of a Bioanalytical Assay for 4β-Hydroxycholesterol, an Emerging Endogenous Biomarker of CYP3A Activity.
abstract: The availability of reliable assays for measuring 4β-hydroxycholesterol (4β-HC), a CYP3A metabolite of cholesterol, is an important step in qualifying this endogenous moiety as a biomarker of CYP3A activity. Liquid and gas chromatographic methods with mass spectrometric detection have been developed with varying sensitivities, with or without derivatization. Care must be taken to chromatographically resolve 4β-HC from the multiple isobaric cholesterol oxidation products present in plasma, including 4α-hydroxycholesterol (4α-HC). Plasma concentrations of 4β-HC are low in humans (10-60 ng/ml), lower than many other cholesterol metabolites and far less than cholesterol itself. Stability of 4β-HC has been established for at least 12 months at -20°C in plasma samples obtained with a typical clinical workflow. Oxidation of plasma cholesterol during storage produces both 4β-HC and 4α-HC, and 4α-HC may be used as assessment of sample quality. As 4β-HC concentrations over time in untreated individuals have low intra-individual variability, assay precision and reproducibility are the key assay attributes in assessing CYP3A4 induction, and potentially inhibition. Assessment of CYP3A4/5 activity with 4β-HC relies on the differences between pre- and post-dose concentrations, in which each subject acts as their own control. To reduce analytical variability, samples from a single subject should be analyzed together to facilitate interpretation of study results. As an endogenous biomarker, 4β-HC offers the opportunity for less invasive assessment of CYP3A induction potential of new drugs during clinical development.
authors.affiliation: Bristol-Myers Squibb Co, Analytical and Bioanalytical Operations, Route 206 and Province Line Road, Princeton, New Jersey, 08540, USA. anne.aubry@bms.com.
authors.forename: Anne-Françoise
authors.initials: AF
authors.lastname: Aubry
authors.affiliation: Genentech, Drug Metabolism and Pharmacokinetics, South San Francisco, California, USA.
authors.forename: Brian
authors.initials: B
authors.lastname: Dean
authors.affiliation: Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Ulf
authors.initials: U
authors.lastname: Diczfalusy
authors.affiliation: Technical Operations, Amicus Therapeutics, Cranbury, New Jersey, USA.
authors.forename: Angela
authors.initials: A
authors.lastname: Goodenough
authors.affiliation: Drug Metabolism and Pharmacokinetics, Vertex Pharmaceuticals, Boston, Massachusetts, USA.
authors.forename: André
authors.initials: A
authors.lastname: Iffland
authors.affiliation: Drug Development, Galleon Pharmaceuticals, Horsham, Pennsylvania, USA.
authors.forename: James
authors.initials: J
authors.lastname: McLeod
authors.affiliation: Pharmacokinetics, Pharmacodynamics and Metabolism, Janssen Research & Development, Spring House, Pennsylvania, USA.
authors.forename: Naidong
authors.initials: N
authors.lastname: Weng
authors.affiliation: Drug Metabolism and Pharmacokinetics, Novartis, East Hanover, New Jersey, USA.
authors.forename: Ziping
authors.initials: Z
authors.lastname: Yang
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods / Q000639:trends*; D015415:Biomarkers / Q000378:metabolism; D051544:Cytochrome P-450 CYP3A / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006888:Hydroxycholesterols / Q000737:chemistry / Q000378:metabolism*; D004364:Pharmaceutical Preparations / Q000737:chemistry / Q000378:metabolism*; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D015415:Biomarkers; D006888:Hydroxycholesterols; D004364:Pharmaceutical Preparations; C099828:cholest-5-ene-3,4-diol; D051544:Cytochrome P-450 CYP3A
keywords: 4β-hydroxycholesterol; CYP3A4; biomarker; drug-drug interaction; mass spectrometry