title: Evaluation of 4-[18F]fluorobenzoyl-FALGEA-NH2 as a positron emission tomography tracer for epidermal growth factor receptor mutation variant III imaging in cancer.
abstract: BACKGROUND
  This study describes the radiosynthesis, in vitro and in vivo evaluation of the novel small peptide radioligand, 4-[(18)F]fluorobenzoyl-Phe-Ala-Leu-Gly-Glu-Ala-NH(2,) ([(18)F]FBA-FALGEA-NH(2)) as a positron emission tomography (PET) tracer for imaging of the cancer specific epidermal growth factor receptor (EGFR) variant III mutation, EGFRvIII.
  
  
  METHODS
  For affinity, stability and PET measurements, H-FALGEA-NH(2) was radiolabelled using 4-[(18)F]fluorobenzoic acid ([(18)F]FBA). The binding affinity of ([(18)F]FBA)-FALGEA-NH(2) was measured on EGFRvIII expressing cells, NR6M. Stability studies in vitro and in vivo were carried out in blood plasma from nude mice. PET investigations of [(18)F]FBA-FALGEA-NH(2) were performed on a MicroPET scanner, using seven nude mice xenografted subcutaneously with human glioblastoma multiforme (GBM) tumours, expressing the EGFRvIII in its native form, and five nude mice xenografted subcutaneously with GBM tumours lacking EGFRvIII expression. Images of [(18)F]FDG were also obtained for comparison. The mice were injected with 5-10 MBq of the radiolabelled peptide or [(18)F]FDG. Furthermore, the gene expression of EGFRvIII in the tumours was determined using quantitative real-time PCR.
  
  
  RESULTS
  Radiolabelling and purification was achieved within 180 min, with overall radiochemical yields of 2.6-9.8% (decay-corrected) and an average specific radioactivity of 6.4 GBq/μmol. The binding affinity (K(d)) of [(18)F]FBA-FALGEA-NH(2) to EGFRvIII expressing cells was determined to be 23 nM. The radiolabelled peptide was moderately stable in the plasma from nude mice where 53% of the peptide was intact after 60 min of incubation in plasma but rapidly degraded in vivo, where no intact peptide was observed in plasma 5 min post-injection. The PET imaging showed that [(18)F]FBA-FALGEA-NH(2) accumulated preferentially in the human GBM xenografts which expressed high amounts of the mutated receptor. The average tumour-to-muscle ratio (T/M) in the EGFRvIII tumours was 7.8 at 60 min post-injection, compared with 4.6 in the wild-type EGFR tumours. Furthermore, there was a strong correlation (R=0.86, P=.007) between the expression of EGFRvIII in the tumours and the tracer uptake expressed as T/M.
  
  
  CONCLUSIONS
  Our results indicate that, despite its rapid metabolism, [(18)F]FBA-FALGEA-NH(2) binds preferentially to EGFRvIII in the tumours in vivo and is a promising lead for further development of EGFRvIII specific peptide radiopharmaceuticals.
authors.affiliation: Department of Clinical Physiology, Nuclear Medicine and PET, Copenhagen University Hospital, Rigshospitalet, 2100 Copenhagen Ø, Denmark. charlotte.lund.denholt@rh.regionh.dk
authors.forename: Charlotte Lund
authors.initials: CL
authors.lastname: Denholt
authors.forename: Tina
authors.initials: T
authors.lastname: Binderup
authors.forename: Marie-Thérése
authors.initials: MT
authors.lastname: Stockhausen
authors.forename: Hans Skovgaard
authors.initials: HS
authors.lastname: Poulsen
authors.forename: Mogens
authors.initials: M
authors.lastname: Spang-Thomsen
authors.forename: Paul Robert
authors.initials: PR
authors.lastname: Hansen
authors.forename: Nic
authors.initials: N
authors.lastname: Gillings
authors.forename: Andreas
authors.initials: A
authors.lastname: Kjær
mesh_terms: D000818:Animals; D001565:Benzoates / Q000737:chemistry; D001692:Biological Transport; D002471:Cell Transformation, Neoplastic; D004355:Drug Stability; D066246:ErbB Receptors / Q000235:genetics* / Q000378:metabolism; D019788:Fluorodeoxyglucose F18; D015972:Gene Expression Regulation, Neoplastic; D005909:Glioblastoma / Q000000981:diagnostic imaging* / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D006801:Humans; D051379:Mice; D009154:Mutation; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism; D049268:Positron-Emission Tomography / Q000379:methods*; D011849:Radioactive Tracers
chemical_list: C560404:4-fluorobenzoyl-phenylalanyl-alanyl-leucyl-glycyl-glutamyl-alaninamide; D001565:Benzoates; D009842:Oligopeptides; D011849:Radioactive Tracers; C410723:epidermal growth factor receptor VIII; D019788:Fluorodeoxyglucose F18; D066246:ErbB Receptors; C022893:4-fluorobenzoic acid