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.
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.
Wängler
Carmen
C
McConnell Brain Imaging Centre, McGill University, Montreal, Canada. Carmen.Waengler@mail.mcgill.ca
Nada
Dina
D
Höfner
Georg
G
Maschauer
Simone
S
Wängler
Björn
B
Schneider
Stephan
S
Schirrmacher
Esther
E
Wanner
Klaus T
KT
Schirrmacher
Ralf
R
Prante
Olaf
O
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
D004337:Drug Carriers; D005462:Fluorine Radioisotopes; D005710:Gallium Radioisotopes; D008024:Ligands; D010455:Peptides; D017448:Receptors, Dopamine D2; D011990:Receptors, Transferrin; D014168:Transferrin
Blood Cells Mol Dis. 2007 Jul-Aug;39(1):82-91
17428703
Neuroimage. 2009 Jun;46(2):447-58
19233294
Neuroimage. 2009 Apr 15;45(3):891-902
19136064
Proc Natl Acad Sci U S A. 1995 Mar 28;92(7):2820-4
7708731
Biomaterials. 2007 Nov;28(33):4978-90
17716725
Pharmacol Rev. 2002 Dec;54(4):561-87
12429868
Methods Enzymol. 1997;289:44-67
9353717
Eur J Pharm Biopharm. 2009 Feb;71(2):251-6
18805484
Bioconjug Chem. 2009 Feb;20(2):317-21
19132825
Bioconjug Chem. 2005 Nov-Dec;16(6):1503-11
16287248
Bioconjug Chem. 2008 Jul;19(7):1327-38
18547095
Pharm Res. 2006 Apr;23(4):790-7
16550469
J Drug Target. 2006 Jan;14(1):45-53
16603451
Bioconjug Chem. 2007 Jan-Feb;18(1):254-62
17226980
Angew Chem Int Ed Engl. 2006 Sep 11;45(36):6047-50
16888726
Keio J Med. 2008 Sep;57(3):125-31
18854664
Mov Disord. 2003 Oct;18 Suppl 7:S39-42
14531045
Eur J Biochem. 2001 Apr;268(7):2004-12
11277922
Pharm Res. 2007 Sep;24(9):1759-71
17619996
Bioconjug Chem. 2007 Nov-Dec;18(6):2085-9
18030993
Cereb Cortex. 2009 Nov;19(11):2499-507
19321655
Neurology. 2009 Feb 10;72(6):528-34
19204262
Neuropsychopharmacology. 2008 Sep;33(10):2435-41
18288096
J Control Release. 2008 Jun 4;128(2):120-7
18436327
Bioorg Med Chem. 2008 Mar 1;16(5):2606-16
18065226
Am J Psychiatry. 2008 Aug;165(8):988-95
18381901
Epilepsia. 2006 Aug;47(8):1392-6
16922886
Eur J Pharmacol. 1996 May 6;303(1-2):123-8
8804920
Eur J Nucl Med Mol Imaging. 2004 Aug;31(8):1128-35
15042325
Eur J Nucl Med Mol Imaging. 2009 Mar;36(3):454-62
19037637
Cancer Lett. 2009 Feb 8;274(1):33-9
18838215
J Neural Transm (Vienna). 2005 Dec;112(12):1635-45
16284908
J Neurochem. 2001 Oct;79(1):119-29
11595764
Annu Rev Pharmacol Toxicol. 2007;47:323-55
16961459