title: A functional radioreceptor assay of alpha-V-beta-3 (alphavbeta3) inhibitors in plasma: application as an ex vivo pharmacodynamic model.
abstract: Development of alphavbeta3-integrin inhibitors has been hampered by a lack of pharmacodynamic endpoints to identify doses that inhibit alphavbeta3 in vivo. To address this need, we developed an alphavbeta3 radioreceptor assay (RRA) that could be performed in 100% plasma. The RRA was based on 125I-echistatin binding to plate-immobilized alphavbeta3. Small molecule alphavbeta3 inhibitors efficiently competed echistatin binding to alphavbeta3 when the assay was carried out in buffer. However, when carried out in 100% plasma, the RRA revealed a 45 to >3000-fold loss in compound potencies. The losses in potency reflected, in part, the high plasma protein binding by the compounds examined. The RRA was adapted as an ex vivo pharmacodynamic model. Echistatin binding was measured in the presence of plasma harvested at timed intervals from rats dosed with select compounds. Using this pharmacodynamic model, compound and dose selection was optimized for further testing in models of corneal angiogenesis. Moderate anti-angiogenic activity was achieved when rats were dosed sufficient to achieve sustained (>50%) plasma inhibition through the trough interval. Thus, the RRA provided a simple technique to rank order compound potency in plasma, and could find general use as an ex vivo pharmacodynamic assay to select compounds and doses for preclinical and clinical proof-of-principle studies.
authors.affiliation: Johnson & Johnson Pharmaceutical Research and Development, 665 Stockton Drive, Exton, PA 19341, USA.
authors.forename: Margery A
authors.initials: MA
authors.lastname: Chaikin
authors.forename: Júan José
authors.initials: JJ
authors.lastname: Marugan
authors.forename: Gerald W
authors.initials: GW
authors.lastname: De Vries
authors.forename: Peter
authors.initials: P
authors.lastname: Baciu
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Edelman
authors.forename: Ming
authors.initials: M
authors.lastname: Ni
authors.forename: Bruce E
authors.initials: BE
authors.lastname: Tomczuk
authors.forename: Wenxi
authors.initials: W
authors.lastname: Pan
authors.forename: Zihong
authors.initials: Z
authors.lastname: Guo
authors.forename: Beth
authors.initials: B
authors.lastname: Anaclerio
authors.forename: Kristi
authors.initials: K
authors.lastname: Leonard
authors.forename: Stephen H
authors.initials: SH
authors.lastname: Eisennagel
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Molloy
authors.forename: Carl L
authors.initials: CL
authors.lastname: Manthey
mesh_terms: D020533:Angiogenesis Inhibitors / Q000493:pharmacokinetics / Q000494:pharmacology; D000818:Animals; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism; D001798:Blood Proteins / Q000378:metabolism; D003315:Cornea / Q000098:blood supply / Q000187:drug effects; D004353:Drug Evaluation, Preclinical; D066298:In Vitro Techniques; D039302:Integrin alphaVbeta3 / Q000037:antagonists & inhibitors* / Q000097:blood*; D036341:Intercellular Signaling Peptides and Proteins; D008297:Male; D009389:Neovascularization, Pathologic / Q000517:prevention & control; D010455:Peptides / Q000097:blood / Q000493:pharmacokinetics; D011485:Protein Binding; D011869:Radioligand Assay / Q000379:methods*; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D020533:Angiogenesis Inhibitors; D001798:Blood Proteins; D039302:Integrin alphaVbeta3; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; C057986:echistatin