In-depth evaluation of the cycloaddition–retro-Diels–Alder reaction for in vivo targeting with [111In]-DTPA-RGD conjugates

Laverman, Peter; Meeuwissen, Silvie A.; van Berkel, Sander S.; Oyen, Wim J.G.; van Delft, Floris L.; Rutjes, Floris P.J.T.; Boerman, Otto C.

doi:10.1016/j.nucmedbio.2009.05.001

« Previous Next »Nuclear Medicine and Biology
Volume 36, Issue 7 , Pages 749-757, October 2009

In-depth evaluation of the cycloaddition–retro-Diels–Alder reaction for in vivo targeting with [¹¹¹In]-DTPA-RGD conjugates

Peter Laverman
- Department of Nuclear Medicine, Radboud University Nijmegen Medical Centre, 6500 HB Nijmegen, The Netherlands
- Corresponding author. Department of Nuclear Medicine, Radboud University Nijmegen Medical Centre, PO Box 9101, 6500 HB Nijmegen, The Netherlands. Tel.: +31 24 3615054; fax: +31 24 3618942.
Silvie A. Meeuwissen
- Department of Nuclear Medicine, Radboud University Nijmegen Medical Centre, 6500 HB Nijmegen, The Netherlands
- Institute for Molecules and Materials, Radboud University Nijmegen, 6500 GL Nijmegen, The Netherlands
Sander S. van Berkel
- Institute for Molecules and Materials, Radboud University Nijmegen, 6500 GL Nijmegen, The Netherlands
Wim J.G. Oyen
- Department of Nuclear Medicine, Radboud University Nijmegen Medical Centre, 6500 HB Nijmegen, The Netherlands
Floris L. van Delft
- Institute for Molecules and Materials, Radboud University Nijmegen, 6500 GL Nijmegen, The Netherlands
Floris P.J.T. Rutjes
- Institute for Molecules and Materials, Radboud University Nijmegen, 6500 GL Nijmegen, The Netherlands
Otto C. Boerman
- Department of Nuclear Medicine, Radboud University Nijmegen Medical Centre, 6500 HB Nijmegen, The Netherlands

Received 22 October 2008; received in revised form 1 May 2009; accepted 2 May 2009. published online 10 July 2009.

Abstract

Introduction

The spontaneous copper-free tandem 1,3-dipolar cycloaddition–retro-Diels–Alder (tandem crDA) reaction between cyclic Arg-Gly-Asp-d-Phe-Orn(N₃) [c(RGDfX)] and oxanorbornadiene-DTPA (o-DTPA) or methyloxanorbornadiene-DTPA (mo-DTPA) into two DTPA-c(RGDfX) regioisomers is characterized. Since there is no information on the stability and reaction rate of the tandem crDA reaction in biological media, we set out to characterize these reaction parameters.

Methods

The effects of concentration of the reactants, temperature, pH and reaction environment (serum, blood) on the kinetics of the reaction were determined using ¹¹¹In-labeled oxanorbornadiene-DTPA analogs. The affinity of the radiolabeled conjugate was determined in a solid-phase α_vβ₃ integrin binding assay. Furthermore, the octanol–water partition coefficient was determined and, finally, the biodistribution of the labeled compounds in mice with subcutaneous α_vβ₃-expressing tumors was determined.

Results

Fifty percent conversion was reached after 26 h. Kinetic experiments furthermore established that the reaction rate of the tandem crDA reaction follows temperature- and concentration-dependent second-order kinetics, but is independent of the pH of the medium. Affinity of the two [¹¹¹In]DTPA-cRGDfX conjugates for α_vβ₃ integrin is 191 nM. Biodistribution studies showed specific (α_vβ₃-mediated) uptake of [¹¹¹In]DTPA-c(RGDfX) in the tumor and in α_vβ₃-expressing tissues.

Conclusion

The tandem crDA reaction using methyl-substituted oxanorbornadiene is a versatile method for a single-step ligation that proceeds independently of pH and also proceeds in serum and blood. Currently, we are further looking into enhancement of reaction kinetics and exploitation of tandem crDA in vivo.

Keywords: Oxanorbornadiene, Cycloadditions, Radiolabeling, Cyclic-RGD, Bioconjugation