title: Production of [F-18]fluoroannexin for imaging apoptosis with PET.
abstract: Recombinant human-annexin-V was conjugated with 4-[F-18]fluorobenzoic acid (FBA) via its reaction with the N-hydroxysuccinimidyl ester (FBA-OSu) at pH 8.5. A series of reactions using varying amounts of annexin-V, unlabeled FBA-OSu, and time produced products with different conjugation levels. Products were characterized by mass spectrometry and a cell-binding assay to assess the effect of conjugation. In each case, the conjugated protein was a mixture of proteins with a range of conjugation. Annexin-V could be conjugated with an average of two FBA mole equivalents without decreasing its affinity for red blood cells (K(d) 6-10 nM) with exposed phosphatidylserine. An average conjugation of 7.7 (range 3-13) diminished the binding 3-fold. Large-scale production and purification of [F-18]FBA-OSu from [F-18]fluoride was accomplished within 90 min and in 77% radiochemical yield (decay-corrected to the end of cyclotron bombardment). The conjugation reaction of annexin with [F-18]FBA-OSu was studied with respect to activity level, protein mass, and concentration. Under the most favorable conditions, >25 mCi [F-18]fluoroannexin (FAN) was isolated in 64% yield (decay-corrected for a 22 min conjugation process) from labeling 1.1 mg of annexin-V. A pilot PET imaging study of [F-18]fluoroannexin in normal rats showed high uptake in the renal excretory system and demonstrated sufficient clearance from most other internal organs within 1 h. [F-18]Fluoroannexin should prove useful in imaging targeted apoptosis.
authors.affiliation: Department of Radiology, University of Washington, Seattle, Washington 91895, USA. grierson@u.washington.edu
authors.forename: John R
authors.initials: JR
authors.lastname: Grierson
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Yagle
authors.forename: Janet F
authors.initials: JF
authors.lastname: Eary
authors.forename: Jonathan F
authors.initials: JF
authors.lastname: Tait
authors.forename: Don F
authors.initials: DF
authors.lastname: Gibson
authors.forename: Barbara
authors.initials: B
authors.lastname: Lewellen
authors.forename: Jeanne M
authors.initials: JM
authors.lastname: Link
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Krohn
mesh_terms: D000818:Animals; D017304:Annexin A5 / Q000032:analysis / Q000138:chemical synthesis* / Q000378:metabolism*; D017209:Apoptosis; D001565:Benzoates / Q000378:metabolism; D004912:Erythrocytes / Q000737:chemistry / Q000378:metabolism; D005462:Fluorine Radioisotopes / Q000378:metabolism*; D006801:Humans; D008297:Male; D049268:Positron-Emission Tomography / Q000379:methods*; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D017304:Annexin A5; D001565:Benzoates; D005462:Fluorine Radioisotopes