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Comparison of in vitro and in vivo properties of 99mTc-RGD peptides for targeting tumour angiogenesis labelled using different novel technetium-cores
Decristoforo, C.; Rey, A.; Künstler, U.; Pietzsch, H.-J.; Galamba, J.; Santos, I.; Smith, J.; Faintuch, B.; Hernandez-Gonzales, I.; Rupprich, M.; Alberto, R.; Haubner, R.;
1. Introduction
Integrins are cell surface transmembrane glycoproteins that are found as αβ heterodimers. The αvβ3 integrin is known to be over-expressed in many tumour types and sprouting blood vessels in the tumour, but expressed at lower levels in normal tissues. Peptides containing the Arg-Gly-Asp (RGD) sequence, bind with high affinity to αvβ3 and have found increasing interest for radiolabelling to target v3 receptors and for imaging angiogenesis. Recently, it was demonstrated using [18F]Galacto-RGD and PET that monitoring of αvβ3 is possible in animal models as well as in humans (1). Recent advances in organometallic chemistry enable 99mTc-labelling of biomolecules with high specific activity and high stability. The aim of this study was to use a cyclic RGD derivative, c(RGDyK), containing a Lys residue for derivatisation with different moieties for labelling with 99mTc using novel technetium-cores, such as HYNIC, Tc(CO)3, and Tc(4+1) approaches and to compare chemical and biological properties in vitro and in vivo.

2. Materials & Methods:
c(RGDyK) (RGD) was derivatised with HYNIC (1), Cys (2), 3, 5–Me2 –pyrazol-1-yl-ethyldiamine –RGD (PZ1, 3) and an isonitrile moiety (L3, 4) at the terminal lysine group of c(RGDyK) using standard fmoc chemistry (examples see Fig1). 99mTc-labelling was performed using both Tricine as well as EDDA as coligands for HYNIC-RGD, [99mTc(CO)3(H2O)3]+ was used for labelling Cys-, PZ1 and HYNIC-RGD and Aminotrithiols as coligangds for L3-RGD at high specific activities. Radiolabelled peptides were characterised concerning lipophilicity (octanol/PBS coefficient, RP-HPLC), protein binding using SEC, stability in buffer, serum and tissue homogenates and tested for stability in PBS, Cystein and Histidine solutions, tissue homogenates and serum up to 6 h. Integrin receptor activity was determined in internalisation assays using v3 receptor positive M21 and negative M21-L melanoma cells. Biodistribution and in vivo tumour uptake was evaluated in nude mice carrying M21 and M21-L tumours for selected compounds.

3. Results
All peptides could be labelled at high specific activities and revealed high stability in various solvents. Both lipophilicity as determined by HPLC retention times and log P values (-0.92 to -3.57) and binding to serum proteins (2-32%) varied considerably All 99mTc-labelled peptides showed specific uptake in αvβ3 positive cells with comparable values for all compounds around 1% of total/mg protein except for 99mTc-L1 and L3-RGD with lower values of less than 0.5%. In a mouse tumour model great variations in pharmacokinetic behaviour was found ranging from predominant renal excretion up to predominant hepatobiliar eliminiation. Highest specific values of tumour uptake were found for 99mTc-EDDA/HYNIC-RGD and 99mTc(CO)3-PZ1-RGD (see Fig. 2) with about 2.5% uptake in M21 receptor positive tumours and <1% in receptor negative M21-L tumours with lower intestinal uptake of 99mTc-EDDA/HYNIC-RGD.

4. Discussion/Conclusion
Our study provides for the first time data on chemical and biological characterisation of the same peptide labelled with 99mTc using different technetium-cores. It shows the decisive influence of labelling approaches on imaging properties of 99mTc-labelled peptides and the necessity to use different in vitro assays to predict targeting properties in vivo.

5. References:
(1) Haubner R, Weber WA, Beer AJ, Vabuliene E, Reim D, Sarbia M, Becker KF, Goebel M, Hein R, Wester HJ, Kessler H, Schwaiger M. [2005] PLoS Medicine. 2(3):e70.

6. Acknowledgements:
This work is part of the CRP “Development of Tc-99m based small bio-molecules using novel 99mTc cores” of the IAEA
  • Lecture (Conference)
    European Symposium on Radiopharmacy and Radiopharmaceuticals - ESRR 06, 30.03.-02.04.2006, Lucca, Italy
  • Abstract in refereed journal
    Quarterly Journal of Nuclear Medicine and Molecular Imaging 50(2006)Suppl. 1, 19

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Publ.-Id: 8389