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. 2020 Aug 25;25(17):3854.
doi: 10.3390/molecules25173854.

Evaluation of Met-Val-Lys as a Renal Brush Border Enzyme-Cleavable Linker to Reduce Kidney Uptake of 68Ga-Labeled DOTA-Conjugated Peptides and Peptidomimetics

Shreya Bendre  1 , Zhengxing Zhang  1 , Hsiou-Ting Kuo  1 , Julie Rousseau  1 , Chengcheng Zhang  1 , Helen Merkens  1 , Áron Roxin  1 , François Bénard  1   2   3 , Kuo-Shyan Lin  1   2   3
Affiliations

Evaluation of Met-Val-Lys as a Renal Brush Border Enzyme-Cleavable Linker to Reduce Kidney Uptake of 68Ga-Labeled DOTA-Conjugated Peptides and Peptidomimetics

Shreya Bendre et al. Molecules. .

Abstract

High kidney uptake is a common feature of peptide-based radiopharmaceuticals, leading to reduced detection sensitivity for lesions adjacent to kidneys and lower maximum tolerated therapeutic dose. In this study, we evaluated if the Met-Val-Lys (MVK) linker could be used to lower kidney uptake of 68Ga-labeled DOTA-conjugated peptides and peptidomimetics. A model compound, [68Ga]Ga-DOTA-AmBz-MVK(Ac)-OH (AmBz: aminomethylbenzoyl), and its derivative, [68Ga]Ga-DOTA-AmBz-MVK(HTK01166)-OH, coupled with the PSMA (prostate-specific membrane antigen)-targeting motif of the previously reported HTK01166 were synthesized and evaluated to determine if they could be recognized and cleaved by the renal brush border enzymes. Additionally, positron emission tomography (PET) imaging, ex vivo biodistribution and in vivo stability studies were conducted in mice to evaluate their pharmacokinetics. [68Ga]Ga-DOTA-AmBz-MVK(Ac)-OH was effectively cleaved specifically by neutral endopeptidase (NEP) of renal brush border enzymes at the Met-Val amide bond, and the radio-metabolite [68Ga]Ga-DOTA-AmBz-Met-OH was rapidly excreted via the renal pathway with minimal kidney retention. [68Ga]Ga-DOTA-AmBz-MVK(HTK01166)-OH retained its PSMA-targeting capability and was also cleaved by NEP, although less effectively when compared to [68Ga]Ga-DOTA-AmBz-MVK(Ac)-OH. The kidney uptake of [68Ga]Ga-DOTA-AmBz-MVK(HTK01166)-OH was 30% less compared to that of [68Ga]Ga-HTK01166. Our data demonstrated that derivatives of [68Ga]Ga-DOTA-AmBz-MVK-OH can be cleaved specifically by NEP, and therefore, MVK can be a promising cleavable linker for use to reduce kidney uptake of radiolabeled DOTA-conjugated peptides and peptidomimetics.

Keywords: cancer imaging and therapy; cleavable linkers; kidney uptake; neutral endopeptidase (NEP); prostate-specific membrane antigen (PSMA); radiopharmaceuticals; renal brush border enzymes.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
DOTA-AmBz-MVK(Targeting vector)-OH inspired by the reported design of NOTA-MVK(Targeting vector)-OH. The amide bond between Met-Val (pointed by an arrow) is recognized and cleaved by NEP.
Figure 2
Figure 2
Chemical structures of (A) HTK01166, (B) DOTA-AmBz-MVK(Ac)-OH, (C) DOTA-AmBz-MVK(HTK01166)-OH, (D) DOTA-AmBz-Met-OH, and (E) DOTA-AmBz-M(O)VK(HTK01166)-OH.
Scheme 1
Scheme 1
Synthesis of Fmoc-l-Lys(pentynoyl)-OtBu (2).
Scheme 2
Scheme 2
Synthesis of (A) DOTA-AmBz-MVK(Ac)-OH and (B) DOTA-AmBz-Met-OH. ● = resin.
Scheme 3
Scheme 3
Synthesis of DOTA-AmBz-MVK(HTK01166)-OH and DOTA-AmBz-M(O)VK(HTK01166)-OH. ● = resin.
Figure 3
Figure 3
Radio-HPLC chromatograms of in vitro enzyme assay samples of [68Ga]Ga-DOTA-AmBz-MVK(Ac)-OH (A) without and (B) with the presence of phosphoramidon. HPLC conditions were 82/18 A/B at a flow rate of 2 mL/min; A: H2O containing 0.1% TFA; B: CH3CN containing 0.1% TFA; HPLC column: Luna C18, 5 µm particle size, 100 Å pore size, 250 × 4.6 mm.
Figure 4
Figure 4
Radio-HPLC chromatograms of in vitro enzyme assay samples of [68Ga]Ga-DOTA-AmBz-MVK(HTK01166)-OH (A) without and (B) with the presence of phosphoramidon. HPLC conditions were 72/28 A/B at a flow rate of 2 mL/min; A: H2O containing 0.1% TFA; B: CH3CN containing 0.1% TFA; HPLC column: Luna C18, 5 µm particle size, 100 Å pore size, 250 × 4.6 mm.
Figure 5
Figure 5
Radio-HPLC chromatograms of in vitro enzyme assay samples of [68Ga]Ga-DOTA-AmBz-M(O)VK(HTK01166)-OH (A) without and (B) with the presence of phosphoramidon. HPLC conditions were 76/24 A/B at a flow rate of 2 mL/min; A: H2O containing 0.1% TFA; B: CH3CN containing 0.1% TFA; HPLC column: Luna C18, 5 µm particle size, 100 Å pore size, 250 × 4.6 mm.
Figure 6
Figure 6
A representative maximum-intensity-projection PET image of [68Ga]Ga-DOTA-AmBz-MVK(Ac)-OH showing its rapid excretion predominantly via the renal pathway with minimal kidney retention (<2.5 %ID/g). The range of color bar is 0–5 %ID/g. k: kidney; b: bladder.
Figure 7
Figure 7
Representative maximum-intensity-projection PET images of [68Ga]Ga-DOTA-AmBz-MVK(HTK01166)-OH acquired at 1 h p.i. from LNCaP tumor-bearing mice (A) without and (B) with the co-injection of 2-PMPA (0.2 mg). The range of color bar is 0–5 %ID/g. t: tumor; k: kidney; b: bladder.
Figure 8
Figure 8
Representative maximum-intensity-projection PET/CT images of [68Ga]Ga-DOTA-AmBz-M(O)VK(HTK01166)-OH acquired at 1 h p.i. from LNCaP tumor-bearing mice (A) without and (B) with the co-injection of 2-PMPA (0.2 mg). The range of color bar is 0–5 %ID/g. t: tumor; k: kidney; b: bladder.
Figure 9
Figure 9
Representative radio-HPLC chromatograms of [68Ga]Ga-DOTA-AmBz-MVK(Ac)-OH obtained from mouse (A) blood and (B) urine samples collected at 5 and 15 min p.i., respectively. HPLC conditions were 82/18 A/B at a flow rate of 2 mL/min; A: H2O containing 0.1% TFA; B: CH3CN containing 0.1% TFA; HPLC column: Luna C18, 5 µm particle size, 100 Å pore size, 250 × 4.6 mm.
Figure 10
Figure 10
Representative radio-HPLC chromatograms of [68Ga]Ga-DOTA-AmBz-MVK(HTK01166)-OH obtained from mouse (A) blood and (B) urine samples collected at 5 and 15 min p.i., respectively. HPLC conditions were 72/28 A/B at a flow rate of 2 mL/min; A: H2O containing 0.1% TFA; B: CH3CN containing 0.1% TFA; HPLC column: Luna C18, 5 µm particle size, 100 Å pore size, 250 × 4.6 mm.
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