title: In vitro and initial in vivo evaluation of (68)Ga-labeled transferrin receptor (TfR) binding peptides as potential carriers for enhanced drug transport into TfR expressing cells.
abstract: OBJECTIVE
  The transferrin receptor (TfR) is one of the most attractive targets to overcome the blood-brain barrier (BBB). It has recently been shown that THRPPMWSPVWP binds to the TfR and is subsequently internalized into TfR-expressing cells. Here, we evaluated the ability of THRPPMWSPVWP to become internalized into human TfR-expressing cells via endocytosis to determine its potential to act as a carrier system for the transport of small molecules across the BBB.
  
  
  METHODS
  To validate the underlying concept of a conjugate consisting of a small brain imaging tracer and a large peptidic carrier molecule, a conjugate of the high affinity D2 receptor ligand fallypride and the TfR targeting peptide THRPPMWSPVWP has been synthesized. Furthermore, two derivatives of THRPPMWSPVWP were labeled with (68)Ga in high radiochemical yields (>96%) and a radiochemical purity of 96-98% and evaluated in vitro and in vivo.
  
  
  RESULTS
  The fallypride-THRPPMWSPVWP conjugate still displayed a K(i) of 27 nM. The uptake of the (68)Ga-labeled peptides into TfR-bearing cells was investigated using U87MG and HT-29 cells to assess the capability of the peptide to act as a carrier molecule targeting the TfR. The in vitro binding studies revealed negligible uptake of the tested (68)Ga-labeled conjugates ranging from 0.08% to 0.66% after 60 min incubation at 37°C. Initial in vivo experiments with (68)Ga-DOTA-S-maleimido-THRPPMWSPVWP in two healthy rats showed a mean brain uptake of 0.037% injected dose per gram, confirming the results obtained in vitro.
  
  
  CONCLUSIONS
  These results suggest that the accumulation of the (68)Ga-radiolabeled conjugates of the TfR-binding peptide THRPPMWSPVWP into TFR expressing human cell lines is nonspecific and too low to render this peptide suitable as a possible carrier molecule for a receptor-mediated transport of compounds across the BBB.
authors.affiliation: McConnell Brain Imaging Centre, McGill University, Montreal, Canada. Carmen.Waengler@mail.mcgill.ca
authors.forename: Carmen
authors.initials: C
authors.lastname: Wängler
authors.forename: Dina
authors.initials: D
authors.lastname: Nada
authors.forename: Georg
authors.initials: G
authors.lastname: Höfner
authors.forename: Simone
authors.initials: S
authors.lastname: Maschauer
authors.forename: Björn
authors.initials: B
authors.lastname: Wängler
authors.forename: Stephan
authors.initials: S
authors.lastname: Schneider
authors.forename: Esther
authors.initials: E
authors.lastname: Schirrmacher
authors.forename: Klaus T
authors.initials: KT
authors.lastname: Wanner
authors.forename: Ralf
authors.initials: R
authors.lastname: Schirrmacher
authors.forename: Olaf
authors.initials: O
authors.lastname: Prante
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D001692:Biological Transport; D045744:Cell Line, Tumor; D004337:Drug Carriers / Q000737:chemistry*; D016503:Drug Delivery Systems / Q000379:methods*; D005260:Female; D005462:Fluorine Radioisotopes; D005710:Gallium Radioisotopes; D006801:Humans; D007553:Isotope Labeling; D008024:Ligands; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley; D017448:Receptors, Dopamine D2 / Q000378:metabolism; D011990:Receptors, Transferrin / Q000378:metabolism*; D014018:Tissue Distribution; D014168:Transferrin / Q000378:metabolism
chemical_list: D004337:Drug Carriers; D005462:Fluorine Radioisotopes; D005710:Gallium Radioisotopes; D008024:Ligands; D010455:Peptides; D017448:Receptors, Dopamine D2; D011990:Receptors, Transferrin; D014168:Transferrin
references.citation: Blood Cells Mol Dis. 2007 Jul-Aug;39(1):82-91
references.pmid: 17428703
references.citation: Neuroimage. 2009 Jun;46(2):447-58
references.pmid: 19233294
references.citation: Neuroimage. 2009 Apr 15;45(3):891-902
references.pmid: 19136064
references.citation: Proc Natl Acad Sci U S A. 1995 Mar 28;92(7):2820-4
references.pmid: 7708731
references.citation: Biomaterials. 2007 Nov;28(33):4978-90
references.pmid: 17716725
references.citation: Pharmacol Rev. 2002 Dec;54(4):561-87
references.pmid: 12429868
references.citation: Methods Enzymol. 1997;289:44-67
references.pmid: 9353717
references.citation: Eur J Pharm Biopharm. 2009 Feb;71(2):251-6
references.pmid: 18805484
references.citation: Bioconjug Chem. 2009 Feb;20(2):317-21
references.pmid: 19132825
references.citation: Bioconjug Chem. 2005 Nov-Dec;16(6):1503-11
references.pmid: 16287248
references.citation: Bioconjug Chem. 2008 Jul;19(7):1327-38
references.pmid: 18547095
references.citation: Pharm Res. 2006 Apr;23(4):790-7
references.pmid: 16550469
references.citation: J Drug Target. 2006 Jan;14(1):45-53
references.pmid: 16603451
references.citation: Bioconjug Chem. 2007 Jan-Feb;18(1):254-62
references.pmid: 17226980
references.citation: Angew Chem Int Ed Engl. 2006 Sep 11;45(36):6047-50
references.pmid: 16888726
references.citation: Keio J Med. 2008 Sep;57(3):125-31
references.pmid: 18854664
references.citation: Mov Disord. 2003 Oct;18 Suppl 7:S39-42
references.pmid: 14531045
references.citation: Eur J Biochem. 2001 Apr;268(7):2004-12
references.pmid: 11277922
references.citation: Pharm Res. 2007 Sep;24(9):1759-71
references.pmid: 17619996
references.citation: Bioconjug Chem. 2007 Nov-Dec;18(6):2085-9
references.pmid: 18030993
references.citation: Cereb Cortex. 2009 Nov;19(11):2499-507
references.pmid: 19321655
references.citation: Neurology. 2009 Feb 10;72(6):528-34
references.pmid: 19204262
references.citation: Neuropsychopharmacology. 2008 Sep;33(10):2435-41
references.pmid: 18288096
references.citation: J Control Release. 2008 Jun 4;128(2):120-7
references.pmid: 18436327
references.citation: Bioorg Med Chem. 2008 Mar 1;16(5):2606-16
references.pmid: 18065226
references.citation: Am J Psychiatry. 2008 Aug;165(8):988-95
references.pmid: 18381901
references.citation: Epilepsia. 2006 Aug;47(8):1392-6
references.pmid: 16922886
references.citation: Eur J Pharmacol. 1996 May 6;303(1-2):123-8
references.pmid: 8804920
references.citation: Eur J Nucl Med Mol Imaging. 2004 Aug;31(8):1128-35
references.pmid: 15042325
references.citation: Eur J Nucl Med Mol Imaging. 2009 Mar;36(3):454-62
references.pmid: 19037637
references.citation: Cancer Lett. 2009 Feb 8;274(1):33-9
references.pmid: 18838215
references.citation: J Neural Transm (Vienna). 2005 Dec;112(12):1635-45
references.pmid: 16284908
references.citation: J Neurochem. 2001 Oct;79(1):119-29
references.pmid: 11595764
references.citation: Annu Rev Pharmacol Toxicol. 2007;47:323-55
references.pmid: 16961459