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

INTRODUCTION

The spontaneous copper-free tandem 1,3-dipolar cycloaddition-retro-Diels-Alder (tandem crDA) reaction between cyclic Arg-Gly-Asp-d-Phe-Orn(N(3)) [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 (111)In-labeled oxanorbornadiene-DTPA analogs. The affinity of the radiolabeled conjugate was determined in a solid-phase alpha(v)beta(3) integrin binding assay. Furthermore, the octanol-water partition coefficient was determined and, finally, the biodistribution of the labeled compounds in mice with subcutaneous alpha(v)beta(3)-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 [(111)In]DTPA-cRGDfX conjugates for alpha(v)beta(3) integrin is 191 nM. Biodistribution studies showed specific (alpha(v)beta(3)-mediated) uptake of [(111)In]DTPA-c(RGDfX) in the tumor and in alpha(v)beta(3)-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.