Evaluation of ^¹¹¹In-Labelled Exendin-4 Derivatives Containing Different Meprin β-Specific Cleavable Linkers

<div><p>Background</p><p>Cleavable linkers, which are specifically cleaved by defined conditions or enzymes, are powerful tools that can be used for various purposes. Amongst other things, they have been successfully used to deliver toxic payloads as prodrugs into target tissues. In this work novel linker sequences targeting meprin β, a metalloprotease expressed in the kidney brush-border membrane, were designed and included in the sequence of three radiolabelled exendin-4 derivatives. As radiolabelled exendin-4 derivatives strongly accumulate in the kidneys, we hypothesised that specific cleavage of the radiolabelled moiety at the kidney brush-border membrane would allow easier excretion of the activity into the urine and therefore improve the pharmacological properties of the peptide.</p><p>Results</p><p>The insertion of a cleavable linker did not negatively influence the <i>in vitro</i> properties of the peptides. They showed a good affinity to the GLP-1 receptor expressed in CHL cells, a high internalisation and sufficiently high stability in fresh human blood plasma. <i>In vitro</i> digestion with recombinant meprin β rapidly metabolised the corresponding linker sequences. After 60 min the majority of the corresponding peptides were digested and at the same time the anticipated fragments were formed. The peptides were also quickly metabolised in CD1 nu/nu mouse kidney homogenates. Immunofluorescence staining of meprin β in kidney sections confirmed the expression of the protease in the kidney brush-border membrane. Biodistribution in GLP-1 receptor positive tumour-xenograft bearing mice revealed high specific uptake of the ¹¹¹In-labelled tracers in receptor positive tissue. Accumulation in the kidneys, however, was still high and comparable to the lead compound ¹¹¹In-Ex4NOD40.</p><p>Conclusion</p><p>In conclusion, we show that the concept of cleavable linkers specific for meprin β is feasible, as the peptides are rapidly cleaved by the enzyme while retaining their biological properties.</p></div

Cleavage sites of In-labelled peptides digested with meprin α and β.

<p>Cleavage sites for meprin α are illustrated with ↑, for meprin β with ↓. The linkers are highlighted in bold. In-labelled Ex4NOD40 was used as control.</p><p>Cleavage sites of In-labelled peptides digested with meprin α and β.</p

The most common amino acids around the cleavage sites of both meprin α and β.

<p>The size of the one letter code of the amino acid represents the frequency of that amino acid in that particular position. The figure was generated using icelogo, based on peptide cleavage assays described previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123443#pone.0123443.ref013" target="_blank">13</a>]. Peptide sequences were aligned on the scissile bond between P1 and P1′ indicated by a black line. Statistically significant amino acid residue occurrences present (P<0.05) were plotted. Those amino acids that were completely absent are shown below in pink.</p

Immunofluorescence picture of murine kidney sections.

<p>(A) Anti-Meprin β antibody was used to detect the protease, visualised by fluorescence detection at 488 nm. (B) For the control goat igG was used.</p

<i>In vitro</i> assessment of the peptides containing cleavable linkers in CHL cells expressing GLP-1R.

<p>(A) IC₅₀ values of ^natIn labelled peptides. 0.1–0.6 pmol ¹¹¹In-Ex4NOD40 was used as tracer. (B) Internalisation kinetics of the ¹¹¹In-labelled probes. 0.2–1 pmol of the respective ¹¹¹In-labelled peptide was used as tracer. Non-specific binding was determined by incubation with 1 μM of ^natIn labelled peptide.</p

Plasma stability of ¹¹¹In-labelled PSI-CLNOD1, PSI-CLNOD2 and PSI-CLNOD3 in fresh human blood plasma.

<p>Plasma stability of ¹¹¹In-labelled PSI-CLNOD1, PSI-CLNOD2 and PSI-CLNOD3 in fresh human blood plasma.</p

Biodistribution of ¹¹¹In-labeled peptides in CD1 nu/nu mice with CHL-GLP1R positive tumour-xenografts.

<p>Values are mean percentages injected dose per gram tissue (<i>n</i> = 4. Error bars SD). Blocking was performed by pre-injection of 100 μg excess of unmodified exendin-4. Mice were sacrificed 4 h after injection. The significance was tested by a two-was ANOVA test corrected for multiple comparisons by Tukey’s honest significance test (**<i>p</i><0.01; ***p<0.001; ****p>0.0001).</p

<i>Ex vivo</i> metabolism of the corresponding peptides in CD1 nu/nu kidney homogenates.

<p><i>Ex vivo</i> metabolism of the corresponding peptides in CD1 nu/nu kidney homogenates.</p

Digestion of In-labelled probes with recombinant meprin β.

<p>A, C, E, and G show the metabolism of the corresponding ¹¹¹In-labelled peptides. Curves B, D, F and H show the degradation of the corresponding ^natIn-labelled peptides and the formation of different fragments over time.</p

Graphical representation of exendin-4 and its derivatives.

<p>The linkers are highlighted in different colours.</p