Combination of the amide-to-triazole substitution strategy with alternative structural modifications for the metabolic stabilization of tumor-targeting, radiolabeled peptides.

Radiolabeled peptides play a key role in nuclear medicine to selectively deliver radionuclides to malignancies for diagnosis (imaging) and therapy. Yet, their efficiency is often compromised by low metabolic stability. The use of 1,4-disubstituted 1,2,3-triazoles (1,4-Tzs) as stable amide bond bioisosteres can increase the half-life of peptides in vivo while maintaining their biological properties. Previously, the amide-to-triazole substitution strategy was used for the stabilization of the pansomatostatin radioligand [111In]In-AT2S, resulting in the mono-triazolo-peptidomimetic [111In]In-XG1, a radiotracer with moderately enhanced stability in vivo and retained ability to bind multiple somatostatin receptor (SSTR) subtypes. However, inclusion of additional 1,4-Tz led to a loss of affinity towards SST2R, the receptor overexpressed by most SSTR-positive cancers. To enhance further the stability of [111In]In-XG1, alternative modifications at the enzymatically labile position Thr10-Phe11 were employed. Three novel 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-peptide conjugates were synthesized with a 1,4-Tz (Asn5-Ψ[Tz]-Phe6) and either a ß-amino acid (ß-Phe11), reduced amide bond (Thr10-Ψ[NH]-Phe11), or N-methylated amino acid (N-Me-Phe11). Two of the new peptidomimetics were more stable in blood plasma in vitro than [111In]In-XG1. Yet none of them retained high affinity towards SST2R. We demonstrate for the first time the combination of the amide-to-triazole substitution strategy with alternative stabilization methods to improve the metabolic stability of tumor-targeting peptides.

Automated solid-phase synthesis of metabolically stabilized triazolo-peptidomimetics.

The use of 1,4-disubstituted 1,2,3-triazoles as trans-amide bond surrogates has become an important tool for the synthesis of metabolically stabilized peptidomimetics. These heterocyclic bioisosters are generally incorporated into the peptide backbone by applying a diazo-transfer reaction followed by CuAAC (click chemistry) with an α-amino alkyne. Even though the manual synthesis of backbone-modified triazolo-peptidomimetics has been reported by us and others, no procedure has yet been described for an automated synthesis using peptide synthesizers. In order to efficiently adapt these reactions to an automated setup, different conditions were explored, putting special emphasis on the required long-term stability of both the diazo-transfer reagent and the Cu(I) catalyst in solution. ISA·HCl is the reagent of choice to accomplish the diazo-transfer reaction; however, it was found instable in DMF, the most commonly used solvent for SPPS. Thus, an aqueous solution of ISA·HCl was used to prevent its degradation over time, and the composition in the final diazo-transfer reaction was adjusted to preserve suitable swelling conditions of the resins applied. The CuAAC reaction was performed without difficulties using [Cu (CH3 CN)4 ]PF6 as a catalyst and TBTA as a stabilizer to prevent oxidation to Cu(II). The optimized automated two-step procedure was applied to the synthesis of structurally diverse triazolo-peptidomimetics to demonstrate the versatility of the developed methodology. Under the optimized conditions, five triazolo-peptidomimetics (8-5 amino acid residues) were synthesized efficiently using two different resins. Analysis of the crude products by HPLC-MS revealed moderate to good purities of the desired triazolo-peptidomimetics (70-85%). The synthesis time ranged between 9 and 12.5 h.

Straightforward Synthesis of DFO* - An Octadentate Chelator for Zirconium-89.

DFO* is an octadentate chelator able to form highly stable chelates with Zirconium-89 (89 Zr) for nuclear medicinal applications in Positron Emission Tomography (PET).[1,2] The synthesis of DFO* and its scale-up remains challenging by reported synthetic protocols. For this reason, we set out to develop a de novo synthesis of a hydroxamate-containing building block suitable for the coupling to the commercially available DFO (desferrioxamine B, mesylate salt) yielding, after deprotection, the desired chelator DFO* in a more efficient procedure. Highlights of the new synthesis of DFO* reported herein are less synthetic steps and the isolation of the desired product DFO* by using solid phase extraction (SPE), thus avoiding tedious HPLC purification. DFO* is obtained in excellent purity (92-98 %) and an overall yield of approximately 29 %. In addition, the isolated trifluoroacetic acid (TFA)-salt of DFO* displays an improved solubility in organic solvents (DMSO, DMF, methanol), which will facilitate its use for the preparation of structurally diverse derivatives suitable for bioconjugation chemistry and the development of 89 Zr-labeled radiotracers.

Amide-to-Triazole Switch in Somatostatin-14-Based Radioligands: Impact on Receptor Affinity and In Vivo Stability.

The use of metabolically stabilized, radiolabeled somatostatin (SST) analogs ([68Ga]Ga/[177Lu]Lu-DOTA-TATE/TOC/NOC) is well established in nuclear medicine. Despite the pivotal role of these radioligands in the diagnosis and therapy of neuroendocrine tumors (NETs), their inability to interact with all five somatostatin receptors (SST1-5R) limits their clinical potential. [111In]In-AT2S is a radiolabeled DOTA-conjugate derived from the parent peptide SST-14 that exhibits high binding affinity to all SSTR subtypes, but its poor metabolic stability represents a serious disadvantage for clinical use. In order to address this issue, we have replaced strategic trans-amide bonds of [111In]In-AT2S with metabolically stable 1,4-disubstituted 1,2,3-triazole bioisosteres. From the five cyclic triazolo-peptidomimetics investigated, only [111In]In-XG1 combined a preserved nanomolar affinity for the SST1,2,3,5R subtypes in vitro and an improved stability in vivo (up to 17% of intact peptide 5 min postinjection (pi) versus 6% for [111In]In-AT2S). The involvement of neprilysin (NEP) in the metabolism of [111In]In-XG1 was confirmed by coadministration of Entresto®, a registered antihypertensive drug, in vivo releasing the selective and potent NEP-inhibitor sacubitrilat. A pilot SPECT/CT imaging study conducted in mice bearing hSST2R-positive xenografts failed to visualize the xenografts due to the pronounced kidney uptake (>200% injected activity (IA)/g at 4 h pi), likely the result of the formation of cationic metabolites. To corroborate the imaging data, the tumors and the kidneys were excised and analyzed with a γ-counter. Even if receptor-specific tumor uptake for [111In]In-XG1 could be confirmed (1.61% IA/g), further optimization is required to improve its pharmacokinetic properties for radiotracer development.

Mini-review: Targeted radiopharmaceuticals incorporating reversible, low molecular weight albumin binders.

The combination of low molecular weight, reversible human serum albumin (HSA) binders with targeted radiopharmaceuticals in dual-targeted radioconjugates holds great promise, in particular for endoradiotherapy. Attachment of HSA-binders to radiopharmaceuticals extends their blood circulation time and results in an enhanced tumour uptake as well as often in an improved pharmacokinetic profile. In this mini-review, an overview of currently pursued approaches of this novel strategy is provided.