PEGylation of 99m Tc-labeled bombesin analogues improves their pharmacokinetic properties Simone Däpp a , Elisa García Garayoa a , Veronique Maes b , Luc Brans b , Dirk A. Tourwé b , Cristina Müller a , Roger Schibli a,c, ⁎ a Paul Scherrer Institute, Center for Radiopharmaceutical Sciences ETH-PSI-USZ, CH-5232 Villigen-PSI, Switzerland b Department of Organic Chemistry, Vrije Universiteit Brussel, 1050 Brussels, Belgium c Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 Zurich, Switzerland Received 14 December 2010; received in revised form 17 February 2011; accepted 27 February 2011 Abstract Introduction: Radiolabeled bombesin (BN) conjugates are promising radiotracers for imaging and therapy of breast and prostate tumors in which BN 2 /gastrin-releasing peptide (GRP) receptors are overexpressed. However, the low in vivo stability of BN conjugates may limit their clinical application. In an attempt to improve their pharmacokinetics and counteract their rapid enzymatic degradation, we prepared a series of polyethylene glycol (PEG)-ylated BN(7-14) analogues for radiolabeling with 99m Tc(CO) 3 and evaluated them in vitro and in vivo. Methods: Derivatization of a stabilized (N α His)Ac-BN(7-14)[Cha 13 ,Nle 14 ] analogue with linear PEG molecules of various sizes [5 kDa (PEG 5 ), 10 kDa (PEG 10 ) and 20 kDa (PEG 20 )] was performed by PEGylation of the ɛ-amino group of a β 3 hLys-βAla-βAla spacer between the stabilized BN sequence and the (N α His)Ac chelator. The analogues were then radiolabeled by employing the 99m Tc-tricarbonyl technique. Binding affinity and internalization/externalization studies were performed in vitro in human prostate carcinoma PC-3 cells. Stability was investigated in vitro in human plasma and in vivo in Balb/c mice. Finally, single photon emission computed tomography (SPECT)/X-ray computed tomography studies were performed in nude mice bearing PC-3 tumor xenografts. Results: PEGylation did not affect the binding affinity of BN analogues, as the binding affinity for BN 2 /GRP receptors remained high (K d b0.9 nM). However, in vitro binding kinetics of the PEGylated analogues were slower. Steady-state condition was reached after 4 h, and the total cell binding was 10 times lower than that for the non-PEGylated counterpart. Besides, PEGylation improved the stability of BN conjugates in vitro and in vivo. The BN derivative conjugated with a PEG 5 molecule showed the best pharmacokinetics in vivo, i.e., faster blood clearance and preferential renal excretion. The tumor uptake of the 99m Tc-PEG 5 -Lys-BN conjugate was slightly higher compared to that of the non-PEGylated analogue (3.91%±0.44% vs. 2.80%±0.28% injected dose per gram 1 h postinjection, p.i.). Tumor retention was also increased, resulting in a threefold higher amount of radioactivity in the tumor at 24 h p.i. Furthermore, decreased hepatobiliary excretion and increased tumor-to-nontarget ratios (tumor-to-blood: 17.1 vs. 2.1; tumor-to-kidney: 1.1 vs. 0.4; tumor-to-liver: 5.8 vs. 1.0, 24 h p.i.) were observed and further confirmed via small-animal SPECT images 1 h p.i. Conclusion: PEGylation proved to be an effective strategy to enhance the tumor-targeting potential of 99m Tc-labeled BN-based radiopharmaceuticals and probably other radiolabeled peptides. © 2011 Elsevier Inc. All rights reserved. Keywords: Gastrin-releasing peptide receptor; Bombesin; Technetium-99m; SPECT/CT imaging; PEGylation; Prostate cancer 1. Introduction The fact that certain tumor types overexpress receptors for peptide hormones provides the basis for successful use of radiolabeled peptide analogues as tumor tracers in nuclear medicine. Peptide-based radiopharmaceuticals exhibit a great potential for diagnostic and therapeutic application in oncology because of their fast clearance, rapid tissue Available online at www.sciencedirect.com Nuclear Medicine and Biology 38 (2011) 997 – 1009 www.elsevier.com/locate/nucmedbio ⁎ Corresponding author. Paul Scherrer Institute, Center for Radiophar- maceutical Sciences ETH-PSI-USZ, CH-5232 Villigen-PSI, Switzerland. Tel.: +41 56 310 28 37; fax: +41 56 310 28 49. E-mail address: roger.schibli@psi.ch (R. Schibli). 0969-8051/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.nucmedbio.2011.02.014