Triazole-based radiopeptidomimetics : novel neurotensin (8-13) analogues with increased metabolic stability

Neurotensin (NT) is a regulatory peptide with a nanomolar affinity towards NT receptors, which are overexpressed by different types clinically relevant tumours (e.g. Ewing’ sarcoma, breast, colon and exocrine pancreatic cancer). Regulatory peptides have been shown to be suitable vectors for the specific delivery of radioactivity to tumours for diagnostic and therapeutic applications in nuclear medicine. Therefore, the binding sequence of Neurotensin, NT (8-13), is a promising vector for the development of peptidic radiotracers for tumour imaging and therapy. A potential drawback of this peptide vector is its instability in vivo as the result of rapid degradation by proteases. It has been shown that stabilization of a peptide against proteases can lead to an increased tumour uptake. However, classical peptide backbone modifications (e.g., N-methylation or reduction of the amide bond) were only partially successful in providing stabilized NT-analogues with improved tumour-targeting properties. It has been suggested that 1,4-disubstituted 1,2,3-triazoles might represent suitable trans-amide bond bioisosters, which are resistant to proteases. To study the effect of 1,4-disubstituted 1,2,3-triazoles on NT (8-13) as amide bond mimics, we performed a ‘triazole scan’ of the amino acid sequence by which every backbone amide bond of the NT (8-13) sequence is substituted with a 1,4-disubstituted 1,2,3-triazole.
The focus of the first part of this thesis was on the investigation of the pharmacological influence of a spacer on a radiometallated DOTA-functionalized NT (8-13) sequence. A NT (8-13)-based peptide conjugate without a spacer to separate the chelator from the tumour-targeting peptide was compared side-by-side with two NT (8-13) peptide conjugates, each having a different spacer, one hydrophilic (tetraethylenglycol, PEG4) and one lipophilic (6-aminohexanoic acid, Ahx). The compound with the PEG4-spacer exhibited the best properties in vitro and was thus selected as reference compound and starting point for future peptide conjugates.
A ‘triazole scan’ of NT (8-13) yielded several triazole backbone-modified NT (8-13) analogues with interesting properties. It was found that a triazole modification was only tolerated between the N-terminal PEG4 spacer and Arg9 and in the position between Arg8 and Arg9. Substitution of these bonds yielded two single-triazole containing compounds as well as one bis-triazole analogue with nanomolar affinities towards NTR1 and moderately improved metabolic stabilities (first generation of triazole-containing NT (8-13) peptidomimetics).
Further enhancement of the metabolic stability of the peptidomimetics of the first generation was achieved by the substitution of Ile12 of NT (8-13) with a Tle12. The triazole insertion was only introduced at the previously identified positions which tolerate the modification. The obtained compounds (second generation of triazole-containing NT (8-13) analogues) were evaluated in vitro and compared side-by-side to the reference compound. Our investigations revealed that the Ile12 to Tle12 residue switch led to a substantial improvement of the metabolic stability of the compounds, however a significant loss of receptor affinity was observed.
The compounds with the most promising properties in vitro (retained nanomolar NTR1 affinity and improved metabolic stability) were selected for biodistribution studies and compared side-by-side with the corresponding reference compounds of the first and second generation. The NT (8-13) analogues with a 1,4-disubstituted 1,2,3-triazole between Arg8 and Arg9 exhibited a 2-fold tumour uptake, in comparison to the reference compounds. Compared to literature data we can report exceptionally good tumour to background ratios in general, especially for the tumour to kidney ratio. A high tumour to background ratio is beneficial for the potential application of peptidic radiopharmaceuticals as imaging or therapeutic agents. In general, no or little correlation between the in vitro behaviour (metabolic stability or receptor binding affinity) and the uptake of the conjugates in the tumour in vivo could be observed, which demonstrates the necessity of preclinical evaluations of novel radiotracers.
In summary, we report the first ‘triazole scan’ of the binding sequence of NT (8-13) and the synthesis and biological evaluation of novel radiolabelled triazole-peptidomimetics. The substitution of amide bonds with 1,4-disubstitued 1,2,3-triazoles provided NT (8-13) peptidomimetics with improved tumour-targeting properties. The NT (8-13) analogues described in this thesis represent interesting candidates for the development of novel tumour-targeting probes with applications in nuclear medicine.